阅读说明：本技术 包含非规范碱基对的rna分子 (RNA molecules comprising non-canonical base pairs ) 是由 N·A·史密斯 M·B·王 D·张 T·J·多兰 M·蒂泽德 A·D·阿鲁 I·K·格里 于 2019-08-02 设计创作，主要内容包括：本发明涉及新的双链RNA(dsRNA)结构及其在基因沉默中的用途。(The present invention relates to novel double stranded rna (dsrna) structures and their use in gene silencing.)

1. A chimeric ribonucleic acid (RNA) molecule comprising a double-stranded RNA (dsRNA) region comprising a first sense ribonucleotide sequence that is at least 20 consecutive nucleotides in length and a first antisense ribonucleotide sequence that is at least 20 consecutive nucleotides in length, whereby the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are capable of hybridizing to each other to form the dsRNA region, wherein

i) The first sense ribonucleotide sequence consisting of a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide that are covalently linked in 5 'to 3' order,

ii) the first antisense ribonucleotide sequence consists of a second 5 'ribonucleotide, a second RNA sequence and a second 3' ribonucleotide being covalently linked in the 5 'to 3' order,

iii) the first 5 'ribonucleotide base-pairing with the second 3' ribonucleotide to form the terminal base pair of the dsRNA region,

iv) the second 5 'ribonucleotide base-pairing with the first 3' ribonucleotide to form the terminal base pair of the dsRNA region,

v) about 5% to about 40% of the ribonucleotides of the first sense ribonucleotide sequence and of the first antisense ribonucleotide sequence as a whole are base-paired or not base-paired in non-canonical base pairs,

vi) the dsRNA region does not comprise 20 consecutive canonical base pairs,

vii) the RNA molecule is capable of being processed in eukaryotic cells or in vitro, thereby cleaving the first antisense ribonucleotide sequence to produce a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length,

viii) the RNA molecule or at least some of the asRNA molecules or both are capable of reducing the expression or activity of a target RNA molecule in a eukaryotic cell, and

ix) the RNA molecule can be prepared enzymatically by transcription in vitro or in cells or both.

2. The chimeric RNA molecule of claim 1, wherein the first sense ribonucleotide sequence is covalently linked to the first antisense ribonucleotide sequence through a first connecting ribonucleotide sequence, the first linked ribonucleotide sequence comprises a loop sequence of at least 4 nucleotides, or 4 to 1000 ribonucleotides, or 4 to 200 ribonucleotides, or 4 to 50 ribonucleotides, or at least 10 nucleotides, or 10 to 1000 ribonucleotides, or 10 to 200 ribonucleotides, or 10 to 50 ribonucleotides in length, whereby the first linked ribonucleotide sequence is covalently linked to the second 3 'ribonucleotide and the first 5' ribonucleotide, or preferably covalently linked to said first 3 'ribonucleotide and said second 5' ribonucleotide, such that said sequence is comprised in a single continuous strand of RNA.

3. The chimeric RNA molecule of claim 2, wherein the loop sequence in the RNA molecule comprises one or more binding sequences complementary to an RNA molecule endogenous to the eukaryotic cell and/or the loop sequence in the RNA molecule comprises an open reading frame encoding a polypeptide or a functional polynucleotide.

4. The chimeric RNA molecule according to any one of claims 1 to 3, wherein about 5% to about 40% of the ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence of the dsRNA as a whole are base-paired in non-canonical base pairs, preferably in G: U base pairs.

5. The chimeric RNA molecule according to any of claims 1 to 4, the first antisense ribonucleotide sequence is fully complementary to a region of the target RNA, and the first sense ribonucleotide sequence differs in sequence from the region of the target RNA in that the C nucleotide in the region of the target RNA is substituted by a U nucleotide.

6. The chimeric RNA molecule according to any of claims 1 to 5, comprising a second sense ribonucleotide sequence, and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are connected by a first connecting ribonucleotide sequence, which comprises a loop sequence of at least 4 nucleotides in length, whereby the first connecting ribonucleotide sequence is covalently linked to the first 3' ribonucleotide and the second 5' ribonucleotide, and the RNA molecule further comprises a second connecting ribonucleotide sequence, which comprises a loop sequence of at least 4 nucleotides in length, and which is covalently linked to the second 3' ribonucleotide and the second sense ribonucleotide sequence.

7. The chimeric RNA molecule according to any of claims 1 to 5, which comprises a second antisense ribonucleotide sequence, and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are connected by a first connecting ribonucleotide sequence, which comprises a loop sequence of at least 4 nucleotides in length, whereby the first connecting ribonucleotide sequence is covalently linked to the second 3' ribonucleotide and the first 5' ribonucleotide, and the RNA molecule further comprises a second connecting ribonucleotide sequence, which comprises a loop sequence of at least 4 nucleotides in length, and which is covalently linked to the second 3' ribonucleotide and the second antisense ribonucleotide sequence.

8. The chimeric RNA molecule according to any of claims 1 to 5, comprising a second sense ribonucleotide sequence and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are capable of hybridizing to each other to form a second dsRNA region, and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are connected by a first connecting ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first connecting ribonucleotide sequence is covalently linked to the first 3' ribonucleotide and the second 5' ribonucleotide, and the RNA molecule optionally comprises a second connecting ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length and which is in contact with the second 3' ribonucleotide and the second sense ribonucleotide The nucleotide sequence is covalently linked, or it is covalently linked, to said second sense ribonucleotide sequence and said second antisense ribonucleotide sequence.

9. The chimeric RNA molecule according to any of claims 1 to 5, comprising a second sense ribonucleotide sequence and a second antisense ribonucleotide sequence, and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are connected by a first linking ribonucleotide sequence, which comprises a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to the second 3' ribonucleotide and the first 5' ribonucleotide, and the RNA molecule further comprises a second linking ribonucleotide sequence, which comprises a loop sequence of at least 4 nucleotides in length and which is covalently linked to the first 3' ribonucleotide and the second antisense ribonucleotide sequence, or which covalently links the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence .

10. The chimeric RNA molecule according to any of claims 6 to 9, wherein the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence each comprise consecutive nucleotides of at least 20 in length.

11. The chimeric RNA molecule according to any of claims 6 to 10, wherein the first and second sense ribonucleotide sequences are covalently linked by an intervening ribonucleotide sequence that is not related in sequence to the target RNA molecule or related in sequence to the target RNA molecule, or the first and second sense ribonucleotide sequences are covalently linked without an intervening ribonucleotide sequence.

12. The chimeric RNA molecule according to any of claims 6-11, wherein the first and second antisense ribonucleotide sequences are covalently linked by an intervening ribonucleotide sequence that is not related in sequence to the complement of the target RNA molecule or is related in sequence to the complement of the target RNA molecule, or the first and second antisense ribonucleotide sequences are covalently linked without an intervening ribonucleotide sequence.

13. The chimeric RNA molecule according to any of claims 6-12, wherein the total of 5% to 40% of the ribonucleotides of the second sense ribonucleotide sequence and of the second antisense ribonucleotide sequence are base-paired or not base-paired in non-canonical base pairs, preferably in a ratio of G: u base pairs, wherein the second dsRNA region does not comprise 20 consecutive canonical base pairs, and wherein the RNA molecule is capable of being processed in a eukaryotic cell or in vitro, whereby the second antisense ribonucleotide sequence is cleaved to produce a short antisense RNA (asrna) molecule of 20-24 ribonucleotides in length.

14. The chimeric RNA molecule according to any of claims 6-13, wherein the length of each linked ribonucleotide sequence is independently between 4 and about 2000 nucleotides, preferably the length of each linked ribonucleotide sequence is independently between 4 and about 1200 nucleotides, more preferably the length of each linked ribonucleotide sequence is independently between 4 and about 200 nucleotides, and most preferably the length of each linked ribonucleotide sequence is independently between 4 and about 50 nucleotides.

15. The chimeric RNA molecule according to any one of claims 6 to 14, further comprising a 5 'leader sequence or a 3' trailer sequence, or both.

16. A chimeric RNA molecule comprising a first RNA component and a second RNA component covalently linked to the first RNA component,

wherein the first RNA component comprises a first double-stranded RNA (dsRNA) region comprising a first sense ribonucleotide sequence and a first antisense ribonucleotide sequence that are capable of hybridizing to each other to form the first dsRNA region, and a first intervening ribonucleotide sequence of at least 4 nucleotides that covalently links the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence,

Wherein the second RNA component comprises a second sense ribonucleotide sequence, a second antisense ribonucleotide sequence and a second intervening ribonucleotide sequence of at least 4 ribonucleotides, which second intervening ribonucleotide sequence covalently links the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence hybridizes to the second antisense ribonucleotide sequence in the RNA molecule,

wherein in the first RNA component,

i) the first sense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides, being a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide, covalently linked in 5 'to 3' order,

ii) the first antisense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides, which are a second 5 'ribonucleotide, a second RNA sequence and a second 3' ribonucleotide, covalently linked in the order 5 'to 3',

iii) the first 5 'ribonucleotide base pairs with the second 3' ribonucleotide,

iv) the second 5 'ribonucleotide base pairs with the first 3' ribonucleotide,

v) the total of 5% to 40% of the ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are base-paired or not base-paired in non-canonical base pairs, and

vi) the first dsRNA region does not comprise 20 consecutive canonical base pairs,

wherein the chimeric RNA molecule is capable of being processed in eukaryotic cells or in vitro, whereby the first antisense ribonucleotide sequence is cleaved to yield a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length, and wherein

(a) The chimeric RNA molecule or at least some of the asRNA molecules, or both, are capable of reducing the expression or activity of a target RNA molecule in the eukaryotic cell, or

(b) The first antisense ribonucleotide sequence comprises a sequence of at least 20 consecutive ribonucleotides having a sequence of at least 50% identity, preferably at least 90% or 100% identity, in sequence with a region of the complement of the target RNA molecule, or

(c) Both (a) and (b).

17. The chimeric RNA molecule according to any of claims 1 to 16, wherein at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence are capable of base pairing with nucleotides of the first region of the target RNA molecule.

18. The chimeric RNA molecule according to any of claims 1 to 17, wherein the RNA molecule comprises two or more antisense ribonucleotide sequences and a sense ribonucleotide sequence base-paired therewith, said antisense sequences each being complementary, preferably fully complementary, to a region of the target RNA molecule.

19. The chimeric RNA molecule of claim 18, wherein the two or more antisense ribonucleotide sequences are complementary to different regions of the same target RNA molecule.

20. The chimeric RNA molecule of claim 18, wherein the two or more antisense ribonucleotide sequences are complementary to regions of different target RNA molecules.

21. The chimeric RNA molecule according to any one of claims 1 to 20, comprising a hairpin RNA (hprna) structure having a 5 'end, a sense ribonucleotide sequence of at least 21 nucleotides in length, an antisense ribonucleotide sequence that is fully base-paired with the sense ribonucleotide sequence over at least 21 consecutive nucleotides, an intervening loop sequence and a 3' end.

22. The chimeric RNA molecule according to any one of claims 1 to 20, comprising a single strand of ribonucleotides having a 5 'end, at least one sense ribonucleotide sequence of at least 21 nucleotides in length, an antisense ribonucleotide sequence that is fully base-paired with each sense ribonucleotide sequence over at least 21 consecutive nucleotides, at least two loop sequences and a 3' end.

23. The chimeric RNA molecule according to any one of claims 1 to 22, wherein about 15% to about 30%, or about 16% to about 25% of the ribonucleotides of the sense ribonucleotide sequence and of the antisense ribonucleotide sequence as a whole are base paired or not base paired in non-canonical base pairs, preferably in non-canonical base pairs, more preferably in G: U base pairs.

24. The chimeric RNA molecule according to any one of claims 1 to 23, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% of the non-canonical base pairs are gavu base pairs.

25. The chimeric RNA molecule according to any one of claims 1 to 24, wherein less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1% or none of the nucleotides in the dsRNA region are not base paired.

26. The chimeric RNA molecule according to any one of claims 1 to 25, wherein each of the 4 ribonucleotides to each of the 6 ribonucleotides in the dsRNA region form a non-canonical base pair or a non-base pairing, preferably a G: U base pair.

27. The chimeric RNA molecule according to any of claims 1 to 26, wherein the dsRNA region does not comprise 8 consecutive canonical base pairs.

28. The chimeric RNA molecule according to any of claims 1 to 27, wherein the dsRNA region comprises at least 8 consecutive canonical base pairs, preferably at least 8 but not more than 12 consecutive canonical base pairs.

29. The chimeric RNA molecule according to any one of claims 1 to 28, wherein all ribonucleotides in the or each dsRNA region are base paired in canonical base pairs or non-canonical base pairs.

30. The chimeric RNA molecule according to any of claims 1-28, wherein one or more ribonucleotides of the sense ribonucleotide sequence or one or more ribonucleotides of the antisense ribonucleotide sequence or both are not base-paired.

31. The chimeric RNA molecule according to any of claims 1-30, wherein the antisense RNA sequence is less than 100% identical in sequence to the complement of a region of the target RNA molecule, or about 80% to 99.9% identical, or about 90% to 98% identical, or about 95% to 98% identical.

32. The chimeric RNA molecule according to any of claims 1-30, wherein the antisense RNA sequence has 100% identity in sequence to a region of the target RNA molecule.

33. The chimeric RNA molecule according to any of claims 1-32, wherein the sense and/or antisense ribonucleotide sequence, preferably both, is at least 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, or about 100 to about 1000, or 20 to about 1000 nucleotides, or 20 to about 500 nucleotides in length.

34. The chimeric RNA molecule according to any of claims 1-33, wherein the number of ribonucleotides in the sense ribonucleotide sequence is from about 90% to about 110% of the number of ribonucleotides in the antisense ribonucleotide sequence.

35. The chimeric RNA molecule according to any of claims 1-34, wherein the number of ribonucleotides in the sense ribonucleotide sequence is the same as the number of ribonucleotides in the antisense ribonucleotide sequence.

36. The chimeric RNA molecule according to any one of claims 1-35, wherein the chimeric RNA molecule further comprises a 5 'extension sequence covalently linked to the first 5' ribonucleotide or a 3 'extension sequence covalently linked to the second 3' ribonucleotide, or both.

37. The chimeric RNA molecule according to any one of claims 1-36, wherein the chimeric RNA molecule further comprises a 5 'extension sequence covalently linked to the second 5' ribonucleotide or a 3 'extension sequence covalently linked to the first 3' ribonucleotide, or both.

38. The chimeric RNA molecule according to any of claims 1 to 37, comprising two or more identical or different dsRNA regions.

39. The chimeric RNA molecule according to any one of claims 1 to 38, wherein when expressed in a eukaryotic cell, a greater number of asRNA molecules of 22 and/or 20 ribonucleotides in length are formed when compared to the processing of similar RNA molecules having a corresponding dsRNA region that is fully base-paired in canonical base pairs.

40. An isolated and/or exogenous polynucleotide encoding a chimeric RNA molecule according to any one of claims 1 to 39.

41. The polynucleotide of claim 40 which is a DNA construct.

42. The polynucleotide of claim 40 or 41, operably linked to a promoter capable of directing expression of said RNA molecule in a host cell or in vitro.

43. The polynucleotide of claim 42, wherein the promoter is an RNA polymerase promoter, such as an RNA polymerase III promoter, an RNA polymerase II promoter, or a promoter that functions in vitro.

44. The polynucleotide of any one of claims 40-43, which encodes an RNA precursor molecule comprising an intron, preferably in the 5' extension sequence or in at least one loop sequence, wherein the intron can be spliced out in a host cell or during in vitro transcription of the polynucleotide.

45. A vector comprising a polynucleotide according to any one of claims 40-44.

46. The vector of claim 45, which is a viral vector.

47. A host cell comprising one or more or all of the following: the chimeric RNA molecule of any one of claims 1 to 39, a small RNA molecule produced by processing said chimeric RNA molecule, the polynucleotide of any one of claims 40-44, or the vector of claim 45 or claim 46.

48. The host cell of claim 47, which is a bacterial cell, a fungal cell such as a yeast cell, a plant cell or an animal cell, preferably a plant cell.

49. A host cell according to claim 47 or claim 48 which is dead and/or unable to reproduce.

50. The polynucleotide of claim 42 or claim 43 or the host cell according to any one of claims 47-49, which encodes and/or comprises a chimeric RNA molecule according to any one of claims 1-39, wherein the promoter region of said polynucleotide has a lower level of methylation, such as less than about 50%, less than about 40%, less than about 30% or less than about 20%, when compared to the promoter of the corresponding polynucleotide encoding an RNA molecule having a corresponding dsRNA region that is fully base-paired in canonical base pairs.

51. The host cell according to claim 50, preferably a plant cell or a fungal cell, comprising said chimeric RNA molecule or a small RNA molecule produced by processing said chimeric RNA molecule or both, wherein said chimeric RNA molecule comprises in 5 'to 3' order a first sense ribonucleotide sequence, a first connecting ribonucleotide sequence comprising a loop sequence and a first antisense ribonucleotide sequence.

52. The host cell according to any one of claims 47-51, which is a eukaryotic cell and comprises at least two copies of a polynucleotide encoding a chimeric RNA molecule according to any one of claims 1 to 39, and wherein

i) The eukaryotic cell has a reduced level of expression or activity of the target RNA molecule that is about the same or greater than the reduced level of expression or activity of the target RNA molecule if the cell has a single copy of the polynucleotide, and/or

ii) a reduced level of expression or activity of the target RNA molecule in the eukaryotic cell is lower when compared to a corresponding cell comprising an RNA molecule having a corresponding dsRNA region that is fully base paired in canonical base pairs.

53. A non-human organism comprising one or more or all of the following: a chimeric RNA molecule according to any one of claims 1 to 39, a small RNA molecule produced by processing said chimeric RNA molecule, a polynucleotide according to any one of claims 40 to 44 or 50, or a vector of claim 45 or claim 46, or a host cell according to any one of claims 47 to 52.

54. The non-human organism of claim 53, which is a transgenic non-human organism, preferably a plant or a fungus, comprising a polynucleotide according to any one of claims 40-44 or 50.

55. The non-human organism of claim 54, wherein said polynucleotide is stably integrated into the genome of said organism.

56. A method of producing the chimeric RNA molecule of any one of claims 1 to 39, the method comprising expressing the polynucleotide according to any one of claims 40-44 or 50 in a host cell or cell-free expression system.

57. The method of claim 56, further comprising at least partially purifying the RNA molecule.

58. A method of producing the non-human organism of claim 54 or claim 55, the method comprising introducing the polynucleotide according to any one of claims 40-44 or 50 into a cell so that it is stably integrated into the genome of the cell, and producing the non-human organism from the cell.

59. An extract of a host cell according to any one of claims 47 to 52, wherein the extract comprises one or more of: a chimeric RNA molecule according to any one of claims 1 to 39, a small RNA molecule produced by processing said chimeric RNA molecule, or a polynucleotide according to any one of claims 40 to 44 or 50.

60. A composition comprising one or more of the following in combination with one or more suitable carriers: a chimeric RNA molecule according to any of claims 1 to 39, a small RNA molecule produced by processing said chimeric RNA molecule, a polynucleotide according to any of claims 40 to 44 or 50, a vector according to claim 45 or claim 46, a host cell according to any of claims 47 to 52, or an extract according to claim 59.

61. The composition of claim 60, which is a pharmaceutical composition.

62. The composition of claim 60, which is suitable for application to plants growing in a field.

63. The composition according to any one of claims 60-62, further comprising at least one compound that enhances the stability of one or more of the following: the chimeric RNA molecule, RNA molecules and polynucleotides produced by processing the chimeric RNA molecule, and/or the at least one compound facilitates uptake of the RNA molecule, chimeric RNA molecule, or polynucleotide by cells of an organism.

64. The composition of claim 63, wherein the compound is a transfection facilitating agent.

65. A method of reducing or down-regulating the level and/or activity of a target RNA molecule in a cell or organism, the method comprising delivering to the cell or organism one or more of: the chimeric RNA molecule according to any one of claims 1 to 39, a small RNA molecule produced by processing said chimeric RNA molecule, a polynucleotide according to any one of claims 40 to 44 or 50, a vector of claim 45 or claim 46, a host cell of any one of claims 47 to 52, an extract of claim 59, or a composition according to any one of claims 60 to 64.

66. The method of claim 65, wherein said chimeric RNA molecule or a small RNA molecule produced by processing said chimeric RNA molecule, or both, is contacted with said cell or organism, preferably a plant cell, plant, fungus or insect, by topical application to said cell or organism, or provided to said organism in feed.

67. A method of reducing damage to a non-human organism caused by a pest or pathogen, the method comprising delivering to or contacting with the pest or pathogen one or more of: the chimeric RNA molecule according to any one of claims 1 to 39, a small RNA molecule produced by processing said chimeric RNA molecule, a polynucleotide according to any one of claims 40 to 44 or 50, a vector of claim 45 or claim 46, a host cell of any one of claims 47 to 52, an extract of claim 59, or a composition according to any one of claims 60 to 64.

68. A method of controlling a non-human organism, the method comprising delivering to the non-human organism one or more of: the chimeric RNA molecule according to any one of claims 1 to 39, the small RNA molecule produced by processing the chimeric RNA molecule, the polynucleotide according to any one of claims 40 to 44 or 50, the vector of claim 45 or claim 46, the host cell according to any one of claims 47 to 52, the extract of claim 59, or the composition according to any one of claims 60 to 64, wherein the RNA molecule small RNA molecule has a deleterious effect on the non-human organism.

69. The method of claim 68, wherein the non-human organism is an arthropod or plant.

70. The method of claim 69 wherein the non-human organism according to any one of claims 53-55 is a plant and the arthropod consumes the plant or portion thereof.

71. A method of preventing or treating a disease in a subject, the method comprising administering to the subject one or more of: the chimeric RNA molecule according to any one of claims 1 to 39, the small RNA molecule produced by processing the chimeric RNA molecule, the polynucleotide according to any one of claims 40 to 44 or 50, the vector of claim 45 or claim 46, the host cell according to any one of claims 47 to 52, the extract of claim 59, or the composition according to any one of claims 60 to 64, wherein the chimeric RNA molecule or the small RNA molecule produced by processing the chimeric RNA molecule, or both, has a beneficial effect on at least one symptom of the disease.

72. The method of claim 71, wherein said chimeric RNA molecule, polynucleotide, vector or composition is administered topically, orally or by injection.

73. The method of claim 71 or 72, wherein the subject is a vertebrate.

74. The method of claim 73, wherein the vertebrate is a mammal, such as a human.

75. A chimeric RNA molecule according to any one of claims 1 to 39, a small RNA molecule produced by processing said chimeric RNA molecule, a polynucleotide according to any one of claims 40 to 44 or 50, a vector of claim 45 or claim 46, a host cell according to any one of claims 47 to 52, an extract of claim 59, or a composition according to any one of claims 60 to 64, for use in preventing or treating a disease in a subject, wherein said chimeric RNA molecule or said small RNA molecule produced by processing said chimeric RNA molecule, or both, has a beneficial effect on at least one symptom of said disease.

76. Use of the chimeric RNA molecule according to any one of claims 1 to 39, a small RNA molecule produced by processing the chimeric RNA molecule, a polynucleotide according to any one of claims 40 to 44 or 50, a vector of claim 45 or claim 46, a host cell according to any one of claims 47 to 52, an extract of claim 59, or a composition according to any one of claims 60 to 64, in the manufacture of a medicament for preventing or treating a disease in a subject, wherein the chimeric RNA molecule or the small RNA molecule produced by processing the chimeric RNA molecule, or both, has a beneficial effect on at least one symptom of the disease.

77. A kit comprising one or more of: a chimeric RNA molecule according to any one of claims 1 to 39, a small RNA molecule produced by processing said chimeric RNA molecule, a polynucleotide according to any one of claims 40 to 44 or 50, a vector of claim 45 or claim 46, a host cell according to any one of claims 47 to 52, an extract of claim 59 or a composition according to any one of claims 60 to 64.

Technical Field

The present invention relates to novel double stranded rna (dsrna) structures and their use in gene silencing.

Background

RNA silencing is an evolutionarily conserved gene silencing mechanism in eukaryotes that is induced by double-stranded RNA (dsrna), which may be in a form known as hairpin RNA (hprna). In the basic RNA silencing pathway, dsRNA is processed by Dicer protein into short 20-25 nucleotide (nt) small RNA duplexes, one of which binds to the argonaute (ago) protein to form the RNA-induced silencing complex (RISC). The silencing complex uses small RNAs as a guide to find and bind complementary single-stranded RNAs, where cleavage of RNA by AGO proteins leads to its degradation.

In plants, there are a number of RNA silencing pathways, including the microrna (mirna), trans-acting small interfering RNA (tassirna), repeat-associated sirna (rasirna), and exogenous (viral and transgenic) sirna (exosirna) pathways. mirnas are small 20-24nt RNAs processed in the nucleus by Dicer-like 1(DCL1) from short stem-loop precursor RNAs transcribed from the MIR gene by RNA polymerase II. tassirna is a staged siRNA of predominantly 21nt in size derived from DCL4 processing of long dsRNA synthesized by RNA-dependent RNA polymerase 6(RDR6) from a miRNA-cleaved TAS RNA fragment. 24-nt rasiRNA is processed by DCL3, and precursor dsRNA is generated from repetitive DNA in the genome by the combined functions of plant-specific DNA-dependent RNA polymerase IV (PolIV) and RDR 2. The exosiRNA and rasiRNA pathways overlap, and both DCL4 and DCL3 are involved in exosiRNA processing. In addition to DCL1, DCL3, and DCL4, the model plant Arabidopsis (Arabidopsis thaliana) and other higher plants encode DCL2 or equivalents, which produce 22-nt siRNAs, including 22-nt exosiRNAs, and play a key role in systemic and transient gene silencing in plants. All these plant small RNAs are methylated at the 2 '-hydroxyl of the 3' terminal nucleotide by the HUA enhancer 1(HEN1) and this 3 'terminal 2' -O-methylation is believed to stabilize small RNAs in plant cells. mirnas, tasrnas, and exosirnas are functionally similar to small RNAs in animal cells, which are involved in post-transcriptional gene silencing or sequence-specific degradation of RNA in animals. However, rasiRNA is unique to plants and functions to direct de novo (de novo) cytosine methylation on the same class of DNA, a transcriptional gene silencing mechanism known as RNA-guided DNA methylation (RdDM).

dsRNA-induced RNA silencing has been widely utilized to reduce gene activity in various eukaryotic systems, and a number of gene silencing techniques have been developed. Different organisms are generally suitable for different gene silencing approaches. For example, long dsrnas (at least 100 base pairs in length) are less suitable for inducing RNA silencing in mammalian cells due to dsRNA-induced interferon responses, and thus shorter dsrnas (less than 30 base pairs) are typically used in mammalian cells, whereas hairpin RNAs (hprnas) with long dsRNA stems are very effective in plants. In plants, different RNA silencing pathways have led to different gene silencing techniques, such as artificial miRNA, artificial tassirna and virus-induced gene silencing techniques. However, to date, successful application of RNA silencing technology in plants has been achieved primarily through the use of long hpRNA transgenes. hpRNA transgene constructs typically comprise inverted repeats comprising fully complementary sense and antisense sequences (when transcribed from the dsRNA stem of the hpRNA) of the target gene sequence separated by a spacer sequence (forming a loop of the hpRNA) interposed between a promoter and a transcription terminator for expression in a plant cell. The function of the spacer sequence is to stabilize the inverted repeat DNA in bacteria during the construct preparation process. The dsRNA stem of the hpRNA transcript produced is processed by the DCL protein into siRNA that directs silencing of the target gene. hpRNA transgenes have been widely used to knock down gene expression, modify metabolic pathways, and enhance disease and pest resistance in plants to improve crops, and many successful applications of this technology in crop improvement have now been reported (Guo et al, 2016; Kim et al, 2019).

However, recent studies have shown that hpRNA transgenes suffer from self-induced transcriptional repression, compromising the stability and efficacy of target gene silencing. Although all transgenes may suffer from location or copy number dependent transcriptional silencing, hpRNA transgenes are unique in that they generate sirnas that can direct DNA methylation to their own sequences via the RdDM pathway and potentially lead to transcriptional self-silencing.

While dsRNA-induced gene silencing has proven to be a valuable tool for altering the phenotype of an organism, there is a need for alternative, preferably improved dsRNA molecules that can be used for RNAi.

Disclosure of Invention

The present inventors contemplate a new design of a genetic construct for producing an RNA molecule comprising one or more double stranded RNA region comprising a plurality of non-canonical base-paired nucleotides or non-base-paired nucleotides, or both, including forms having two or more loop sequences, referred to herein as loop-terminal dsrna (ledrna). These RNA molecules have one or more of the following characteristics: they are readily synthesized, accumulate to higher levels in cells following transcription of the genetic constructs encoding them, they more readily form dsRNA structures and induce efficient silencing of target RNA molecules in eukaryotic cells, and may form circular RNA molecules following processing in plant cells. RNA molecules are also effective when applied topically to plants or fed to animals (e.g., insects).

In a first aspect, the present invention provides a chimeric ribonucleic acid (RNA) molecule comprising a double-stranded RNA (dsRNA) region comprising a first sense ribonucleotide sequence being at least 20 consecutive nucleotides in length and a first antisense ribonucleotide sequence being at least 20 consecutive nucleotides in length, whereby the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are capable of hybridizing to each other to form the dsRNA region, wherein

i) The first sense ribonucleotide sequence consists of a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide that are covalently linked in 5 'to 3' order,

ii) the first antisense ribonucleotide sequence consists of a second 5 'ribonucleotide, a second RNA sequence and a second 3' ribonucleotide being covalently linked in the order 5 'to 3',

iii) the first 5 'ribonucleotide base-pairs with the second 3' ribonucleotide to form the terminal base pair of the dsRNA region,

iv) the second 5 'ribonucleotide base-pairing with the first 3' ribonucleotide to form the terminal base pair of the dsRNA region,

v) about 5% to about 40% of the ribonucleotides of the first sense ribonucleotide sequence and of the first antisense ribonucleotide sequence as a whole are base-paired or not base-paired in non-canonical base pairs,

vi) the dsRNA region does not contain 20 consecutive canonical base pairs,

vii) the RNA molecule can be processed in eukaryotic cells or in vitro to cleave the first antisense ribonucleotide sequence to produce a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length,

viii) the RNA molecule or at least some of the asRNA molecules or both are capable of reducing the expression or activity of a target RNA molecule in a eukaryotic cell, and

ix) RNA molecules can be prepared enzymatically by transcription in vitro or in cells or both.

In a preferred embodiment of the first aspect, the first sense ribonucleotide sequence is covalently linked to the first antisense ribonucleotide sequence by a first linking ribonucleotide sequence, said first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides, or 4 to 1000 ribonucleotides, or 4 to 200 ribonucleotides, or 4 to 50 ribonucleotides, or at least 10 nucleotides, or 10 to 1000 ribonucleotides, or 10 to 200 ribonucleotides, or 10 to 50 ribonucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to the first 3 'ribonucleotide and the second 5' ribonucleotide, or to the second 3 'ribonucleotide and the first 5' ribonucleotide, such that said sequences are comprised in a single continuous strand of RNA. In another embodiment, the first linked ribonucleotide sequence is covalently linked to the second 3 'ribonucleotide and the first 5' ribonucleotide, or preferably to the first 3 'ribonucleotide and the second 5' ribonucleotide, such that the sequence is comprised in a single continuous strand of RNA.

In its simplest form, such RNA molecules are referred to as hairpin RNA (hprna). In a more preferred embodiment, about 5% to about 40% of the ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence of the dsRNA are base-paired in non-canonical base pairs (preferably G: U base pairs). That is, all ribonucleotides of the first sense ribonucleotide sequence base pair with ribonucleotides of the first antisense ribonucleotide sequence in either regular base pairs or non-regular base pairs, whereby the dsRNA region comprises 20 consecutive base pairs, including some non-regular base pairs. The dsRNA region therefore does not contain 20 consecutive canonical base pairs. In a more preferred embodiment of the hpRNA of the invention, the first antisense ribonucleotide sequence is fully complementary to a region of the target RNA. In this embodiment, the first sense ribonucleotide sequence differs in sequence from the region of the target RNA in that the C nucleotide in the region of the target RNA is substituted with the U nucleotide in the hpRNA. Such molecules are exemplified in examples 6-11 by the inclusion of G: hairpin RNA of U base pairs. In preferred embodiments, the length of the first antisense ribonucleotide sequence is from 20 to about 1000 nucleotides, or from 20 to about 500 nucleotides, or other lengths as described herein. More preferably, the hpRNA is produced in or introduced into a plant cell or a fungal cell. In these embodiments, the target RNA can be a transcript of an endogenous gene in the cell, or a transcript of an endogenous gene of a plant pathogen, or a transcript of an endogenous gene of a pest (such as an insect pest).

In a more preferred embodiment, the RNA molecule comprises a second sense ribonucleotide sequence, and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are linked by a first linking ribonucleotide sequence, said first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to a first 3' ribonucleotide and a second 5' ribonucleotide, and the RNA molecule further comprises a second linking ribonucleotide sequence, said second linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, and which is covalently linked to the second 3' ribonucleotide and the second sense ribonucleotide sequence, thereby forming a ledRNA structure. In an alternative preferred embodiment, the RNA molecule comprises a second antisense ribonucleotide sequence, and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are connected by a first linking ribonucleotide sequence, said first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to the second 3' ribonucleotide and the first 5' ribonucleotide, and the RNA molecule further comprises a second linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, and which is covalently linked to the second 3' ribonucleotide and the second antisense ribonucleotide sequence, thereby forming a ledRNA structure.

In another preferred embodiment, the RNA molecule comprises a second sense ribonucleotide sequence and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are capable of hybridizing to each other to form a second dsRNA region and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are connected by a first connecting ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first connecting ribonucleotide sequence is covalently linked to a first 3' ribonucleotide and a second 5' ribonucleotide and the RNA molecule further or optionally comprises a second connecting ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length and which is covalently linked to the second 3' ribonucleotide and the second sense ribonucleotide sequence, or which covalently links the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, thereby forming a ledRNA structure. In another preferred embodiment, the RNA molecule comprises a second sense ribonucleotide sequence and a second antisense ribonucleotide sequence, and the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are linked by a first linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length, whereby the first linking ribonucleotide sequence is covalently linked to a second 3' ribonucleotide and a first 5' ribonucleotide, and the RNA molecule further or optionally comprises a second linking ribonucleotide sequence comprising a loop sequence of at least 4 nucleotides in length and which is covalently linked to the first 3' ribonucleotide and the second antisense ribonucleotide sequence, or which covalently links the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, thereby forming a ledRNA structure. In a more preferred embodiment, the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, if present in an RNA molecule, each comprise consecutive nucleotides of at least 20 in length. In these embodiments, the first and second sense ribonucleotide sequences can be covalently linked by an intervening ribonucleotide sequence that is not related or associated in sequence with the target RNA molecule, or the first and second sense ribonucleotide sequences can be covalently linked without an intervening nucleotide sequence. The first and second sense ribonucleotide sequences can form a contiguous sense ribonucleotide region that is at least 50% identical in sequence to the target RNA molecule. In further embodiments, the first and second antisense ribonucleotide sequences can be covalently linked by an intervening ribonucleotide sequence that is not related in sequence or related in sequence to the complement of the target RNA molecule, or the first and second antisense ribonucleotide sequences are covalently linked without an intervening nucleotide sequence. The first and second antisense sense ribonucleotide sequences can form a contiguous antisense ribonucleotide region that is at least 50% identical in sequence to the complement of the target RNA molecule. In these embodiments, the RNA molecule comprises a second sense ribonucleotide sequence and a second antisense ribonucleotide sequence that hybridize by base pairing, preferably that between 5% and 40% of the total ribonucleotides of the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are base paired in non-canonical base pairs or not base paired (preferably in G: U base pairs), wherein the second dsRNA region does not comprise 20 consecutive canonical base pairs, and wherein the RNA molecule is capable of being processed in a eukaryotic cell or in vitro, whereby the second antisense ribonucleotide sequence is cleaved to produce a short antisense RNA (asrna) molecule of 20-24 ribonucleotides in length.

In a most preferred embodiment, each sense ribonucleotide sequence and in total 5% to 40% of the ribonucleotides of its corresponding antisense ribonucleotide sequence that hybridizes by base pairing, are base-paired or not base-paired in non-canonical base pairs, and the entire RNA molecule does not comprise 20 consecutive canonical base pairs in any of its dsRNA regions, and wherein the RNA molecule is capable of being processed in eukaryotic cells or in vitro, whereby each antisense ribonucleotide sequence is cleaved to produce a short antisense RNA (asrna) molecule of 20-24 ribonucleotides in length, is considered for each dsRNA region within the RNA molecule.

In a preferred embodiment, each linked ribonucleotide sequence is independently between 4 and about 2000 nucleotides, preferably between 4 and about 1200 nucleotides, more preferably between 4 and about 200 nucleotides, and most preferably between 4 and about 50 nucleotides in length. In one embodiment, the RNA molecule further comprises a 5 'leader sequence or a 3' trailer sequence, or both.

In a second aspect, the present invention provides a chimeric ribonucleic acid (RNA) molecule comprising a double-stranded RNA (dsRNA) region comprising a sense ribonucleotide sequence and an antisense ribonucleotide sequence that are capable of hybridizing to each other to form a dsRNA region, wherein

i) The sense ribonucleotide sequence consists of a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide being covalently linked in the order of 5 'to 3',

ii) the antisense ribonucleotide sequence consists of a second 5 'ribonucleotide, a second RNA sequence and a second 3' ribonucleotide being covalently linked in the order 5 'to 3',

iii) the first 5 'ribonucleotide base-pairs with the second 3' ribonucleotide to form the terminal base pair of the dsRNA region,

iv) the second 5 'ribonucleotide base-pairing with the first 3' ribonucleotide to form the terminal base pair of the dsRNA region,

v) about 5% to about 40% of the ribonucleotides of the sense ribonucleotide sequence and the antisense ribonucleotide sequence as a whole are base-paired or not base-paired in non-canonical base pairs,

vi) the dsRNA region does not contain 20 consecutive canonical base pairs,

vii) RNA molecules capable of being processed in eukaryotic cells or in vitro, thereby cleaving the antisense ribonucleotide sequence to produce short antisense RNA (asRNA) molecules of 20-24 ribonucleotides in length,

viii) the RNA molecule or at least some of the asRNA molecules or both are capable of reducing the expression or activity of a target RNA molecule in a eukaryotic cell, and

ix) RNA molecules can be prepared enzymatically by transcription in vitro or in cells or both.

As will be appreciated by the skilled person, each of the embodiments relating to the first aspect apply to the second aspect, except for the case where the length of the sense ribonucleotide sequence and the antisense ribonucleotide sequence is less than 20 consecutive nucleotides.

In a third aspect, the invention provides a ribonucleic acid (RNA) molecule comprising a first RNA component, a second RNA component covalently linked to the first RNA component, and optionally one or more or all of: (i) a linking ribonucleotide sequence covalently linked to the first and second RNA components, (ii) a 5 'leader sequence and (iii) a 3' trailer sequence,

wherein the first RNA component consists of, in 5 'to 3' order, a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide, wherein the first 5 'and 3' ribonucleotides in the RNA molecule are base-paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 consecutive ribonucleotides, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence of at least 20 consecutive ribonucleotides, wherein the first antisense ribonucleotide sequence is hybridized to the first sense ribonucleotide sequence in the RNA molecule, wherein the first antisense ribonucleotide sequence is capable of hybridizing to a first region of a target RNA molecule,

Wherein, if a linking ribonucleotide sequence is present, via said linking ribonucleotide sequence, or if a linking ribonucleotide sequence is not present, directly, said second RNA component is covalently linked to said first 5 'ribonucleotide or to said first 3' ribonucleotide,

wherein the second RNA component consists of, in 5 'to 3' order, a second 5 'ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide, wherein the second 5 'and 3' ribonucleotides are base-paired with each other in the RNA molecule, wherein the second RNA sequence comprises a second sense ribonucleotide sequence, a second loop sequence of at least 4 ribonucleotides, and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence hybridizes to the second antisense ribonucleotide sequence in the RNA molecule;

wherein the 5' leader sequence, if present, consists of a ribonucleotide sequence that is covalently linked to the first 5' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide or to the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide, and

Wherein the 3' trailer sequence, if present, consists of a ribonucleotide sequence that is covalently linked to the second 3' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or to the first 3' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide.

In a fourth aspect, the present invention provides an RNA molecule comprising a first RNA component, a second RNA component covalently linked to the first RNA component, and optionally one or more or all of: (i) a linking ribonucleotide sequence covalently linked to the first and second RNA components, (ii) a 5 'leader sequence and (iii) a 3' trailer sequence,

wherein the first RNA component consists of a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide in the 5 'to 3' order, wherein the first 5 'and 3' ribonucleotides are in base pair, wherein the first RNA sequence comprises a first sense ribonucleotide sequence, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence, wherein the first sense ribonucleotide sequence and first antisense ribonucleotide sequence each consist of at least 20 consecutive ribonucleotides, wherein at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are fully base paired with at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence, wherein at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence or at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence are in sequence with the target RNA component, respectively The first regions of either or both the daughter and its complement are the same,

Wherein, if the linking ribonucleotide sequence is present, the second RNA component is covalently linked to the first 5 'ribonucleotide or the first 3' ribonucleotide via the linking ribonucleotide sequence,

wherein the second RNA component consists of a second 5 'ribonucleotide, a second RNA sequence and a second 3' ribonucleotide in the 5 'to 3' order, wherein the second 5 'and 3' ribonucleotides are base-paired, wherein the second RNA sequence comprises a second sense ribonucleotide sequence, a second loop sequence of at least 4 ribonucleotides and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence is base-paired with the second antisense ribonucleotide sequence,

wherein the 5' leader sequence, if present, consists of a ribonucleotide sequence that is covalently linked to the first 5' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide or to the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide, and

Wherein the 3' trailer sequence, if present, consists of a ribonucleotide sequence that is covalently linked to the second 3' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or to the first 3' ribonucleotide if the second RNA component is linked to the first 5 ' ribonucleotide.

In a preferred embodiment of the above aspect, the RNA molecule of the invention is a chimeric RNA molecule.

In a fifth aspect, the present invention provides a chimeric RNA molecule comprising a first RNA component and a second RNA component covalently linked to the first RNA component,

wherein the first RNA component comprises a first double-stranded RNA (dsRNA) region comprising a first sense ribonucleotide sequence and a first antisense ribonucleotide sequence that are capable of hybridizing to each other to form a first dsRNA region, and a first intervening ribonucleotide sequence of at least 4 nucleotides that covalently links the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence,

Wherein the second RNA component comprises a second sense ribonucleotide sequence, a second antisense ribonucleotide sequence and a second intervening ribonucleotide sequence of at least 4 ribonucleotides, which second intervening ribonucleotide sequence covalently links the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence in the RNA molecule hybridizes to the second antisense ribonucleotide sequence,

wherein in the first RNA component,

i) the first sense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides, being a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide, covalently linked in 5 'to 3' order,

ii) the first antisense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides, which are a second 5 'ribonucleotide, a second RNA sequence and a second 3' ribonucleotide, covalently linked in 5 'to 3' order,

iii) the first 5 'ribonucleotide base pairs with the second 3' ribonucleotide,

iv) the second 5 'ribonucleotide base pairs with the first 3' ribonucleotide,

v) the total of 5% to 40% of the ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are base-paired or not base-paired in non-canonical base pairs, and

vi) the first dsRNA region does not comprise 20 consecutive canonical base pairs,

wherein the chimeric RNA molecule is capable of being processed in eukaryotic cells or in vitro, whereby the first antisense ribonucleotide sequence is cleaved to yield a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length, and wherein

(a) The chimeric RNA molecule or at least some of the asRNA molecules, or both, are capable of reducing the expression or activity of a target RNA molecule in the eukaryotic cell, or

(b) The first antisense ribonucleotide sequence comprises a sequence of at least 20 consecutive ribonucleotides having a sequence of at least 50% identity, preferably at least 90% or 100% identity, in sequence with a region of the complement of the target RNA molecule, or

(c) Both (a) and (b).

In embodiments where the RNA molecule has a first RNA component, the first 5 'ribonucleotide and the first 3' ribonucleotide of the first RNA component base pair with each other. This base pair is defined herein as the terminal base pair of the dsRNA region formed by self-hybridization of the first RNA component. In embodiments where the first sense ribonucleotide sequence is covalently linked to the first 5 'ribonucleotide without any intervening nucleotides and the first antisense ribonucleotide sequence is covalently linked to the first 3' ribonucleotide without any intervening nucleotides, the first 5 'ribonucleotide is directly linked to one of the sense sequence and the antisense sequence and the first 3' ribonucleotide is directly linked to the other of the sense sequence and the antisense sequence.

In a preferred embodiment, at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence are capable of base pairing with nucleotides of the first region of the target RNA molecule. In this case, base pairing may be canonical or non-canonical, e.g., having at least some G: u base pairs. Independently for each G: u base pairs, G may be in the first region of the target RNA molecule, or preferably in the first antisense ribonucleotide sequence. Optionally, at least 20 consecutive ribonucleotides of not the first antisense ribonucleotide sequence are each base paired with a nucleotide of the first region of the target RNA molecule. For example, 1, 2, 3, 4, or 5 ribonucleotides of at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence are not base paired with the first region of the target RNA molecule. In one embodiment, the first sense ribonucleotide sequence is covalently linked to the first 5 'ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is covalently linked to the first 3' ribonucleotide without any intervening nucleotides, or both.

In one embodiment of the above aspect, the RNA molecule comprises one or more linking ribonucleotide sequence, wherein the linking ribonucleotide sequence is related in sequence to the target RNA molecule, at least in part identical to a region of the target RNA molecule or at least in part identical to the complement of a region of the target RNA molecule. In preferred embodiments, the linking ribonucleotide sequence forms part of a continuous sense sequence together with the sense sequence in the first and second RNA component or forms part of a continuous antisense sequence together with the antisense sequence in the first and second RNA component. In one embodiment, the RNA molecule comprises a linked ribonucleotide sequence, wherein the linked ribonucleotide sequence is less than 20 ribonucleotides. In one embodiment, the linking ribonucleotide sequence hybridizes to a target RNA molecule. In one embodiment, the linking ribonucleotide sequence is the same as a portion of the complement of the target RNA molecule. In one embodiment, the linked ribonucleotide sequence is between 1 and 10 ribonucleotides.

In embodiments of the above aspect, the RNA molecule comprises one or more or all of: (i) a linking ribonucleotide sequence covalently linking the first and second RNA components, (ii) a 5 'extension sequence and (iii) a 3' extension sequence, wherein, respectively, if a 5 'extension sequence is present, it consists of a ribonucleotide sequence covalently linked to the first RNA component or the second RNA component, and wherein if a 3' extension sequence is present, it consists of a ribonucleotide sequence covalently linked to the second RNA component or the first RNA component. In one embodiment, the first RNA component and the second RNA component are covalently linked by a linking ribonucleotide sequence. In another embodiment, the first RNA component and the second RNA component are directly linked without any linking ribonucleotide sequence.

In an embodiment of the first to fifth aspect, the RNA molecule comprises two or more sense ribonucleotide sequences, each sense ribonucleotide sequence being identical in sequence to a region of the target RNA molecule, and the RNA molecule comprises one or more antisense ribonucleotide sequences based on pairing with the sense ribonucleotide sequence, wherein the one or more antisense sequences are complementary, preferably fully complementary, to the region of the target molecule. In one embodiment, the two or more sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule, which may or may not be contiguous in the target RNA molecule. In one embodiment, the two or more sense ribonucleotide sequences are identical in sequence to regions of different target RNA molecules. In one embodiment, the two or more sense ribonucleotide sequences have no intervening loop sequence, i.e., they are contiguous with respect to the target RNA molecule.

In a preferred embodiment of the first to fifth aspect, the RNA molecule comprises two or more antisense ribonucleotide sequences and a sense ribonucleotide sequence base-paired therewith, said antisense sequences each being complementary, preferably fully complementary, to a region of the target RNA molecule. The region to which the target RNA molecule is complementary may or may not be contiguous in the target RNA molecule. In one embodiment, the two or more antisense ribonucleotide sequences are complementary to different regions of the same target RNA molecule. In one embodiment, the second of the two or more antisense ribonucleotide sequences is complementary to a region of the target RNA molecule that is different from the first of the two or more antisense ribonucleotide sequences. In a preferred embodiment, the two or more antisense ribonucleotide sequences have no intervening loop sequence, i.e.they are contiguous with respect to the complement of the target RNA molecule. In preferred embodiments, one or both of the two or more antisense and sense ribonucleotide sequences base pair along their entire length by canonical base pairs, or by some canonical and some non-canonical base pairs, preferably G: U base pairs.

In a preferred embodiment of the first to fifth aspects, the RNA molecule is a single-stranded ribonucleotide. In its simplest form, an RNA molecule comprises a hairpin RNA (hprna) structure having a 5 'end, a sense ribonucleotide sequence that is at least 21 nucleotides in length, an antisense ribonucleotide sequence that is fully base paired to the sense ribonucleotide sequence over at least 21 consecutive nucleotides, an intervening loop sequence, and a 3' end. The RNA molecule may comprise a 5 '-leader sequence and/or a 3' -trailer sequence. In another form, the RNA molecule comprises a single-stranded ribonucleotide having a 5 'end, at least one sense ribonucleotide sequence that is at least 21 nucleotides in length, an antisense ribonucleotide sequence that is fully base-paired with each sense ribonucleotide sequence over at least 21 consecutive nucleotides, at least two loop sequences and a 3' end. The 5 'to 3' order can be a sense ribonucleotide sequence and then an antisense ribonucleotide sequence, or vice versa. In one embodiment, the 5 'ribonucleotide and the 3' ribonucleotide are contiguous, each base pairing and not directly covalently bound, see, e.g., FIG. 1.

In another embodiment of the first to fifth aspects, the RNA molecule comprises a first antisense ribonucleotide sequence that hybridizes to a first region of the target RNA; a second antisense ribonucleotide sequence that hybridizes to a second region of a target RNA that is different from the first region of the target RNA; and said RNA molecule comprises only one sense ribonucleotide sequence having at least 50% sequence identity to said target RNA, wherein said two antisense sequences are not contiguous in said RNA molecule. In one embodiment, the first and second regions of the target RNA are contiguous in the target RNA. Alternatively, they are not continuous.

In another embodiment of the first to fifth aspect, the RNA molecule comprises a first sense ribonucleotide sequence that is at least 60% identical to a first region of the target RNA; and a second sense ribonucleotide sequence that is at least 60% identical to a second region of a target RNA that is different from the first region of the target RNA; and said RNA molecule comprises only one antisense ribonucleotide sequence that hybridizes to said target RNA, wherein said two sense sequences are not contiguous in said RNA molecule. In one embodiment, the first and second regions of the target RNA are contiguous in the target RNA molecule. Alternatively, they are not continuous. In preferred embodiments, the first and second sense ribonucleotide sequences are each independently at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the corresponding region of the target RNA, i.e., the first sense sequence can have at least 70% identity to its target region and the second sequence at least 80% identity to its target sequence, and so on.

In a preferred embodiment of the first to fourth aspect, the RNA molecule is a single-stranded ribonucleotide having a 5 'end, at least one sense ribonucleotide sequence being at least 21 nucleotides in length, an antisense ribonucleotide sequence being fully base-paired with each sense ribonucleotide sequence over at least 21 consecutive nucleotides, at least two loop sequences and a 3' end. In a more preferred embodiment, the base pairing in the RNA molecule is comprised in a double-stranded region of at least 21 consecutive base pairs in length, which double-stranded region comprises some non-canonical base pairs, most preferably some G: U base pairs, said double-stranded region comprising at least one sense ribonucleotide sequence of at least 21 nucleotides in length.

In a preferred embodiment of the third and fourth aspects, the second RNA component is characterized by:

i) the second sense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides, being a second 5 'ribonucleotide, a third RNA sequence and a third 3' ribonucleotide, covalently linked in the order 5 'to 3',

ii) the second antisense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides, which are a third 5 'ribonucleotide, a fourth RNA sequence and a second 3' ribonucleotide, covalently linked in the order 5 'to 3',

iii) the second 5 'ribonucleotide base pairs with the second 3' ribonucleotide,

iv) the third 3 'ribonucleotide base-pairing with the third 5' ribonucleotide,

wherein the chimeric RNA molecule is capable of being processed in eukaryotic cells or in vitro, whereby the second antisense ribonucleotide sequence is cleaved to yield a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length. Most preferably, the asRNA molecule produced from the second antisense sequence is capable of reducing the expression of the target RNA without combining with the asRNA produced from the first antisense sequence of the first RNA component or combining with the asRNA produced from the first antisense sequence of the first RNA component.

In a preferred embodiment of the fifth aspect, the second RNA component is characterized by:

i) the second sense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides, being a third 5 'ribonucleotide, a third RNA sequence and a third 3' ribonucleotide, covalently linked in the order 5 'to 3',

ii) the second antisense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides, which are a fourth 5 'ribonucleotide, a fourth RNA sequence and a fourth 3' ribonucleotide, covalently linked in the order 5 'to 3',

iii) the third 5 'ribonucleotide base pairs with the fourth 3' ribonucleotide,

iv) the third 3 'ribonucleotide base-pairing with the third 5' ribonucleotide,

wherein the chimeric RNA molecule is capable of being processed in eukaryotic cells or in vitro, whereby the second antisense ribonucleotide sequence is cleaved to yield a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length.

In each of the above preferred embodiments, it is more preferred that the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence, and/or the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, and/or that each sense ribonucleotide sequence and its hybridized corresponding antisense ribonucleotide sequence collectively have 5% to 40% of the ribonucleotides base-paired or not base-paired in non-regular base pairs, and/or that the dsRNA region formed between the complementary sense and antisense sequences does not comprise 20 consecutive regular base pairs. More preferably, the ribonucleotides of the sense ribonucleotide sequence and its corresponding antisense ribonucleotide sequence (preferably for each dsRNA region of an RNA molecule) are collectively about 12%, about 15%, about 18%, about 21%, about 24%, about 27%, about 30%, or 10% to 30%, or 15% to 30%, or even more preferably 16% to 25% base paired, or not base paired, in non-canonical base pairs. Even more preferably, generally about 12%, about 15%, about 18%, about 21%, about 24%, about 27%, about 30%, 10% to 30%, or 15% to 30%, or even more preferably 16% to 25% of the ribonucleotides of one or more dsRNA regions in an RNA molecule are base paired in non-canonical base pairs, or all other ribonucleotides of one or more dsRNA regions in an RNA molecule are base paired in canonical base pairs. In preferred embodiments, the first or second dsRNA region, or the sum of the non-canonical base pairs in all dsRNA regions, is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% G: U base pairs.

Most preferably, in these embodiments,

(a) the chimeric RNA molecule or at least some of the asRNA molecules, or both, are capable of reducing the expression or activity of a target RNA molecule in the eukaryotic cell, or

(b) The first or second antisense ribonucleotide sequence in an RNA molecule, preferably each antisense ribonucleotide sequence, comprises a sequence of at least 20 consecutive ribonucleotides having a sequence of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% or 100% identity to a region of the complement of a target RNA molecule, or both (a) and (b).

In an embodiment of the first to fifth aspect, the RNA molecule comprises a 5 'leader sequence or a 5' extension sequence. In one embodiment, the RNA molecule comprises a 3 'trailer sequence or a 3' extension sequence. In a preferred embodiment, the RNA molecule comprises a 5 'leader/extension sequence and a 3' trailer/extension sequence.

In an embodiment of the first to fifth aspect, each ribonucleotide of the RNA molecule is covalently linked to two other nucleotides, i.e. it is a covalently closed loop. Alternatively, the RNA molecule may be represented as dumbbell-shaped (fig. 1), but with a gap or nick in a portion of the double-stranded structure.

In an embodiment of the first to fifth aspect, at least one or all loop sequences of the RNA molecule are longer than 20 nucleotides. In preferred embodiments, at least one loop of the RNA molecule is 4 to 1200 ribonucleotides or 4 to 1000 ribonucleotides in length. In a more preferred embodiment, all loops are 4 to 1000 ribonucleotides in length. In a more preferred embodiment, at least one loop of the RNA molecule is 4 to 200 ribonucleotides in length. In an even more preferred embodiment, all loops are 4 to 200 ribonucleotides in length. In an even more preferred embodiment, at least one loop of the RNA molecule is 4 to 50 ribonucleotides in length. In a most preferred embodiment, all loops are 4-50 ribonucleotides in length. In embodiments, the minimum length of the loop is 20 nucleotides, 30 nucleotides, 40 nucleotides, or 50 nucleotides. In one embodiment, the eukaryotic cell is a vertebrate or plant cell and each loop of the RNA molecule is independently 20 to 50 ribonucleotides, or 20 to 40 ribonucleotides, or 20 to 30 ribonucleotides in length.

In a preferred embodiment, at least one loop sequence in the RNA molecule comprises one or more binding sequences that are complementary to an RNA molecule that is endogenous to the eukaryotic cell (e.g., miRNA or other regulatory RNA in the eukaryotic cell). As will be readily appreciated, this feature may be combined with any of the loop length features, non-canonical base pairing, and any other features described above for RNA molecules. In one embodiment, at least one loop sequence comprises multiple binding sequences for a miRNA, or both. In one embodiment, at least one loop sequence in the RNA molecule comprises an open reading frame encoding a polypeptide or functional polynucleotide. The open reading frame is preferably operably linked to a translation initiation sequence, whereby the open reading frame is capable of translation in a eukaryotic cell of interest. For example, the translation initiation sequence comprises or is contained in an Internal Ribosome Entry Site (IRES). The IRES is preferably a eukaryotic IRES. The translated polypeptide is preferably 50-400 amino acid residues in length, or 50-300 or 50-250 or 50-150 amino acid residues in length. When such RNA molecules are produced in a plant cell, they can be processed to form a circular RNA molecule comprising most or all of the loop sequence, and can be translated to provide high levels of the polypeptide.

In embodiments of the first to fifth aspects, the RNA molecule has no, or one or two or more bulges (bulks) in the double-stranded region. Herein, a protuberance is a nucleotide in a sense or antisense ribonucleotide sequence, or two or more contiguous nucleotides that are not base paired in a dsRNA region and that do not have mismatched nucleotides at corresponding positions in the complementary sequence of the dsRNA region. The dsRNA region of the RNA molecule can comprise a sequence of more than 2 or 3 nucleotides in the sense or antisense sequence or both, which loops from the dsRNA region when the dsRNA structure is formed. The looped-out sequence may itself form some internal base pairing, for example it may itself form a stem-loop structure.

In embodiments of the first to fifth aspects, the RNA molecule has no, or one or two or more bulges in the double-stranded region. Herein, a protuberance is a nucleotide in a sense or antisense ribonucleotide sequence, or two or more contiguous nucleotides that are not base paired in a dsRNA region and that do not have mismatched nucleotides at corresponding positions in the complementary sequence of the dsRNA region. The dsRNA region of the RNA molecule can comprise a sequence of more than 2 or 3 nucleotides in the sense or antisense sequence or both, which loops from the dsRNA region when the dsRNA structure is formed. The looped-out sequence may itself form some internal base pairing, for example it may itself form a stem-loop structure.

In one embodiment, the RNA molecule has three, four or more loops. In a preferred embodiment, the RNA molecule has only two loops. In one embodiment, the first duplex region or the first and second dsRNA regions, or each dsRNA region, of the RNA molecule comprises one or two or more nucleotides that are not base paired in the duplex region, or independently comprises at most 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the nucleotides that are not base paired in the duplex region.

In a preferred embodiment of the first to fifth aspect, the target RNA molecule or the RNA molecule or both of the invention is in a eukaryotic cell. For example, the eukaryotic cell may be a plant cell, an animal cell, or a fungal cell. In one embodiment, the eukaryotic cell is a fungal cell, e.g., a cell of a fungal pathogen of one or more plant species, such as Fusarium (Fusarium) species, Verticillium (Verticillium) species, or a powdery mildew (powdery mildew) causing fungus. In one embodiment, the eukaryotic cell is an arthropod cell, such as an insect cell. Preferred insects are sap-sucking insects, such as aphids. For example, the insect may be a Lepidopteran (Lepidopteran) insect, a Coleopteran (Coleopteran) insect, or a Dipteran insect. In one embodiment, the RNA molecules of the invention are produced in a cell, e.g. a bacterial cell or other microbial cell, which is different from the cell comprising the target RNA. In a preferred embodiment, the microbial cell is a cell in which an RNA molecule is produced by transcription from a genetic construct encoding the RNA molecule, wherein the RNA molecule is substantially or preferably not predominantly processed in the microbial cell by cleavage within one or more loop sequences, one or more dsRNA regions, or both. For example, the microbial cell is a yeast cell or other fungal cell that does not have a Dicer enzyme. A highly preferred cell for producing RNA molecules is a Saccharomyces cerevisiae cell. The microbial cells may be viable or may be killed by some treatment, such as heat treatment, or may be in the form of a dry powder. Similarly, in one embodiment, in producing the RNA molecule of the invention, the RNA molecule of the invention is produced in a eukaryotic cell that does not contain the target RNA, but a eukaryotic cell that contains the RNA molecule of the invention and/or its processed RNA product may become the host for the target RNA, for example if the target RNA is viral RNA or other introduced RNA. Such cells can be prophylactically protected against viruses or other introduced RNA.

In a preferred embodiment of the first to fifth aspect, the RNA molecule can be enzymatically produced by transcription in vitro or in cells or both. In one embodiment, the RNA molecule of the invention is expressed in a cell, i.e. produced in a cell by transcription from one or more nucleic acids encoding the RNA molecule. The one or more nucleic acids encoding an RNA molecule are preferably DNA molecules, which may be present on a vector in a cell or integrated into the genome of a cell, the nuclear genome of a cell or the plastid DNA of a cell. The one or more nucleic acids encoding the RNA molecule can also be an RNA molecule, such as a viral vector.

Thus, in another aspect, the invention provides a cell comprising an RNA molecule as described herein. In a preferred embodiment, the present invention provides an RNA molecule as described herein that is expressed in a cell and has been isolated and/or purified from the cell. Accordingly, the present invention provides a preparation of isolated RNA molecules according to one or more of the first to fifth aspects and any embodiments described above or below, which is suitable for administration to a cell comprising a target RNA or potentially comprising a target RNA.

In one embodiment, the one or more target RNAs encode a protein. Alternatively, the one or more target RNAs do not encode a protein, e.g., rRNA, tRNA, snoRNA, or miRNA.

In embodiments of the first to fifth aspects, the total of about 12%, about 15%, about 18%, about 21%, about 24%, or about 15% to about 30%, or preferably about 16% to about 25% of the ribonucleotides forming the sense ribonucleotide sequence and its corresponding antisense ribonucleotide sequence of the dsRNA region are base-paired or not base-paired in non-canonical base pairs. In preferred embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% of the non-canonical base pairs in a dsRNA region or all dsRNA regions in an RNA molecule are G: U base pairs. The G nucleotides in each G.u base pair can be independently in the sense ribonucleotide sequence or preferably in the antisense ribonucleotide sequence. With respect to the G nucleotides in the G: U base pairs of the dsRNA region, preferably at least 50% is in the antisense ribonucleotide sequence, more preferably at least 60% or 70%, even more preferably at least 80% or 90%, and most preferably at least 95% is in the antisense ribonucleotide sequence of the dsRNA region. This feature can be applied independently to one or more or all dsRNA regions in an RNA molecule. In one embodiment, there is generally less than 25%, less than 20%, less than 15%, less than 10%, preferably less than 5%, more preferably less than 1%, or most preferably no ribonucleotide base pairing in the RNA molecule. In preferred embodiments, every 4 to every 1 of the 6 ribonucleotides in or overall in the dsRNA region form a non-canonical base pair or do not base pair within the RNA molecule. In preferred embodiments, the dsRNA region or dsRNA region does not generally comprise 10 or 9 or preferably 8 consecutive canonical base pairs. In another embodiment, the dsRNA region comprises at least 8 contiguous canonical base pairs, such as 8-12 or 8-14 or 8-10 contiguous canonical base pairs. In preferred embodiments, all ribonucleotides in a dsRNA region or in all dsRNA regions in an RNA molecule are base paired in canonical base pairs or non-canonical base pairs. In one embodiment, one or more ribonucleotides of the sense ribonucleotide sequence or one or more ribonucleotides of the antisense ribonucleotide sequence, or both, are not base paired. In one embodiment, in the RNA molecule of the invention, the one or more ribonucleotides of each sense ribonucleotide sequence and the one or more ribonucleotides of each antisense ribonucleotide sequence are not base paired.

In one embodiment, one or more or all of the antisense ribonucleotide sequences of an RNA molecule are less than 100% identical in sequence to the complement of a region of a target RNA molecule or to two such regions (which may or may not be contiguous in the target RNA molecule), or about 80% to 99.9% identical, or about 90% to 98% identical, or about 95% to 98% identical, preferably 98% to 99.9% identical. In preferred embodiments, the one or more antisense RNA sequences are 100% identical in sequence to a region of the complement of the target RNA molecule (e.g., to a region comprising 21, 23, 25, 27, 30, or 32 contiguous nucleotides). In one embodiment, the sense or antisense ribonucleotide sequence, preferably both, is at least 40, at least 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, or from about 100 to about 1,000 contiguous nucleotides in length. When using RNA molecules in plant cells or fungal cells or for non-vertebrate cells, a length of at least 100 nucleotides is preferred. When the RNA molecule is used in a vertebrate cell, the length of the sense and antisense ribonucleotide sequences in the dsRNA is 50 nucleotides or less, e.g. 31 to 50 nucleotides is preferred. However, RNA molecules having more than 50 base pairs in the dsRNA region, e.g., up to 100 or even 200 base pairs, can be used in vertebrate cells, provided that the dsRNA region has 10-30% of the nucleotides in the dsRNA region base-paired in G: U base pairs. In one embodiment, the number of ribonucleotides in the sense ribonucleotide sequence is between about 90% and about 110%, preferably between 95% and 105%, more preferably between 98% and 102%, even more preferably between 99% and 101% of the number of ribonucleotides in the corresponding antisense ribonucleotide sequence to which it hybridizes. In a most preferred embodiment, the number of ribonucleotides in the sense ribonucleotide sequence is the same as the number of ribonucleotides in the corresponding antisense ribonucleotide sequence. These features are applicable to each dsRNA region in an RNA molecule.

In an embodiment of the first to fifth aspect, the first 3 'ribonucleotide and the second 5' ribonucleotide in the RNA molecule are covalently linked by a loop sequence consisting of at least 4 ribonucleotides, or 4 to 1,000 ribonucleotides, or preferably 4 to 200 ribonucleotides, more preferably 4 to 50 ribonucleotides. In one embodiment, the RNA molecule further comprises a 5 'extension sequence covalently linked to the first 5' ribonucleotide or a 3 'extension sequence covalently linked to the second 3' ribonucleotide, or both. In one embodiment, the chimeric RNA molecule further comprises a 5 'extension sequence covalently linked to a second 5' ribonucleotide or a 3 'extension sequence covalently linked to a first 3' ribonucleotide, or both. In this embodiment, the RNA molecule comprises two separate RNA strands that hybridize to form the RNA molecule, although it may have been produced by transcription from a nucleic acid molecule into a single RNA transcript and subsequent processing to comprise both RNA strands.

The total length of the RNA molecule of the invention produced as single-stranded RNA after splicing out of any intron but before any processing of the RNA molecule by Dicer or other rnases is typically between 50 and 2000 ribonucleotides, preferably between 60 or 70 and 2000 ribonucleotides, more preferably between 80 or 90 and 2000 ribonucleotides, even more preferably between 100 or 110 and 2000 ribonucleotides. In preferred embodiments, the RNA molecule has a minimum length of 120, 130, 140, 150, 160, 180, or 200 nucleotides and a maximum length of 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, or 2000 ribonucleotides. Each combination of the minimum and maximum lengths described above may be considered. The production of RNA molecules of such length can be readily achieved by in vitro transcription or transcription in cells such as bacteria or other microbial cells, preferably saccharomyces cerevisiae cells, or in eukaryotic cells in which the target RNA molecule is down-regulated.

In an embodiment of the first to fifth aspect, the chimeric RNA molecule comprises two or more dsRNA regions which are identical or preferably different in sequence.

In a preferred embodiment of the first to fifth aspect, the RNA molecule is expressed in a eukaryotic cell, i.e. produced by transcription in the cell. In these embodiments, a greater proportion of dsRNA molecules are formed by processing RNA molecules of 22 and/or 20 ribonucleotides in length than by processing similar RNA molecules with corresponding dsRNA regions that are fully base-paired in canonical base pairs. That is, the RNA molecules of these embodiments are more readily processed than similar RNA molecules whose dsRNA regions are fully base-paired in canonical base pairs to provide 22-and/or 20-ribonucleotide short antisense RNAs as part of a total of 20-24 nucleotide asrnas produced by the RNA molecule. In other words, a lesser proportion of dsRNA molecules are formed by processing RNA molecules of 23 and/or 21 ribonucleotides in length than by processing similar RNA molecules with the corresponding dsRNA region fully base-paired in canonical base pairs. That is, depending on the ratio of the total number of 20-24 nucleotide asRNAs produced from the RNA molecule, the RNA molecules of these embodiments are not readily processed to provide short antisense RNAs of 23-and/or 21-ribonucleotides, as compared to similar RNA molecules in which the dsRNA region is fully base-paired in canonical base pairs. Preferably, at least 50% of the RNA transcripts transcribed from the genetic construct and produced in the cell are not processed by Dicer. In one embodiment, a larger proportion of short antisense RNA molecules formed by processing RNA molecules have more than one phosphate covalently linked at the 5' end when the RNA molecules are expressed in eukaryotic cells, i.e., produced by transcription in the cell, when compared to processing similar RNA molecules with the corresponding dsRNA region fully base-paired in canonical base pairs. That is, a greater proportion of short antisense RNA molecules have an altered charge, which can be observed in gel electrophoresis experiments as a change in mobility of the molecules.

In one embodiment, the RNA molecule of the invention comprises a combination of two or more features of the RNA molecule described herein.

In another aspect, the invention provides polynucleotides encoding the RNA molecules described herein, preferably the chimeric RNA molecules described herein. In one embodiment, the polynucleotide is a DNA construct that can be integrated into a larger DNA molecule, such as a chromosome. In one embodiment, the polynucleotide is operably linked to a promoter capable of directing expression of the RNA molecule in a host cell. The host cell may be: bacterial cells, such as E.coli; fungal cells, such as yeast cells (e.g., saccharomyces cerevisiae); or eukaryotic cells, such as plant cells or animal cells. In one embodiment, the promoter is heterologous with respect to the polynucleotide. The polynucleotide encoding the RNA molecule may be a chimeric or recombinant polynucleotide, or an isolated and/or exogenous polynucleotide. In one embodiment, the promoter may function in vitro, for example a phage promoter, such as the T7 RNA polymerase promoter or the SP6RNA polymerase promoter. In one embodiment, the promoter is an RNA polymerase III promoter, such as the U6 promoter or the H1 promoter. In one embodiment, the promoter is an RNA polymerase II promoter, which may be a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, or an inducible promoter. In one embodiment, the polynucleotide encodes an RNA precursor molecule comprising an intron in at least one loop sequence which is capable of being spliced out during or after transcription of the polynucleotide in a host cell. In one embodiment, the invention provides a vector comprising a polynucleotide described herein. In one embodiment, the vector is a viral vector. In one embodiment, the vector is a plasmid vector, such as a binary vector suitable for use with Agrobacterium tumefaciens (Agrobacterium tumefaciens).

In one embodiment, the polynucleotide is a chimeric DNA comprising, in order, a promoter capable of initiating transcription of an RNA molecule in a host cell, the promoter operably linked to a DNA sequence encoding an RNA molecule, preferably hpRNA, and a transcription termination and/or polyadenylation region. In a preferred embodiment, the RNA molecule comprises a hairpin RNA structure comprising a sense ribonucleotide sequence, a loop sequence and an antisense ribonucleotide sequence, more preferably wherein the sense and antisense ribonucleotide sequences base pair to form a dsRNA region wherein about 5% to about 40% of the ribonucleotides in the dsRNA region are base paired in non-canonical base pairs (preferably G: U base pairs). In a preferred embodiment, the host cell is a plant cell or a fungal cell.

In one embodiment, wherein the polynucleotide or vector of the invention is in a eukaryotic host cell, preferably in a plant or fungal cell, the promoter region of the polynucleotide or vector is operably linked to a region encoding an RNA molecule of the invention, said promoter region having a lower level of methylation than the promoter of a corresponding polynucleotide or vector encoding an RNA molecule having a corresponding dsRNA region with full base pairing in canonical base pairs. In one embodiment, the lower level of methylation is less than 50%, less than 40%, less than 30% or less than 20% when compared to the promoter of the corresponding polynucleotide or vector. In one embodiment, the host cell comprises at least two copies of a polynucleotide or vector encoding an RNA molecule of the invention. In this embodiment:

i) The reduced level of expression and/or activity of the target RNA molecule in the eukaryotic cell is at least the same relative to a corresponding eukaryotic cell having a single copy of the polynucleotide or vector, and/or

ii) a reduced level of expression and/or activity of a target RNA molecule in said eukaryotic cell when compared to a corresponding cell comprising an RNA molecule having a corresponding dsRNA region that is fully base paired in canonical base pairs.

In another aspect, the invention provides a host cell comprising an RNA molecule described herein, a small RNA molecule (20-24 nt in length) produced by processing a chimeric RNA molecule, a polynucleotide described herein, or a vector comprising the same. In one embodiment, the host cell is a non-human cell, such as a bacterial cell, a fungal cell, e.g., a yeast cell such as a saccharomyces cerevisiae cell, a plant cell or a non-human animal cell, preferably a plant cell. In one embodiment, the cell is a non-human cell or a human cell in cell culture. In one embodiment, the cell is a eukaryotic cell, e.g., a cell other than an animal cell. In one embodiment, the cell is a microbial cell such as a prokaryotic cell. In one embodiment, the host cell is living. In another embodiment, the host cell is dead and/or unable to reproduce. The host cell may be a cell in which the RNA molecule is produced by transcription and/or processing, or the cell may be a cell other than a cell in which the RNA molecule is produced by transcription and/or processing, for example a cell comprising a target RNA molecule.

In one embodiment, the host cell is preferably a plant cell or a fungal cell comprising a chimeric RNA molecule or a small RNA molecule produced by processing a chimeric RNA molecule or both, wherein said chimeric RNA molecule comprises in the 5 'to 3' direction a first sense ribonucleotide sequence, a first connecting ribonucleotide sequence comprising a loop sequence and a first antisense ribonucleotide sequence.

In another embodiment, the host cell is a eukaryotic cell comprising at least two copies of a polynucleotide encoding the chimeric RNA molecule of any one of claims 1 to 39, and wherein

i) The eukaryotic cell has a reduced level of expression or activity of the target RNA molecule that is about the same or greater than the reduced level of expression or activity of the target RNA molecule if the cell has a single copy of the polynucleotide, and/or

ii) a reduced level of expression and/or activity of the target RNA molecule in the eukaryotic cell is lower when compared to a corresponding cell comprising an RNA molecule having a corresponding dsRNA region that is fully base paired in canonical base pairs.

In another aspect, the present invention provides a non-human organism, preferably an animal or a plant, comprising an RNA molecule of the invention, preferably a chimeric RNA molecule as described herein, or a small RNA molecule (20-24 nt in length) produced by processing said chimeric RNA molecule, or a polynucleotide or a vector of the invention comprising the same, or a host cell comprising the same. In one embodiment, the non-human organism, preferably a plant or fungus, is transgenic in that it comprises a polynucleotide of the invention. In one embodiment, the polynucleotide is stably integrated into the genome of the non-human organism. The invention also includes animal and plant parts, as well as products obtained therefrom, which comprise an RNA molecule or a small RNA molecule (20-24 nt in length) produced by processing said chimeric RNA molecule, or both, and/or a polynucleotide or vector of the invention, such as seeds, crops, harvested products and post-harvest products produced therefrom.

In another aspect, the invention provides a method of producing an RNA molecule of the invention, the method comprising expressing a polynucleotide of the invention in a host cell or a cell-free expression system. Preferably, the polynucleotide is a chimeric DNA molecule encoding an RNA molecule. In this embodiment, the method may further comprise at least partially purifying or not purifying the RNA molecule.

In another aspect, the invention provides a method of producing a cell or non-human organism, preferably a plant cell, a plant or a fungus, the method comprising introducing a polynucleotide or vector or an RNA molecule of the invention into a cell, preferably an animal cell, a plant cell or a fungus, preferably such that the polynucleotide or vector or a part thereof encoding the RNA molecule is stably integrated into the genome of the cell. In one embodiment, the cell is an animal cell, e.g., a human cell, which may be an animal cell in culture. In one embodiment, the non-human organism is produced from a cell or progeny cell (e.g., by regenerating a transgenic plant and optionally producing a progeny plant therefrom). In one embodiment, the non-human organism is produced by introducing the cell or one or more daughter cells into the non-human organism. As an alternative to stable integration of the polynucleotide or vector into the genome of the cell, the polynucleotide or vector may be introduced into the cell without integration of the polynucleotide or vector into the genome, e.g., to transiently produce an RNA molecule in the cell or organism. In one embodiment, the non-human organism (e.g., an animal or a plant) is resistant to a pest or pathogen (e.g., an animal pest or pathogen, a plant pest or pathogen, preferably an insect pest or fungal pathogen). In one embodiment, the method comprises the step of testing one or more non-human organisms, preferably plants, comprising a polynucleotide or vector or RNA molecule of the invention for resistance to a pest or pathogen. The non-human organism (e.g. plant) being tested may be a progeny of the non-human organism (preferably a plant) into which the polynucleotide or vector or RNA molecule of the invention has first been introduced, and the method may therefore comprise the step of obtaining such progeny. The method may further comprise the step of identifying and/or selecting a non-human organism (e.g. an animal or a plant) that is resistant to the pest or pathogen. For example, a plurality of non-human organisms (e.g., animals or plants), each comprising a polynucleotide or vector or RNA molecule of the invention, can be tested to identify non-human organisms that are resistant to the pest or pathogen, and to identify progeny obtained from the identified non-human organisms, animals, or plants. .

In another aspect, the invention provides an extract of a host cell or organism or portion thereof of the invention, wherein the extract comprises an RNA molecule of the invention, a small RNA molecule (20-24 nt in length) produced by processing the chimeric RNA molecule, or both, and/or a polynucleotide or vector of the invention. In one embodiment, the invention provides a composition comprising one or more RNA molecules of the invention, small RNA molecules (20-24 nt in length) produced by processing chimeric RNA molecules, or both; a polynucleotide of the invention; a vector of the invention; a host cell of the invention; or an extract produced by the method of the invention; and one or more suitable carriers. In one embodiment, the composition is a pharmaceutical composition, such as a composition suitable for administration to a human or other animal. The pharmaceutical composition may be suitable for the prevention or treatment of a disease, or for topical application, such as cosmetic application. In one embodiment, the composition is suitable for application to plants, preferably plants or plant populations in a field, for example as a topical spray, or to insects or insect populations. In one embodiment, the composition is suitable for application to a crop, for example by spraying to crop plants in a field.

In one embodiment, the extract or composition comprising an RNA molecule of the invention or a small RNA molecule (20-24 nt in length) produced by processing the chimeric RNA molecule, or both, further comprises at least one compound that enhances the stability of the RNA molecule or polynucleotide and/or the vector, wherein the at least one compound facilitates the uptake of the RNA molecule, polynucleotide or vector by, for example, cells of a cell of an organism. In one embodiment, the compound is a transfection facilitating agent, such as a lipid-containing compound.

In another aspect, the invention provides a method for reducing or down-regulating the level and/or activity of a target RNA molecule in a cell or organism (e.g. in a part thereof), the method comprising delivering to the cell or organism one or more RNA molecules of the invention or small RNA molecules (20-24 nt in length) or both produced by processing the chimeric RNA molecule, a polynucleotide of the invention, a vector of the invention or a composition of the invention. In this context, delivery can be by feeding, contacting, exposing, transforming, or otherwise introducing an RNA molecule or small RNA molecule or mixtures thereof, or a polynucleotide or vector of the invention, into a cell or organism. Introduction may be enhanced by the use of an agent that increases uptake of the RNA molecule, polynucleotide or vector of the invention, for example by means of a transfection facilitating agent, a DNA-binding polypeptide or an RNA-binding polypeptide, or may be performed without the addition of such an agent, for example, by planting transgenic seed of the polynucleotide or vector of the invention and allowing the seed to grow into a transgenic plant expressing the RNA molecule of the invention. In one embodiment, the target RNA molecule encodes a protein. In one embodiment, the method reduces the level and/or activity of more than one target RNA molecule, which target RNA molecules are different, e.g., reduces the level and/or activity of two or more target RNAs that are related in sequence (e.g., from a gene family). Thus, in one embodiment, the chimeric RNA molecule or the small RNA molecule produced by processing said chimeric RNA molecule, or both, is contacted with a cell or organism, preferably a plant cell, plant, fungus or insect, by topical application to the cell or organism, or provided to the organism as a feed.

In another aspect, the invention provides a method of controlling a non-human organism (e.g., an animal pest or pathogen or a plant pest or pathogen), the method comprising delivering to the non-human organism one or more RNA molecules of the invention or a small RNA molecule (20-24 nt in length) produced by processing the chimeric RNA molecule, or both; a polynucleotide or vector of the invention; a host cell of the invention; an extract produced by the method of the invention; or the composition of the invention, wherein the RNA molecule and/or small RNA molecule has a deleterious effect on the non-human organism. In one embodiment, the non-human organism is an arthropod, such as an insect; or plants, such as weeds. In one embodiment, the non-human organism is a plant and the insect ingests the plant or a part thereof, thereby controlling the insect. Control may include a decrease in survival of the pest or pathogen, or a decrease in adaptability or reproduction of the pest or pathogen, or both. Control may encompass reducing the survival and/or reproduction of progeny of the pest or pathogen into which the RNA molecule was first introduced.

Another aspect of the invention relates to a method of reducing damage to a non-human organism, such as an animal or a plant, caused by a pest or a pathogen, comprising delivering to or contacting the pest or pathogen one or more RNA molecules of the invention or a small RNA molecule (20-24 nt in length) or both produced by processing the chimeric RNA molecule, a polynucleotide or vector of the invention, a host cell of the invention, an extract produced by a method of the invention, or a composition of the invention. In one embodiment, the method comprises sowing seeds that are transgenic for a polynucleotide of the invention, whereby the resulting plant expresses the transgene to produce an RNA molecule of the invention, thereby reducing damage caused by the pest or pathogen. Thus, the present invention provides means for farmers to control pests or pathogens of animals or plants. The invention extends to cells and organisms, e.g., animals or plants or parts thereof, comprising an RNA molecule, polynucleotide or vector of the invention provided to the cell or organism, and to pests or pathogens comprising an RNA molecule or a small RNA molecule (20-24 nt in length) or both produced by processing the chimeric RNA molecule, or a polynucleotide or vector of the invention. The pest or pathogen is live or dead. The invention also relates to progeny cells or organisms comprising an RNA molecule or a small RNA molecule or both.

In one embodiment, the present invention provides a method of preventing or treating a disease in a subject, the method comprising administering to the subject one or more RNA molecules of the invention or small RNA molecules (20-24 nt in length) or both produced by processing the chimeric RNA molecules, the polynucleotides or vectors of the invention, the host cells of the invention, the extracts produced by the methods of the invention, or the compositions of the invention, wherein the RNA molecules or small RNA molecules or both have a beneficial effect on at least one symptom of the disease. In one embodiment, the RNA molecule or small RNA molecule, polynucleotide, vector or composition is administered topically, orally or by injection. In one embodiment, the subject is a vertebrate. In one embodiment, the vertebrate is a mammal, such as a human, livestock such as cattle or sheep, or birds, such as chickens and other poultry.

In another aspect, the invention provides the use of an RNA molecule of the invention, a polynucleotide or vector of the invention, a host cell of the invention, an extract produced by a method of the invention, or a composition of the invention for preventing or treating a disease in a subject, wherein the RNA molecule or small RNA molecule or both has a beneficial effect on at least one symptom of the disease. In one embodiment, the present invention provides the use of an RNA molecule of the invention or a small RNA molecule produced therefrom, a polynucleotide or vector of the invention, a host cell of the invention, an extract produced by a method of the invention, or a composition of the invention for the manufacture of a medicament for the prevention or treatment of a disease in a subject, wherein the RNA molecule or the small RNA molecule produced therefrom, or both, has a beneficial effect on at least one symptom of the disease.

In another aspect, the invention provides a kit comprising one or more of: an RNA molecule of the invention or a small RNA molecule produced therefrom, a polynucleotide or vector of the invention, a host cell of the invention, an extract produced by a method of the invention, or a composition of the invention. The kit may further comprise instructions for using the kit.

Although more widely used in transgenic expression systems, as discussed herein, there are applications of dsRNA technology that rely on the need for large scale production of dsRNA molecules, such as spraying crops for disease and/or pest control. The present inventors have identified s.cerevisiae as an organism suitable for use in large scale production processes because the dsRNA molecules expressed therein are not cleaved. Thus, in another aspect, the invention provides a process for producing a dsRNA molecule, said process comprising

a) Culturing Saccharomyces cerevisiae expressing one or more polynucleotides encoding one or more dsRNA molecules, and

b) collecting the Saccharomyces cerevisiae or dsRNA molecules from Saccharomyces cerevisiae producing dsRNA molecules,

wherein the culture volume of Saccharomyces cerevisiae is at least 1L.

The dsRNA may have any structure, such as shRNA, miRNA, or the dsRNA of the invention.

In one embodiment, the culture volume of saccharomyces cerevisiae is at least 10 liters, at least 100 liters, at least 1000 liters, at least 10000 liters or at least 100000 liters.

In one embodiment, the process produces at least 0.1 g/liter, at least 0.5 g/liter, or at least 1 g/liter of the RNA molecule of the invention.

The saccharomyces cerevisiae produced using the process or dsRNA molecules isolated therefrom (in a purified or partially purified (e.g., extract) state) may be used in the methods described herein, such as, but not limited to, methods of reducing or down-regulating the level and/or activity of a target RNA molecule in a cell or organism, methods of reducing damage caused by a pest or pathogen to a non-human organism, methods of controlling a non-human organism, or methods of preventing or treating a disease in a subject.

Any embodiment herein should be understood as applying to any other embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as exemplary only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of matter shall be taken to include one or more (i.e., one or more) of those steps, compositions of matter, groups of steps or group of matter.

The invention is described below by way of the following non-limiting examples and with reference to the accompanying drawings.

Drawings

FIG. 1 schematic representation of two ledRNA molecules. (A) The ledRNA molecule comprises a sense sequence, which can be considered as two adjacent sense sequences, covalently linked without an intervening spacer sequence, and having identity to the target RNA, an antisense sequence complementary to the sense sequence and divided into two regions, a 5 'region and a 3' region, and two loops separating the sense sequence from the antisense sequence. (B) The ledRNA molecule comprises an antisense sequence, a sense sequence, and two loops: the antisense sequence can be considered to be two adjacent antisense sequences, covalently linked without an intervening spacer sequence, and having identity to the complement of the target RNA, the sense sequence being complementary to the antisense sequence and divided into two regions, the two loops separating the sense sequence from the antisense sequence. The RNA molecule is produced by transcription, for example by in vitro transcription from a promoter such as the T7 or Sp6 promoter, self-annealing by base pairing between the complementary sense and antisense sequences to form a double-stranded region having a loop at each end and a "split" in the antisense or sense sequence. Additional sequences may be attached to the 5 'and/or 3' ends as 5 'or 3' extension sequences.

FIG. 2 ledRNA is more effective in forming dsRNA than sense/antisense annealing or hairpin RNA. Schematic representations of three forms of double stranded RNA molecules are shown: a, a conventional dsRNA formed by annealing of two separate strands; b, a hairpin RNA having a 5 'or 3' extension sequence; and C, ledRNA molecules. The lower panel shows photographs after gel electrophoresis of RNA transcripts targeting three types of RNA molecules of either the GUS gene or the GFP gene.

FIG. 3 Northern blot hybridization of treated (A and B) and untreated distal (C and D) tissues showed that ledRNA is more stable than dsRNA and spreads through tobacco leaf tissue. In contrast to the strong ledRNA signal, no dsRNA signal could be detected in the distal tissues (C and D, top panel).

FIG. 4 LedRNA treatment induced GUS down-regulation in both the treated region (1) and the untreated region (3) described above.

FIG. 5 LedRNA induces FAD2.1 gene silencing in leaves of Nicotiana benthamiana (N.benthamiana).

FIG. 6 Northern blot hybridization demonstrates that FAD2.1 mRNA was strongly down-regulated by treatment with ledFAD2.1 at 6 and 24 hours.

FIG. 7 alignment of the nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) with the sense sequence of the hpGUS [ G: U ] construct (nucleotides 9 to 208 of SEQ ID NO: 11). 52 cytosine (C) nucleotides are substituted with thymine (T) nucleotides. The remaining nucleotides are marked with an asterisk and the substituted C's are not marked with an asterisk.

FIG. 8 alignment of the nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) with the sense sequence of the hpGUS [1:4] construct (nucleotides 9 to 208 of SEQ ID NO: 12). Every fourth nucleotide in hpGUS [1:4] is substituted relative to the corresponding wild-type sense sequence, whereby for every fourth nucleotide C is changed to G, G is changed to C, A is changed to T, and T is changed to A. The remaining nucleotides are marked with an asterisk, substituted G and C are not marked with an asterisk, and substituted A and T are marked with a semicolon.

FIG. 9 alignment of the nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) with the sense sequence of the hpGUS [2:10] construct (nucleotides 9 to 208 of SEQ ID NO: 13). Every "9" and "10" nucleotide in each segment of 10 nucleotides in hpGUS [2:10] is substituted relative to the corresponding wild-type sense sequence, whereby for every "9" and "10" nucleotide, C is changed to G, G is changed to C, A is changed to T, and T is changed to A. The remaining nucleotides are marked with an asterisk, substituted G and C are not marked with an asterisk, and substituted A and T are marked with a semicolon.

Figure 10. schematic shows the structure of a genetic construct encoding a modified hairpin RNA targeting the GUS mRNA.

FIG. 11 schematic representation of the vector pWBPPGH used to transform tobacco plants providing GUS target genes. The T-DNA extends from the Right Border (RB) to the Left Border (LB) of the vector. The selectable marker gene on the T-DNA is the 35S-HPT-tm1' gene encoding hygromycin resistance.

FIG. 12 GUS activity in plants transformed with a construct encoding a modified hairpin RNA for reducing GUS target gene expression. No hp: control PPGH11 and PPGH24 plants, without hpGUS constructs. The number of plants showing less than 10% GUS activity compared to the corresponding control PPGH11 or PPGH24 plants and the percentage of such plants relative to the number of test plants are given in parentheses.

Fig. 13 (a) average GUS activity of all transgenic plants: for the hpGUS [ wt ], 59, for the hpGUS [ G: U ]74, for the hpGUS [1:4], 33, for the hpGUS [2:10], 41. (B) Average GUS activity of all silenced plants (32 for hpGUS [ wt ], 71 for hpGUS [ G: U ], 33 for hpGUS [1:4], 28 for hpGUS [2:10 ]).

FIG. 14 GUS activity in transgenic progeny plants containing hpGUS [ wt ], hpGUS [ G: U ] or hpGUS [1:4 ].

FIG. 15. autoradiogram of Southern blots of DNA from 16 plants transformed with the hpGUS [ G: U ] construct. DNA was digested with HindIII and probed with an OCS-T probe prior to gel electrophoresis. Lane 1: size markers (HindIII digested lambda DNA); lanes 2 and 3, DNA of parental plants PPGHII and PPGH 24; lanes 4-19: DNA of 16 different transgenic plants.

FIG. 16. autoradiogram of Northern blot hybridization experiments to detect sense (top panel) and antisense (bottom panel) sRNA from hairpin RNA expressed in transgenic tobacco plants. Lanes 1 and 2 contain RNA obtained from the parental plants PPGHII and PPGH24 lacking the hpGUS construct. Lanes 3-11 contain RNA from the hpGUS [ wt ] plant, and lanes 12-20 contain RNA from the hpGUS [ G: U ] plant.

FIG. 17. autoradiogram of Northern blot hybridization to detect antisense sRNA from transgenic plants. Lanes 1-10 are from the hpGUS [ wt ] plant, and lanes 11-19 are from the hpGUS [ G: U ] plant. Antisense srnas have a mobility corresponding to a length of 20-24 nt. The blot was re-probed with antisense RNA to U6RNA as a lane loading control.

FIG. 18. autoradiogram of duplicate Northern blot hybridizations detecting antisense sRNA from transgenic plants.

FIG. 19 DNA methylation analysis of the junction region of the 35S promoter and sense GUS region in hpGUS constructs in transgenic plants. The ligated fragments were PCR amplified with (+) or without (-) prior to treatment of plant DNA with McrBC enzyme.

FIG. 20 DNA methylation analysis of the 35S promoter region in hpGUS constructs in transgenic plants. The 35S fragment was PCR amplified with (+) or without (-) prior to treatment of plant DNA with McrBC enzyme.

FIG. 21 size distribution and abundance of processed RNA. (A) The EIN2 construct. (B) A GUS construct.

FIG. 22 alignment of the sense sequence (upper sequence, nucleotides 17 to 216 of SEQ ID NO: 22) and the nucleotide sequence (lower sequence, SEQ ID NO: 27) corresponding to the cDNA region of the Arabidopsis thaliana (A. thaliana) EIN2 target gene for the hpEIIN 2[ G: U ] construct. The sense sequence was obtained by substituting 43 cytosine (C) nucleotides in the wild-type sequence with thymine (T) nucleotides. The remaining nucleotides are marked with an asterisk and the substituted C's are not marked with an asterisk.

FIG. 23 alignment of the sense sequence (top sequence, nucleotides 13 to 212 of SEQ ID NO: 24) of the hpCHS [ G: U ] construct and the nucleotide sequence (SEQ ID NO: 28, reverse) corresponding to the cDNA region of the Arabidopsis (A. thaliana) CHS target gene. The sense sequence was obtained by substituting 65 cytosine (C) nucleotides in the wild-type sequence with thymine (T) nucleotides. The remaining nucleotides are marked with an asterisk and the substituted C's are not marked with an asterisk.

FIG. 24 alignment of antisense sequence (upper sequence, nucleotides 8 to 207 of SEQ ID NO: 25) of hpEIN2[ G: U/U: G ] construct and nucleotide sequence (lower sequence, SEQ ID NO: 29) of region corresponding to the complement of the Arabidopsis thaliana (A. thaliana) EIN2 target gene. Substitution of 49 cytosine (C) nucleotides in the wild type sequence with thymine (T) nucleotides gave the antisense sequence. The remaining nucleotides are marked with an asterisk and the substituted C's are not marked with an asterisk.

FIG. 25 alignment of antisense sequence (upper sequence, nucleotides 13 to 212 of SEQ ID NO: 26) of hpCHS [ G: U/U: G ] construct and nucleotide sequence (lower sequence, SEQ ID NO: 30) of region corresponding to the complement of Arabidopsis (A. thaliana) CHS target gene. Substitution of 49 cytosine (C) nucleotides in the wild type sequence with thymine (T) nucleotides gave the antisense sequence. The remaining nucleotides are marked with an asterisk and the substituted C's are not marked with an asterisk.

FIG. 26 schematic representation of ethylene insensitive 2(EIN2) and chalcone synthase (CHS) hpRNA constructs. 35S: the CaMV 35S promoter; EIN2 and CHS regions are shown as wild type sequences (wt) or G: U modified sequences (G: U). The arrow indicates the orientation of the DNA fragment and the arrow from right to left indicates the antisense sequence. Restriction enzyme sites are also shown.

FIG. 27 hypocotyl length of transgenic Arabidopsis (A. thaliana) seedlings containing hpEIN2[ wt ] or hpEIN2[ G: U ] was determined in EIN 2.

FIG. 28. for CHS mRNA in transgenic Arabidopsis (A. thaliana), qRT-PCR normalized the transgene of the hpCHs [ wt ] or hpCHs [ G: U ] constructs to Actin (Actin)2RNA levels. Col-0 is wild-type (non-transgenic) arabidopsis (a. thaliana).

FIG. 29. autoradiogram of Northern blot hybridization of RNA from plants transformed with hpEIN2[ wt ] or hpEIN2[ G: U ]. The upper panel shows hypocotyl length of the line. The autoradiogram shows a Northern blot probed with the EIN2 sense probe to detect antisense sRNA. The same blot was re-probed with U6 RNA probe as a loading control (U6 RNA).

Figure 30 DNA methylation analysis of 35S promoter and 35S-sense EIN2 sequences in genomic DNA in transgenic arabidopsis (a. thaliana) plants.

FIG. 31 DNA methylation levels in the promoter and 5' region of hairpin RNA constructs.

FIG. 32. 35S promoter in the least methylated line of the hpEIN2[ wt ] population still showed significant methylation.

FIG. 33G 35S promoter in U hpEIN2 line showed only weak methylation (P < 10%).

FIG. 34.72 h has G.U gene silenced ledRNA and hpRNA in CHO and Vero cells.

FIG. 35.48 h dumbbell-shaped plasmids were tested in Hela cells.

Figure 36. examples of possible modifications of dsRNA molecules.

FIG. 37 shows that aphids performance decreases after feeding ledRNA-supplemented artificial feed to down-regulate the expression of MpC002 or MpRack-1 genes in green Myzus persicae. Top panel (a): average nymph number per adult aphid after ten day cycle with 100 μ l of 50 ng/. mu.l ledRNA. Panel (B) below: percentage of aphids surviving over the course of a five day period after feeding 100. mu.l of 200 ng/. mu.l ledRNA containing MpC002, MpRack-1 or control ledGFP.

FIG. 38 Northern blot hybridization using full length sense GUS transcripts as probes to detect ledGUS and hpGUS RNA. The bottom "+" indicates high GUS expression; "-" indicates low/no GUS expression, i.e.strong GUS silencing.

FIG. 39 Northern blot hybridization detection of long hpEIN2 and ledEIN2 RNA (upper panel) and siRNA derived from both constructs (lower panel).

FIG. 40 schematic representation of the stem-loop structure of transcripts expressed from GUS hpRNA constructs. The transcripts have complementary sense and antisense sequences that base pair to form a GUS sequence specific dsRNA stem, have a length in base pairs (bp) for the stem, and a number of nucleotides (nt) in the loop. The transcripts encoded by the GFP hpRNA constructs form GFP-specific dsRNA stems with fully canonical base pairing (GFPhp [ WT ] or dsRNA stems with approximately 25% base pairs of G: U base pairs (GFPhp [ G: U ], loops with regions derived from the GUS coding sequence the loop sequences of GFPhp transcripts each contain two sequences complementary to miR165/miR166 and thus provide binding sites for these miRNAs.

Northern blot hybridization analysis showed that the hpRNA-encoding transgene produced different loop sequence fragments when expressed in plant cells. (A) GUS target Gene (GUS) and expression of long hpRNA transgene GUShp1100 with 1100nt spacer/loop sequence. A construct encoding a cucumber mosaic virus 2b RNA silencing inhibitor (CMV2b) was included to enhance transgene expression. (B) Northern blot analysis revealed RNA expressed from two short hpRNA transgenes, GUSHp93-1 and GUSHp93-2, in stably transformed Arabidopsis plants. RNA samples were treated (+) or untreated (-) with RNAse I. Both northern blots were hybridized with a loop-specific antisense RNA probe.

FIG. 42. the loop of GUSHP1100 was accumulated to a high level in Nicotiana benthamiana cells and was resistant to RNase R digestion.

FIG. 43A transgenic s.cerevisiae expressing the GUSHP1100 construct shows a single RNA molecular species corresponding to the full-length hairpin RNA transcript. The lower panel shows Northern blot hybridization of RNA samples from transgenic Saccharomyces cerevisiae.

FIG. 44 GUSHP1100 transcript expressed in Saccharomyces cerevisiae remained full-length and did not form circular loop RNA. The first four lanes used in vitro transcripts of either the full length of GUShp1100 or the dsRNA stem, supplemented with total RNA isolated from wild type nicotiana benthamiana leaves.

FIG. 45.hpRNA loop can be used as an effective sequence-specific repressor of miRNA. (A) The GFPhp [ G: U ] construct induces a strong miR165/166 inhibition phenotype in transgenic Arabidopsis plants. (B) Northern blot hybridization to determine the abundance of GFPhp transcripts in RNA from transgenic Arabidopsis plants. (C) RT-qPCR analysis of the circular RNA of the GFPhp loop.

Keywords of sequence Listing

SEQ ID NO: 1-GFP ledRNA ribonucleotide sequence.

SEQ ID NO: 2-GUS ledRNA ribonucleotide sequence.

SEQ ID NO: 3-ribonucleotide sequence of Nicotiana benthamiana (N.benthamiana) FAD2.1 ledRNA.

SEQ ID NO: 4-nucleotide sequence encoding GFP ledRNA.

SEQ ID NO: 5-a nucleotide sequence encoding GUS ledRNA.

SEQ ID NO: 6-nucleotide sequence encoding tobacco (N.benthamiana) FAD2.1 ledRNA.

SEQ ID NO: 7-nucleotide sequence encoding GFP.

SEQ ID NO: 8-a nucleotide sequence encoding GUS.

SEQ ID NO: 9-nucleotide sequence encoding tobacco (N.benthamiana) FAD 2.1.

SEQ ID NO: 10-nucleotide sequence for providing a construct encoding a hairpin RNA molecule targeting the GUS mRNA with a GUS sense region.

SEQ ID NO: 11-nucleotide sequence for providing a GUS sense region for a construct encoding a hairpin RNA molecule hpGUS [ G: U ].

SEQ ID NO: 12-nucleotide sequence for providing a GUS sense region for a construct encoding a hairpin RNA molecule hpGUS [1:4 ].

SEQ ID NO: 13-nucleotide sequence for providing a GUS sense region for a construct encoding the hairpin RNA molecule hpGUS [2:10 ].

SEQ ID NO: the nucleotide sequence of nucleotide 781-1020 of the protein coding region of 14-GUS gene.

SEQ ID NO: 15-hpGUS [ wt ] RNA hairpin structure (including its loop) ribonucleotide sequence.

SEQ ID NO: ribonucleotides of the hairpin structure of 16-hpGUS [ G: U ] RNA, including its loop.

SEQ ID NO: 17-hpGUS [1:4] RNA hairpin structure (including its loop) ribonucleotides.

SEQ ID NO: ribonucleotides of the hairpin structure of 18-hpGUS [2:10] RNA, including its loop.

SEQ ID NO: 19-nucleotide sequence of cDNA corresponding to arabidopsis thaliana (a. thaliana) EIN2 gene, accession No. NM — 120406.

SEQ ID NO: 20-nucleotide sequence of cDNA corresponding to the CHS gene of Arabidopsis thaliana (A. thaliana), accession No. NM-121396, 1703 nt.

SEQ ID NO: 21-nucleotide sequence of a DNA fragment comprising a 200nt sense sequence from the cDNA corresponding to the Arabidopsis thaliana (A. thaliana) EIN2 gene flanked by restriction enzyme sites.

SEQ ID NO: 22-nucleotide sequence of a DNA fragment comprising the 200nt sense sequence of EIN2, corresponding to SEQ ID NO: 21 same, except that 43C 'were replaced by T', was used to construct hpEIN2[ G: U ].

SEQ ID NO: 23-nucleotide sequence of a DNA fragment comprising the 200nt sense sequence from the cDNA corresponding to the Arabidopsis thaliana (A. thaliana) CHS flanked by restriction enzyme sites.

SEQ ID NO: 24-nucleotide sequence of a DNA fragment comprising the 200nt sense sequence of CHS, identical to SEQ ID NO: 23 same, except that 65C 'were replaced by T', for the construction of hpCHs [ G: U ].

SEQ ID NO: 25-nucleotide sequence of DNA fragment comprising antisense sequence of 200nt of EIN2, except that T 'was replaced by 50C' for the construction of hpEIN2[ G: U/U: G ].

SEQ ID NO: 26-nucleotide sequence of DNA fragment comprising 200nt antisense sequence of CHS, except that T 'was replaced by 49C's for constructing hpCHS [ G: U/U: G ].

SEQ ID NO: 27-nucleotide sequence of nucleotide 601-900 corresponding to the cDNA of the Arabidopsis thaliana (A. thaliana) EIN2 gene (accession NM-120406).

SEQ ID NO: 28-nucleotide sequence corresponding to nucleotide 813-1112 of the cDNA of the CHS gene (accession NM-121396) of Arabidopsis thaliana (A. thaliana).

SEQ ID NO: 29-nucleotide sequence corresponding to the complement of nucleotide 652-891 of the cDNA of the Arabidopsis thaliana (A. thaliana) EIN2 gene (accession NM-120406).

SEQ ID NO: 30-nucleotide sequence corresponding to the complement of nucleotide 804-1103 of the cDNA of the Arabidopsis thaliana (A. thaliana) CHS gene.

SEQ ID NO: 31-Arabidopsis thaliana (Arabidopsis thaliana), the FANCM I protein coding region of the cDNA of accession No. NM-001333162. Target region nucleotide 675-1174(500 nucleotides)

SEQ ID NO: 32-the FANCM I protein coding region of the cDNA of Brassica napus (Brassica napus). Target region nucleotide 896-1395(500bp)

SEQ ID NO: 33-nucleotide sequence encoding hpFANCMM-At [ wt ], which targets the FANCM I protein coding region of Arabidopsis thaliana (A. thaliana). The FANCM sense sequence, nucleotides 38-537; loop sequence, nucleotides 538-1306; the antisense sequence of FANCM, nucleotide 1307-1806.

SEQ ID NO: 34-nucleotide sequence encoding hpFANCM-At [ G: U ] which targets the FANCM I protein coding region of Arabidopsis thaliana (A. thaliana). The FANCM sense sequence, nucleotides 38-537; loop sequence, nucleotides 538-1306; the antisense sequence of FANCM, nucleotide 1307-1806.

SEQ ID NO: 35-nucleotide sequence encoding hpFANCMM-Bn [ wt ], which targets the FANCM I protein coding region of Brassica napus (B.napus). The FANCM sense sequence, nucleotides 34-533; loop sequence, nucleotide 534-1300; the FANCM antisense sequence, nucleotide 1301- & 1800.

SEQ ID NO: 36-nucleotide sequence encoding hpFANCMM-Bn [ G: U ], which targets the FANCM I protein coding region of Brassica napus (B.napus). The FANCM sense sequence, nucleotides 34-533; loop sequence, nucleotide 534-1300; the FANCM antisense sequence, nucleotide 1301- & 1800.

SEQ ID NO: 37-corresponds to the brassica napus (b.napus) DDMl gene; the nucleotide sequence of the protein coding region of the cDNA of accession No. XR _ 001278527.

SEQ ID NO: 38-nucleotide sequence of DNA encoding hpDDM1-Bn [ wt ], which targets the DDM1 protein coding region of Brassica napus (B.napus).

SEQ ID NO: 39-nucleotide sequence encoding hpDDM1-Bn [ G: U ], which targets the DDM1 protein coding region of Brassica napus (B.napus). DDM1 sense sequence, nucleotides 35-536; the loop sequence, nucleotides 537-1304; DDM1 antisense sequence, nucleotides 1305-.

SEQ ID NO：40-EGFPcDNA。

SEQ ID NO: the nucleotide sequence of the coding region of 41-hpeGFP [ wt ], is antisense/loop/sense with respect to the promoter sequence.

SEQ ID NO: nucleotide sequence of the coding region of 42-hpeGFP [ G: U ], having 157C to T substitutions in the EGFP sense sequence.

SEQ ID NO: 43-ledEGFP [ wt ] coding region, which has no C to T substitutions in the EGFP sense sequence.

SEQ ID NO: nucleotide sequence of the coding region of 44-ledEGFP [ G: U ], with 162C to T substitutions in the EGFP sense sequence.

SEQ ID NO: 45-nucleotide sequence for providing GUS sense region for constructs encoding hairpin RNA molecules hpGUS [ G: U ] not flanked by restriction enzyme sites.

SEQ ID NO: 46-nucleotide sequence for providing a GUS sense region for a construct encoding a hairpin RNA molecule hpGUS [1:4] not flanked by restriction enzyme sites.

SEQ ID NO: 47-nucleotide sequence used to provide the GUS sense region for constructs encoding hairpin RNA molecules hpGUS [2:10] not flanked by restriction enzyme sites.

SEQ ID NO: 48-nucleotide sequence of a DNA fragment comprising the 200nt sense sequence of EIN2, corresponding to SEQ ID NO: 21 same, except that 43C 'were replaced by T', was used to construct hpEIN2[ G: U ] without flanking sequences.

SEQ ID NO: 49-nucleotide sequence of a DNA fragment comprising the 200nt sense sequence of CHS, which is identical to the nucleotide sequence of SEQ ID NO: 23 same, except that 65C 'were replaced by T', was used to construct hpCHs [ G: U ] without flanking sequences.

SEQ ID NO: 50-nucleotide sequence of DNA fragment comprising 200nt antisense sequence of EIN2, except that T 'was replaced by 50C' for the construction of hpEIN2[ G: U/U: G ] without flanking sequences.

SEQ ID NO: 51-nucleotide sequence of DNA fragment comprising 200nt antisense sequence of CHS, except that T 'was replaced by 49C' for constructing hpCHS [ G: U/U: G ] sequence without flanking sequence.

SEQ ID No: 52-oligonucleotide primer for amplifying 200bp GUS sense sequence (GUS-WT-F)

SEQ ID No: 53-oligonucleotide primer for amplifying 200bp GUS sense sequence (GUS-WT-R)

SEQ ID NO: 54-oligonucleotide primers for the production of hpGUS [ G: U ] fragments (Forward) in which each C is replaced by a T (GUS-GU-F)

SEQ ID NO: 55-oligonucleotide primers (reverse) for the production of hpGUS [ G: U ] fragments, in which each C is replaced by a T (GUS-GU-R)

SEQ ID NO: 56-oligonucleotide primers for the production of hpGUS [1:4] fragments (Forward) in which every 4 th nucleotide is substituted (GUS-4M-F)

SEQ ID NO: 57-oligonucleotide primers (reverse) for the production of hpGUS [1:4] fragments, in which every 4 th nucleotide is substituted (GUS-4M-R)

SEQ ID NO: 58-oligonucleotide primer for the production of hpGUS [2:10] fragment (Forward) in which every 9 th and 10 th nucleotides are substituted (GUS-10M-F)

SEQ ID NO: 59-oligonucleotide primers for the production of hpGUS [2:10] fragments (reverse) in which every 9 th and 10 th nucleotide is substituted (GUS-10M-R)

SEQ ID No: 60-nucleotide sequence encoding the Forward primer (35S-F3)

SEQ ID NO: 61-nucleotide sequence encoding a reverse primer (GUGUGUWT-R2)

SEQ ID NO: 62-nucleotide sequence encoding the Forward primer (GUGUGU-R2)

SEQ ID NO: 63-nucleotide sequence encoding reverse primer (GUS4m-R2)

SEQ ID No: 64-nucleotide sequence encoding the Forward primer (35S-F2)

SEQ ID NO: 65-nucleotide sequence encoding reverse primer (35S-R1)

SEQ ID NO: 66-oligonucleotide primers for amplifying wild type 200bp EIN2 sense sequence (EIN2wt-F)

SEQ ID NO: 67-oligonucleotide primers for amplifying wild type 200bp EIN2 sense sequence (EIN2wt-R)

SEQ ID No: 68-oligonucleotide primer for amplifying wild-type 200bp CHS sense sequence (CHSwt-F)

SEQ ID No: 69-oligonucleotide primer for amplifying wild-type 200bp CHS sense sequence (CHSwt-R)

SEQ ID NO: 70-oligonucleotide primers for the production of the hpEIN2[ G: U ] fragment (Forward) in which each C is replaced by a T (EIN2gu-F)

SEQ ID NO: 71-oligonucleotide primers (reverse) for the production of the hpEIN2[ G: U ] fragment, in which each C is replaced by a T (EIN2gu-R)

SEQ ID NO: 72-oligonucleotide primers for the production of hpCHS [ G: U ] fragments (Forward), in which each C is replaced by a T (CHSgu-F)

SEQ ID NO: 73-oligonucleotide primers (reverse) for the production of a fragment of hpCHS [ G: U ], in which each C is substituted by a T (CHSgu-R)

SEQ ID NO: 74-oligonucleotide primers for generating a fragment of hpEIN2[ G: U/U: G ] (Forward), in which each C is replaced by a T (aseIN2gu-F)

SEQ ID NO: 75-oligonucleotide primers for the production of the hpEIN2[ G: U/U: G ] fragment (reverse), in which each C is substituted by a T (aseIN2gu-R)

SEQ ID NO: 76-oligonucleotide primers for the production of a fragment of hpCHS [ G: U/U: G ] (Forward), in which each C is substituted by a T (asCHSgu-F)

SEQ ID NO: 77-oligonucleotide primers for the production of the hpCHS [ G: U/U: G ] fragment (reverse), in which each C is substituted by a T (asCHSgu-R)

SEQ ID No: 78-nucleotide sequence encoding the Forward primer (CHS-200-F2)

SEQ ID NO: 79-nucleotide sequence encoding the reverse primer (CHS-200-R2)

SEQ ID No: 80-nucleotide sequence coding For Forward primer (Actin2-For)

SEQ ID NO: 81-nucleotide sequence encoding the reverse primer (Actin2-Rev)

SEQ ID NO: 82-nucleotide sequence encoding the Forward primer (Top-35S-F2)

SEQ ID NO: 83-nucleotide sequence encoding the reverse primer (Top-35S-R2)

SEQ ID NO: 84-nucleotide sequence encoding the Forward primer (Link-35S-F2)

SEQ ID NO: 85-nucleotide sequence encoding a reverse primer (Link-EIN2-R2)

SEQ ID NO: 86-sense si 22-ribonucleotide sequence

SEQ ID NO: 87-antisense si 22-ribonucleotide sequence

SEQ ID NO: ribonucleotide sequence of 88-forward primer

SEQ ID NO: ribonucleotide sequence of 89-reverse primer

SEQ ID NO: ribonucleotide sequence of the 90-forward primer

SEQ ID NO: ribonucleotide sequence of the 91-reverse primer

SEQ ID NO: possible modifications of 92-dsRNA molecules

SEQ ID NO: 93-nucleotide sequence corresponding to cDNA of the Brassica napus (B.napus) DDM1 gene (accession number XR-001278527).

SEQ ID NO: 94-nucleotide sequence encoding a chimeric DNA of a hairpin RNAi (hpRNA) construct targeting the Brassica napus (B.napus) DDM1 gene.

SEQ ID NO: 95-nucleotide sequence encoding a chimeric DNA targeting the Brassica napus (B.napus) DDM1 gene, a hairpin RNAi (hpRNA) construct with G: U base pairs.

SEQ ID NO: 96-nucleotide sequence encoding a chimeric DNA of the ledRNA construct targeting the Gene of Brassica napus (B.napus) DDM 1.

SEQ ID NO: 97-nucleotide sequence of cDNA corresponding to the FANCM gene (accession NM-001333162) of Arabidopsis thaliana (A. thaliana).

SEQ ID NO: 98-nucleotide sequence encoding a chimeric DNA targeting the hairpin RNAi (hpRNA) construct of the Arabidopsis thaliana (A. thaliana) FANCM gene.

SEQ ID NO: 99-nucleotide sequence encoding a chimeric DNA targeting the arabidopsis thaliana (a. thaliana) FANCM gene, a hairpin rnai (hprna) construct with G: U base pairs.

SEQ ID NO: 100-nucleotide sequence encoding a chimeric DNA targeting the ledRNA construct of the Arabidopsis thaliana (A. thaliana) FANCM gene.

SEQ ID NO: 101-nucleotide sequence corresponding to the cDNA of the brassica napus (b.napus) FANCM gene (accession number XM _ 022719486.1).

SEQ ID NO: 102-nucleotide sequence encoding a chimeric DNA of a hairpin rnai (hprna) construct targeting the brassica napus (b.napus) FANCM gene.

SEQ ID NO: 103-nucleotide sequence encoding a chimeric DNA targeting the brassica napus (b.napus) FANCM gene, a hairpin rnai (hprna) construct with G: U base pairs.

SEQ ID NO: 104-nucleotide sequence encoding a chimeric DNA of a ledRNA construct targeting the FANCM gene of brassica napus (b.napus).

SEQ ID NO: 105-nucleotide sequence corresponding to the protein-coding region of cDNA of TOR gene of Nicotiana benthamiana (Nicotiana benthamiana).

SEQ ID NO: 106-nucleotide sequence encoding a chimeric DNA targeting the ledRNA construct of the TOR gene of Nicotiana benthamiana (Nicotiana benthamiana).

SEQ ID NO: 107: the nucleotide sequence corresponding to the protein coding region of the cDNA of the barley (Hordeum vulgare) (accession number LT601589) acetolactate synthase (ALS) gene.

SEQ ID NO: 108-nucleotide sequence encoding a chimeric DNA of ledRNA construct targeting barley (h.vulgare) ALS gene.

SEQ ID NO: 109: the nucleotide sequence corresponding to the protein coding region of the cDNA of the barley (Hordeum vulgare) HvNCED1 gene (accession No. AK 361999).

SEQ ID NO: 110-nucleotide sequence corresponding to the protein coding region of the cDNA corresponding to the barley (Hordeum vulgare) HvNCEDD2 gene (accession number DQ 145931).

SEQ ID NO: 111-nucleotide sequence of a chimeric DNA encoding the ledRNA construct targeting the NCED1 gene of barley (Hordeum vulgare) and wheat (Triticum aestivum).

SEQ ID NO: 112-nucleotide sequence of a chimeric DNA encoding the ledRNA construct targeting the NCED2 gene of barley (Hordeum vulgare) and wheat (Triticum aestivum).

SEQ ID NO: 113: the nucleotide sequence corresponding to the protein coding region of the cDNA of the barley gene encoding ABA-OH-2 (accession number DQ 145933).

SEQ ID NO: 114-nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the ABA-OH-2 gene of barley (Hordeum vulgare) and wheat (Triticum aestivum).

SEQ ID NO: 115-nucleotide sequence corresponding to the cDNA protein coding region of the Arabidopsis thaliana (A. thaliana) gene (At5g03280) encoding EIN 2.

SEQ ID NO: 116-nucleotide sequence encoding a chimeric DNA of ledRNA construct targeting the arabidopsis thaliana (a. thaliana) EIN2 gene.

SEQ ID NO: 117: a nucleotide sequence corresponding to the protein coding region of the cDNA of the arabidopsis thaliana (a. thaliana) gene (accession No. NM — 121396) encoding CHS.

SEQ ID NO: 118-nucleotide sequence encoding a chimeric DNA of ledRNA construct targeting the CHS gene of arabidopsis thaliana (a. thaliana).

SEQ ID NO: 119: a nucleotide sequence corresponding to the protein coding region of a cDNA encoding the N-like gene of lupin angustifolius (l.angustifolius) (accession XM _ 019604347).

SEQ ID NO: 120-nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a Lupinus angustifolia (L.angustifolius) N-like gene.

SEQ ID NO: 121-nucleotide sequence corresponding to the protein coding region of cDNA of the Vitis vinifera (Vitis pseudoreticulata) MLO gene (accession number KR 362912).

SEQ ID NO: 122-nucleotide sequence encoding a chimeric DNA of a first ledRNA construct targeting the MLO gene of Vitis castanea.

SEQ ID NO: 123-nucleotide sequence corresponding to the protein coding region of the cDNA of the Myzus persicae (Myzus persicae) MpC002 gene.

SEQ ID NO: 124-nucleotide sequence corresponding to the protein coding region of cDNA of the MpRack-1 gene of Myzus persicae (Myzus persicae).

SEQ ID NO: 125-nucleotide sequence encoding a chimeric construct targeting ledRNA of the C002 gene of Myzus persicae (M.persicae).

SEQ ID NO: 126-nucleotide sequence encoding a chimeric construct targeting ledRNA of the myzus persicae (M.persicae) Rack-1 gene.

SEQ ID NO: 127-nucleotide sequence corresponding to cDNA of the cotton bollworm (Helicoverpa armigera) ABCwhite gene.

SEQ ID NO: 128-nucleotide sequence of a chimeric DNA encoding the ledRNA construct targeting the ABC transporter white gene of cotton bollworm (Helicoverpa armigera).

SEQ ID NO: 129-nucleotide sequence of cDNA corresponding to Formica argentata (Linepihema humile) PBAN-type neuropeptide-like (accession XM-012368710).

SEQ ID NO: 130: a nucleotide sequence encoding a chimeric DNA of a ledRNA construct targeting a PBAN gene (accession XM _012368710) in argentina ants.

SEQ ID NO: 131: a nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding the V-type proton atpase catalytic subunit a of lucilia cuprina (l.cuprina) (accession XM _ 023443547).

SEQ ID NO: 132-nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the gene encoding RNAse 1/2 from copperna fly (l.

SEQ ID NO: 133-nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding chitin synthase of lucilia cuprina (l.

SEQ ID NO: 134-nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding the ecdysone receptor (EcR) of lucilia cuprina (l.

SEQ ID NO: 135-nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the gene encoding gamma-tubulin 1/1 of lucilia cuprina (l.

SEQ ID NO: 136-TaMlo target gene (AF 384144).

SEQ ID NO: 137-nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding TaMlo.

SEQ ID NO: 138-nucleotide sequence corresponding to the protein coding region of the cDNA of the Vitis vinifera (Vitis pseudoreticulata) MLO gene (accession number KR 362912).

SEQ ID NO: 139-nucleotide sequence encoding the chimeric DNA of the first ledRNA construct targeting the MLO gene of Vitis castanea.

140-Cyp 51 homolog 1 (accession number KK764651.1, locus RSAG8_00934) of SEQ ID NO.

141-Cyp 51 homolog 2 (accession number KK764892.1, locus number RSAG 8-12664) SEQ ID NO.

142-nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the gene encoding Cyp 51.

143-CesA 3 target gene (accession JN 561774.1).

144-nucleotide sequence of chimeric DNA encoding ledRNA construct targeting the gene encoding CesA 3.

Detailed Description

General techniques and definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be considered to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, gene silencing, protein chemistry, and biochemistry).

Unless otherwise indicated, recombinant proteins, cell cultures and immunological techniques for use in the invention are standard procedures well known to those skilled in the art. Such techniques are described throughout the literature from sources such as: J.though.A.practical Guide to Molecular Cloning, John Wiley and Sons (1984), J.Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring harbor Laboratory Press (1989), T.A.Brown (edition), Essential Molecular Biology: A Practical Aproach, Volumes 1and 2, IRL Press (1991), D.M.Glover and B.D.Hames (editors), DNA Cloning: A Practical Aproach, Volumes 1-4, IRL Press (1995and 1996), and F.M.Imual et al (edition), Current reagent in Molecular Cloning, Audio 1-4, IRL Press (1995and 1996), and F.M.Imual et al (edition), and wild vegetable reagent in biological, mineral Laboratory J.S.J..

The term "antisense regulatory element" or "antisense ribonucleic acid sequence" or "antisense RNA sequence" as used herein refers to an RNA sequence that is at least partially complementary to at least a portion of the target RNA molecule to which it hybridizes. In certain embodiments, the antisense RNA sequence modulates (increases or decreases) the expression or amount of the target RNA molecule or its activity, e.g., by decreasing translation of the target RNA molecule. In certain embodiments, the antisense RNA sequence alters splicing of the target pre-mRNA, resulting in a different splice variant. Exemplary components of antisense sequences include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these.

The term "antisense activity" is used in the context of the present invention to refer to any detectable and/or measurable activity attributed to the hybridization of an antisense RNA sequence to its target RNA molecule. Such detection and/or measurement may be direct or indirect. In one embodiment, antisense activity is assessed by detecting and/or measuring the amount of a transcript of the target RNA molecule. Antisense activity can also be detected as a phenotypic change associated with a target RNA molecule. As used herein, the term "target RNA molecule" refers to a gene transcript that is regulated by an antisense RNA sequence according to the invention. Thus, a "target RNA molecule" can be any RNA molecule whose expression or activity can be modulated by an antisense RNA sequence. Exemplary target RNA molecules include, but are not limited to, RNA (including, but not limited to, pre-mRNA and mRNA or portions thereof) transcribed from DNA encoding a target protein, rRNA, tRNA, small nuclear RNA, and miRNA, including their precursor forms. The target RNA may be a genomic RNA of a pathogen such as a virus or a pest, or an RNA molecule derived therefrom, such as a replicative form of a viral pathogen, or a transcript thereof. For example, the target RNA molecule can be RNA from an endogenous gene (or mRNA transcribed from the gene), or a gene introduced or can be introduced into a eukaryotic cell whose expression is associated with a particular phenotype, trait, disorder, or disease state, or a nucleic acid molecule from an infectious agent. In one embodiment, the target RNA molecule is in a eukaryotic cell. In another embodiment, the target RNA molecule encodes a protein. In this context, antisense activity can be assessed by detecting and/or measuring the amount of the target protein, for example by its activity, e.g. enzymatic activity, or a function different from the enzyme, or by a phenotype associated with its function. As used herein, the term "target protein" refers to a protein regulated by an antisense RNA sequence according to the invention.

In certain embodiments, antisense activity is assessed by detecting and/or measuring the amount of target RNA molecules and/or cleaved target RNA molecules and/or alternatively spliced target RNA molecules.

Antisense activity can be detected or measured using various methods. For example, antisense activity can be detected or assessed by comparing activity in a particular sample and comparing that activity to the activity of a control sample.

The term "targeting" is used in the context of the present invention to refer to the association of an antisense RNA sequence with a specific target RNA molecule or a specific region of nucleotides within a target RNA molecule. In one embodiment, the antisense RNA sequence according to the invention shares complementarity with at least one region of the target RNA molecule. As used herein, the term "complementarity" refers to a ribonucleotide sequence that can base pair with a ribonucleotide sequence on a target RNA molecule through hydrogen bonds between the bases on the ribonucleotide. For example, in RNA, adenine (A) is complementary to uracil (U) and guanine (G) is complementary to cytosine (C).

In certain embodiments, "complementary bases" refers to ribonucleotides of an antisense RNA sequence that are capable of base pairing with ribonucleotides of a sense RNA sequence in an RNA molecule of the invention or its target RNA molecule. For example, if a ribonucleotide at a position of an antisense RNA sequence is capable of hydrogen bonding with a ribonucleotide at a position of a target RNA molecule, the hydrogen bonded position between the antisense RNA sequence and the target RNA molecule is considered to be complementary at that ribonucleotide. In contrast, the term "non-complementary" refers to a pair of ribonucleotides that do not form hydrogen bonds with each other or otherwise support hybridization. The term "complementary" may also be used to refer to the ability of an antisense RNA sequence to hybridize by complementarity to another nucleic acid. In certain embodiments, the RNA sequence and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by ribonucleotides that can bind to each other to allow stable association between the antisense and sense RNA sequences in the RNA molecule and/or the target RNA molecule of the invention. One skilled in the art recognizes that mismatches can be included without eliminating the ability of the antisense RNA sequence to remain associated with the target. Thus, described herein are antisense RNA sequences that may comprise up to about 20% mismatched nucleotides (i.e., not complementary to the corresponding nucleotides of the target sequence). Preferably the antisense compound contains no more than about 15%, more preferably no more than about 10%, most preferably no more than 5% or no mismatches. The remaining ribonucleotides complement or do not disrupt hybridization (e.g., G: U or A: G pairs) between the antisense RNA sequence and the sense RNA sequence or the target RNA molecule. One of ordinary skill in the art will recognize that the antisense RNA sequences described herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (fully) complementary to at least one region of the target RNA molecule.

As used herein, "chimeric RNA molecule" refers to any RNA molecule that does not occur in nature. In one embodiment, the chimeric RNA molecules disclosed herein have been modified to produce mismatches in a region of the dsRNA. For example, the chimeric RNA molecule can be modified to convert cytosine to uracil. In one embodiment, the chimeric RNA molecule has been modified by treatment with bisulfite for a time and under conditions sufficient to convert unmethylated cytosines to uracil.

One skilled in the art will appreciate that various ribonucleotide combinations can be base paired. Both canonical and non-canonical base pairs are contemplated by the present invention. In one embodiment, base pairing may comprise A: T or G: C in a DNA molecule or U: A or G: C in an RNA molecule. In another embodiment, base pairing can comprise A: G or G: T or U: G.

The term "canonical base pairing" as used in the present invention means base pairing between two nucleotides, a: T or G: C for deoxyribonucleotides or a: U or G: C for ribonucleotides.

The term "non-canonical base pairing" as used in the present invention means an interaction between the bases of two nucleotides in the context of two DNA or two RNA sequences, other than canonical base pairing. For example, non-canonical base pairing includes pairing between G and U (G: U) or A and G (A: G). Examples of non-canonical base pairing include purine-purine or pyrimidine-pyrimidine. In the context of the present invention, the most common non-canonical base pairing is G: U. Other examples of non-canonical base pairs are less preferred to A: C, G: T, G: G and A: A.

The present invention relates to RNA components that "hybridize" across a series of ribonucleotides. Those skilled in the art will appreciate that terms such as "hybridize" and "hybridizing" are used to describe molecules that anneal based on complementary nucleic acid sequences. Such molecules do not require 100% complementarity in order to hybridize (i.e., they do not require "full basesPair ". For example, one or more mismatches in sequence complementarity may be present. In one embodiment, an RNA component as defined herein hybridizes under stringent hybridization conditions. The term "stringent hybridization conditions" refers to parameters familiar to the art, including the variation of hybridization temperature with the length of an RNA molecule. Ribonucleotide hybridization parameters can be found in the references compiled for these methods, Sambrook et al (see above), and Ausubel et al (see above). For example, stringent hybridization conditions as used herein can refer to hybridization buffer (3.5 XSSC, 0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5mM NaH) at 65 deg.C₂PO₄(pH7), 0.5% SDS, 2mM EDTA) and then washed one or more times in 0.2 XSSC, 0.01% BSA at 50 ℃. Shorter RNA components, such as RNA sequences 20-24 nucleotides in length, hybridize under less stringent conditions. The term "low stringency hybridization conditions" refers to parameters familiar to the art, including variations in hybridization temperature with the length of an RNA molecule. For example, as used herein, low stringency hybridization conditions can refer to hybridization buffers (3.5 XSSC, 0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5mM NaH) at 42 deg.C ₂PO₄(pH7), 0.5% SDS, 2mM EDTA) and then washed one or more times in 0.2 XSSC, 0.01% BSA at 30 ℃.

The invention also encompasses RNA components that span "full base pairs" of consecutive ribonucleotides. The term "complete base pair" is used in the context of the present invention to refer to a series of consecutive ribonucleotide base pairs. A fully base-paired series of consecutive ribonucleotides does not comprise gaps or non-base-paired nucleotides within the series. The term "contiguous" is used to refer to a series of ribonucleotides. A sequence comprising a consecutive series of ribonucleotides will be linked by a series of consecutive phosphodiester bonds, each ribonucleotide being directly bonded to the next.

The RNA molecules of the invention comprise a sense sequence and a corresponding antisense sequence. The relationship between these sequences is defined herein. The sequence relationship and activity of antisense sequences to target RNA molecules is also defined herein.

The term "covalent linkage" is used in the context of the present invention to refer to the linkage between the first and second RNA components or any RNA sequence or ribonucleotide. As will be understood by those skilled in the art, a covalent link or bond is a chemical bond involving the sharing of electron pairs between atoms. In one embodiment, the first and second RNA components or the sense RNA sequence and the antisense RNA sequence are covalently linked as part of a single RNA strand that can be folded back upon itself by self-complementarity. In this embodiment, the components are covalently linked across one or more ribonucleotides by phosphodiester bonds.

In the context of the present invention, the term "hybridization" means the pairing of complementary polynucleotides by base pairing of complementary bases. While not limited to a particular mechanism, the most common pairing mechanism involves hydrogen bonding between complementary ribonucleotides, which may be watson-crick hydrogen bonding.

As used herein, the phrase "an RNA molecule has a deleterious effect on a non-human organism" or similar phrases means that the target RNA molecule of the molecule is present in the non-human organism and that exposing a cell expressing the target RNA molecule to the target RNA molecule results in a decrease in the level and/or activity of the target RNA molecule when compared to the same cell lacking the RNA molecule. In one embodiment, the target RNA molecule encodes a protein that is important for growth, reproduction, or survival. As an example, if the non-human organism is a crop pest or pathogen, or an animal pest or pathogen, the RNA molecule can have a deleterious effect on feeding, apoptosis, cell differentiation and development, ability or desire to reproduce sexually, muscle formation, muscle twitch, muscle contraction, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, maintenance of cellular membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antenna formation, wing formation, leg formation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, larval phase transition, pupation, emergence from pupation, cell division, energy metabolism, respiration, chitin metabolism, formation of cytoskeletal structure of the pest or pathogen. In another embodiment, the non-human organism is a weed and the RNA molecule has a deleterious effect on amino acid biosynthesis, photosynthesis, fatty acid synthesis, cell membrane integrity, pigment synthesis, or growth.

As used herein, the phrase "an RNA molecule has a beneficial effect on at least one symptom of a disease" or similar phrases means that the target RNA of the molecule is present in a subject and exposure of a cell expressing the target RNA to the RNA molecule results in a decrease in the level and/or activity of the target RNA when compared to the same cell lacking the RNA molecule. In one embodiment, the target RNA encodes a protein that functions in the presence of disease. In one embodiment, the disease is cancer or a cancerous disease, an infectious disease, a cardiovascular disease, a neurological disease, a prion disease, an inflammatory disease, an autoimmune disease, a pulmonary disease, a renal disease, a liver disease, a mitochondrial disease, an endocrine disease, a reproduction-related disease and disorder, and any other indication that may be responsive to the level of a gene product expressed in a cell or organism.

The RNA molecules according to the invention, as well as compositions comprising the RNA molecules, can be administered to a subject. Terms such as "subject", "patient" or "individual" are terms that may be used interchangeably in the present invention in context. In one embodiment, the subject is a mammal. The mammal may be a pet, such as a dog or cat, or a domestic animal, such as a horse or cow. In one embodiment, the object is a person. For example, the subject may be an adult. In another embodiment, the object may be a child. In another embodiment, the subject may be a teenager. In another embodiment, the RNA molecules according to the invention and compositions comprising them can be administered to insects. In another embodiment, the RNA molecules according to the invention and compositions comprising said RNA molecules can be applied to plants. In another embodiment, the RNA molecules according to the invention, and compositions comprising them, can be administered to a fungal cell or population.

As used herein, "resistance" or variants thereof are relative terms in which the presence of an RNA molecule increases resistance, e.g., the reproduction of a pest or pathogen is reduced, or the level of damage to an organism is reduced.

As used herein, the term "not related in sequence to a target" refers to a molecule that is less than 50% identical along the entire length of an intervening RNA sequence. In another aspect, the term "related in sequence to a target" is a molecule that has 50% or more identity along the entire length of the intervening RNA sequence.

The term "and/or", such as "X and/or Y", is understood to mean "X and Y" or "X or Y", and is understood to provide explicit support for both meanings or both meanings.

As used herein, unless otherwise specified to the contrary, the term "about" means +/-20%, more preferably +/-10% of the specified value.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Non-canonical base pairing

In one embodiment, the RNA molecule of the invention comprises a sense ribonucleotide sequence and an antisense ribonucleotide sequence, which are capable of hybridizing to each other to form a double stranded (ds) RNA region having some non-canonical base pairing, i.e. having a combination of canonical and non-canonical base pairing. In one embodiment, the RNA molecule of the invention comprises two or more sense ribonucleotide sequences, each of which is capable of hybridizing to a region of one (contiguous) antisense ribonucleotide sequence to form a dsRNA region with some non-canonical base pairing. See, for example, fig. 1B. In one embodiment, the RNA molecule of the invention comprises two or more antisense sense ribonucleotide sequences, each of which is capable of hybridizing to a region of one (continuous) sense ribonucleotide sequence to form a dsRNA region with some non-canonical base pairing. See, for example, fig. 1A. In one embodiment, the RNA molecule of the invention comprises two or more antisense sense ribonucleotide sequences and two or more sense ribonucleotide sequences, wherein each antisense ribonucleotide sequence is capable of hybridizing to an antisense ribonucleotide sequence to form two or more dsRNA regions, one or both dsRNA regions comprising some non-canonical base pairing.

In the following embodiments, the full length of the dsRNA region (i.e. the entire dsRNA region) of the RNA molecule of the invention is considered to be a context of the features if only one (continuous) dsRNA region is present, or of each dsRNA region of the RNA molecule if two or more dsRNA regions are present in the RNA molecule. In one embodiment, at least 5% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 6% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 7% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 8% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 9% or 10% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 11% or 12% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 15% or about 15% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 20% or about 20% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 25% or about 25% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, at least 30% or about 30% of the base pairs in the dsRNA region are non-canonical base pairs. In each of these embodiments, it is preferred that at most 40% of the base pairs in the dsRNA region are non-canonical base pairs, more preferably at most 35% of the base pairs in the dsRNA region are non-canonical base pairs, and still more preferably at most 30% of the base pairs in the dsRNA region are non-canonical base pairs. In a less preferred embodiment, about 35% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, even more less preferably, about 40% of the base pairs in the dsRNA region are non-canonical base pairs. In each of the above embodiments, the dsRNA region may or may not comprise one or more non-base-paired ribonucleotides in the sense sequence or the antisense sequence or both.

In one embodiment, 10% to 40% of the base pairs in the dsRNA region of the RNA molecule of the invention are non-canonical base pairs. In one embodiment, 10% to 35% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 10% to 30% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 10% to 25% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 10% to 20% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 10% to 15% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 15% to 30% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 15% to 25% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 15% to 20% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 5% to 30% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 5% to 25% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 5% to 20% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 5% to 15% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 5% to 10% of the base pairs in the dsRNA region are non-canonical base pairs. In each of the above embodiments, the dsRNA region may or may not comprise one or more non-base-paired ribonucleotides in the sense sequence or the antisense sequence or both.

In one embodiment, the dsRNA region of the RNA molecule of the invention comprises 20 consecutive base pairs, wherein at least one of the 20 consecutive base pairs is a non-canonical base pair. In one embodiment, the dsRNA region comprises 20 contiguous base pairs, wherein at least 2 base pairs of the 20 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 20 contiguous base pairs, wherein at least 3 base pairs of the 20 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 20 contiguous base pairs, wherein at least 4 base pairs of the 20 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 20 contiguous base pairs, wherein at least 5 base pairs of the 20 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 20 contiguous base pairs, wherein at least 6 base pairs of the 20 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 20 contiguous base pairs, wherein at least 7 base pairs of the 20 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 20 contiguous base pairs, wherein at least 8 base pairs of the 20 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 20 contiguous base pairs, wherein at least 9 base pairs of the 20 contiguous base pairs are non-canonical base pairs. In each of these embodiments, preferably up to 10 of the 20 consecutive base pairs in the dsRNA region are non-canonical base pairs, more preferably up to 9 of the dsRNA region are non-canonical base pairs, still more preferably up to 8 of the base pairs in the dsRNA region are non-canonical base pairs, even more preferably up to 7 of the base pairs in the dsRNA region are non-canonical base pairs, and most preferably up to 6 of the base pairs in the dsRNA region are non-canonical base pairs. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably all non-canonical base pairs are G: U base pairs. Preferably, the features of the above embodiments apply to each of the 20 consecutive base pairs present in the RNA molecule of the invention.

In one embodiment, the dsRNA region of the RNA molecule of the invention comprises 21 consecutive base pairs, wherein at least one of the 21 consecutive base pairs is a non-canonical base pair. In one embodiment, the dsRNA region comprises 21 contiguous base pairs, wherein at least 2 base pairs of the 21 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 21 contiguous base pairs, wherein at least 3 base pairs of the 21 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 21 contiguous base pairs, wherein at least 4 base pairs of the 21 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 21 contiguous base pairs, wherein at least 5 base pairs of the 21 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 21 contiguous base pairs, wherein at least 6 base pairs of the 21 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 21 contiguous base pairs, wherein at least 7 base pairs of the 21 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 21 contiguous base pairs, wherein at least 8 base pairs of the 21 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 21 contiguous base pairs, wherein at least 9 base pairs of the 21 contiguous base pairs are non-canonical base pairs. In each of these embodiments, preferably up to 10 non-canonical base pairs of 21 consecutive base pairs in the dsRNA region, more preferably up to 9 non-canonical base pairs in the dsRNA region, still more preferably up to 8 non-canonical base pairs in the dsRNA region, even more preferably up to 7 non-canonical base pairs in the dsRNA region, and most preferably up to 6 non-canonical base pairs in the dsRNA region. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably all non-canonical base pairs are G: U base pairs. Preferably, the features of the above embodiments apply to each of the 21 consecutive base pairs present in the RNA molecule of the invention.

In one embodiment, the dsRNA region of the RNA molecule of the invention comprises 22 consecutive base pairs, wherein at least one of the 22 consecutive base pairs is a non-canonical base pair. In one embodiment, the dsRNA region comprises 22 contiguous base pairs, wherein at least 2 base pairs of the 22 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 22 contiguous base pairs, wherein at least 3 base pairs of the 22 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 22 contiguous base pairs, wherein at least 4 base pairs of the 22 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 22 contiguous base pairs, wherein at least 5 base pairs of the 22 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 22 contiguous base pairs, wherein at least 6 base pairs of the 22 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 22 contiguous base pairs, wherein at least 7 base pairs of the 22 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 22 contiguous base pairs, wherein at least 8 base pairs of the 22 contiguous base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 22 contiguous base pairs, wherein at least 9 base pairs of the 22 contiguous base pairs are non-canonical base pairs. In each of these embodiments, preferably up to 10 of the 22 consecutive base pairs in the dsRNA region are non-canonical base pairs, more preferably up to 9 of the dsRNA region are non-canonical base pairs, still more preferably up to 8 of the base pairs in the dsRNA region are non-canonical base pairs, even more preferably up to 7 of the base pairs in the dsRNA region are non-canonical base pairs, and most preferably up to 6 of the base pairs in the dsRNA region are non-canonical base pairs. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably all non-canonical base pairs are G: U base pairs. Preferably, the features of the above embodiments apply to each of the 22 consecutive base pairs present in the RNA molecule of the invention.

In one embodiment, the dsRNA region of the RNA molecule of the invention comprises 23 consecutive base pairs, wherein at least one of the 23 consecutive base pairs is a non-canonical base pair. In one embodiment, the dsRNA region comprises 23 consecutive base pairs, wherein at least 2 base pairs of the 23 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 23 consecutive base pairs, wherein at least 3 base pairs of the 23 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 23 consecutive base pairs, wherein at least 4 base pairs of the 23 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 23 consecutive base pairs, wherein at least 5 base pairs of the 23 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 23 consecutive base pairs, wherein at least 6 base pairs of the 23 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 23 consecutive base pairs, wherein at least 7 base pairs of the 23 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 23 consecutive base pairs, wherein at least 8 base pairs of the 23 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 23 consecutive base pairs, wherein at least 9 base pairs of the 23 consecutive base pairs are non-canonical base pairs. In each of these embodiments, preferably up to 10 non-canonical base pairs of 23 consecutive base pairs in the dsRNA region, more preferably up to 9 non-canonical base pairs in the dsRNA region, still more preferably up to 8 non-canonical base pairs in the dsRNA region, even more preferably up to 7 non-canonical base pairs in the dsRNA region, and most preferably up to 6 non-canonical base pairs in the dsRNA region. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably all non-canonical base pairs are G: U base pairs. Preferably, the features of the above embodiments apply to each of the 23 consecutive base pairs present in the RNA molecule of the invention.

In one embodiment, the dsRNA region of the RNA molecule of the invention comprises 24 consecutive base pairs, wherein at least one of the 24 consecutive base pairs is a non-canonical base pair. In one embodiment, the dsRNA region comprises 24 consecutive base pairs, wherein at least 2 base pairs of the 24 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 24 consecutive base pairs, wherein at least 3 base pairs of the 24 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 24 consecutive base pairs, wherein at least 4 base pairs of the 24 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 24 consecutive base pairs, wherein at least 5 base pairs of the 24 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 24 consecutive base pairs, wherein at least 6 base pairs of the 24 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 24 consecutive base pairs, wherein at least 7 base pairs of the 24 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 24 consecutive base pairs, wherein at least 8 base pairs of the 24 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 24 consecutive base pairs, wherein at least 9 base pairs of the 24 consecutive base pairs are non-canonical base pairs. In each of these embodiments, preferably up to 10 non-canonical base pairs of 24 consecutive base pairs in the dsRNA region, more preferably up to 9 non-canonical base pairs in the dsRNA region, still more preferably up to 8 non-canonical base pairs in the dsRNA region, even more preferably up to 7 non-canonical base pairs in the dsRNA region, and most preferably up to 6 non-canonical base pairs in the dsRNA region. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably all non-canonical base pairs are G: U base pairs. Preferably, the features of the above embodiments apply to each of the 24 consecutive base pairs present in the RNA molecule of the invention.

In the following embodiments, the full length of the dsRNA region (i.e. the entire dsRNA region) of the RNA molecule of the invention is considered to be a context of the features if only one (continuous) dsRNA region is present, or of each dsRNA region of the RNA molecule if two or more dsRNA regions are present in the RNA molecule. In one embodiment, the dsRNA region does not comprise 20 contiguous canonical base pairs, i.e., each sub-region of 20 contiguous base pairs comprises at least one non-canonical base pair, preferably at least one G: U base pair. In one embodiment, the dsRNA region does not comprise 19 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 18 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 17 consecutive canonical base pairs. In one embodiment, the dsRNA region does not comprise 16 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 15 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 14 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 13 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 12 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 11 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 10 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 9 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 8 contiguous canonical base pairs. In one embodiment, the dsRNA region does not comprise 7 contiguous canonical base pairs. In the above embodiments, it is preferred that the dsRNA region of the RNA molecule or the longest sub-region of consecutive canonical base pairings of each dsRNA region in the RNA molecule is 5, 6 or 7 consecutive canonical base pairings, i.e. towards the shorter length. Each feature of the above embodiments is preferably combined with the following features in an RNA molecule. In one embodiment, the dsRNA region comprises 10-19 or 20 consecutive base pairs. In a preferred embodiment, the dsRNA region comprises 12-19 or 20 consecutive base pairs. In one embodiment, the dsRNA region comprises 14-19 or 20 consecutive base pairs. In these embodiments, the dsRNA region comprises 15 contiguous base pairs. In one embodiment, the dsRNA region comprises 16, 17, 18, or 19 consecutive base pairs. In one embodiment, the dsRNA region comprises 20 contiguous base pairs. Preferably, in the above embodiments, the continuous base pairs comprise at least one non-canonical base pair including at least one G: U base pair, and more preferably, all non-canonical base pairs in the continuous base pair region are G: U base pairs.

In one embodiment, the dsRNA region comprises a 4 canonical base pair subregion flanked by non-canonical base pairs, i.e., at least 1, preferably 1 or 2 (no more than 2) non-canonical base pairs adjacent each end of the 4 canonical base pairs. In one embodiment, the dsRNA region comprises 2 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 3 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 4 or 5 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 6 or 7 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 8 to 10 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 11 to 15 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 50 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 40 subregions, each subregion being 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 30 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 20 sub-regions, each of which is 4 standard base pairs flanked by non-canonical base pairs. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably, all non-canonical base pairs that flank a consecutive canonical base pair in a subregion are G: U base pairs. In variants of the above embodiments, for some or all of the sub-regions, one or both flanking non-canonical base pairs are replaced in the sense sequence, the antisense sequence, or in both sequences by non-base-pairing ribonucleotides. It will be readily appreciated that in the above embodiments, the maximum number of sub-regions is determined by the length of the dsRNA region in the RNA molecule.

In one embodiment, the dsRNA region comprises a 5 canonical base pair sub-region flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 3 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 4 or 5 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 6 or 7 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 8 to 10 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 11 to 15 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 50 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 50 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 30 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 20 sub-regions, each of which is 5 standard base pairs flanked by non-canonical base pairs. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably, all non-canonical base pairs that flank a consecutive canonical base pair in a subregion are G: U base pairs. In variants of the above embodiments, for some or all of the sub-regions, one or both flanking non-canonical base pairs are replaced in the sense sequence, the antisense sequence, or in both sequences by non-base-pairing ribonucleotides. It will be readily appreciated that in the above embodiments, the maximum number of sub-regions is determined by the length of the dsRNA region in the RNA molecule.

In one embodiment, the dsRNA region comprises a 6 canonical base pair sub-region flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 sub-regions, each of which is 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 3 sub-regions, each of which is 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 4 or 5 sub-regions, each of which is 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 6 or 7 sub-regions, each of which is 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 8 to 10 sub-regions, each of which is 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 11 to 16 sub-regions, each of which is 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 60 subregions, each subregion being 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 60 subregions, each subregion being 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 30 sub-regions, each of which is 6 standard base pairs flanked by non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 to 20 sub-regions, each of which is 6 standard base pairs flanked by non-canonical base pairs. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably, all non-canonical base pairs that flank a consecutive canonical base pair in a subregion are G: U base pairs. In variants of the above embodiments, for some or all of the sub-regions, one or both flanking non-canonical base pairs are replaced in the sense sequence, the antisense sequence, or in both sequences by non-base-pairing ribonucleotides. It will be readily appreciated that in the above embodiments, the maximum number of sub-regions is determined by the length of the dsRNA region in the RNA molecule.

In one embodiment, the dsRNA region comprises a 10 contiguous base pair sub-region, wherein 2-4 base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 sub-regions, each of which is 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 3 sub-regions, each of which is 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 4 sub-regions, each of which is 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 5 sub-regions, each of which is 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 10 sub-regions, each of which is 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 4 sub-regions, each of which is 15 consecutive base pairs, wherein 2-6 of the 15 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 2-50 sub-regions, each sub-region comprising 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 2-40 sub-regions, each sub-region comprising 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 2-30 sub-regions, each sub-region comprising 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 2-20 sub-regions, each sub-region comprising 10 consecutive base pairs, wherein 2-4 of the 10 consecutive base pairs are non-canonical base pairs. In one embodiment, the non-canonical base pairs in one (contiguous) or more or all dsRNA regions of the RNA molecule are not adjacent to non-base pairs. In another embodiment, the non-canonical base pairs are at least 2 consecutive base pairs from non-base pairs. In another embodiment, the non-canonical base pairs are at least 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive base pairs from non-base pairs. In one embodiment, the non-canonical base pairs in one (contiguous) or more or all dsRNA regions of the RNA molecule are not adjacent to the loop sequence. In another embodiment, the non-canonical base pairs are at least 2 consecutive base pairs from the loop sequence. In another embodiment, the non-canonical base pairs are at least 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive base pairs from the loop sequence. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G: U base pair, more preferably all non-canonical base pairs in the sub-region are G: U base pairs. In variants of the above embodiments, for some or all of the sub-regions, one or more of the 2-4 or 2-6 non-canonical base pairs are replaced in the sense sequence, the antisense sequence, or both sequences by non-base-paired ribonucleotides. It will be readily appreciated that in the above embodiments, the maximum number of sub-regions is determined by the length of the dsRNA region in the RNA molecule.

In one embodiment, the ratio of canonical to non-canonical base pairs in the dsRNA region is between 2.5:1 and 3.5:1, e.g., about 3: 1. In one embodiment, the ratio of canonical to non-canonical base pairs in the dsRNA region is between 3.5:1 and 4.5:1, e.g., about 4: 1. In one embodiment, the ratio of canonical to non-canonical base pairs in the dsRNA region is between 4.5:1 and 5.5:1, e.g., about 5: 1. In one embodiment, the ratio of canonical to non-canonical base pairs in the dsRNA region is between 5.5:1 and 6.5:1, e.g., about 6: 1. Different dsRNA regions in an RNA molecule can have different ratios.

In the above embodiments, the non-canonical base pairs in the dsRNA region of the RNA molecule are preferably all G: U base pairs. In one embodiment, at least 99% of the non-canonical base pairs are G: U base pairs. In one embodiment, at least 98% of the non-canonical base pairs are G: U base pairs. In one embodiment, at least 97% of the non-canonical base pairs are G: U base pairs. In one embodiment, at least 95% of the non-canonical base pairs are G: U base pairs. In one embodiment, at least 90% of the non-canonical base pairs are G: U base pairs. In one embodiment, 90-95% of the non-canonical base pairs are G: U base pairs. For example, if there are 10 non-canonical base pairs, then at least 9 (90%) are G: U base pairs.

In another embodiment, 3% to 50% of the non-canonical base pairs are G: U base pairs. In another embodiment, 5% to 30% of the non-canonical base pairs are G: U base pairs. In another embodiment, 10% to 30% of the non-canonical base pairs are G: U base pairs. In another embodiment, 15% to 20% of the non-canonical base pairs are G: U base pairs.

In one example of the above embodiments, there is at least 3G: U base pairing in one (contiguous) or more or all dsRNA regions of the RNA molecule. In another example, there are at least 4, 5, 6, 7, 8, 9, or 10 GuU base pairs. In another example, there are at least 3 to 10 GuU base pairs. In another example, there are at least 5 to 10 G.U base pairs.

The dsRNA region comprising non-canonical base pairing comprises an antisense sequence of 20 contiguous nucleotides that serves as an antisense regulatory element. In one embodiment, the antisense regulatory element is at least 80%, preferably at least 90%, more preferably at least 95% or most preferably 100% complementary to a target RNA molecule in a eukaryotic cell. In one embodiment, the dsRNA region comprises 2, 3, 4 or 5 antisense regulatory elements that are complementary to the same target RNA molecule (i.e., complementary to different regions of the same target RNA molecule) or complementary to different target RNA molecules.

In one embodiment, when the sense and antisense sequences hybridize, one or more ribonucleotides of the sense ribonucleotide sequence or one or more ribonucleotides of the antisense ribonucleotide sequence, or both, are not base-paired in the dsRNA region. In this embodiment, the dsRNA region does not include any loop sequence covalently linking the sense and antisense sequences. One or more ribonucleotides of a dsRNA region or sub-region may not be base-paired. Thus, in this embodiment, the sense strand of the dsRNA region is incompletely base paired with its corresponding antisense strand.

In one embodiment, the chimeric RNA molecule does not comprise non-canonical base pairs at the bases of the loops of the molecule. In another embodiment, one, two, three, four, five or more or all non-canonical base pairs are flanked by canonical base pairs.

In one embodiment, the chimeric RNA molecule comprises at least one plant DCL-1 cleavage site.

In one embodiment, the target RNA molecule is not a viral RNA molecule.

In one embodiment, the target RNA molecule is not a South African cassava mosaic virus (South African cassava mosaic virus) RNA molecule.

In one embodiment, the chimeric RNA molecule comprises at least one non-base pair or at least one stretch of non-base pairs flanked by canonical base pairs, non-canonical base pairs, or both canonical and non-canonical base pairs. This may be, for example, a bump as described herein.

In one embodiment, the chimeric RNA molecule does not comprise a double-stranded region having greater than 11 canonical base pairs.

Furthermore, in one embodiment and optionally in combination with any feature of the embodiments above, the total number of ribonucleotides in the sense sequence and the total number of ribonucleotides in the antisense sequence may not be the same, although preferably they are the same. In one embodiment, the total number of ribonucleotides in the sense ribonucleotide sequence of the dsRNA region is 90% -110% of the total number of ribonucleotides in the antisense ribonucleotide sequence. In one embodiment, the total number of ribonucleotides in the sense ribonucleotide sequence is 95% -105% of the total number of ribonucleotides in the antisense ribonucleotide sequence. In one embodiment, the chimeric RNA molecules of the invention may comprise one or more structural elements, such as internal or terminal bulges or loops. Various embodiments of the ridges and rings are discussed above. In one embodiment, the dsRNA regions are separated by structural elements such as ridges or loops. In one embodiment, the dsRNA regions are separated by intervening (spacer) sequences. Some of the ribonucleotides of the spacer sequence may base pair with other ribonucleotides in the RNA molecule, e.g. with other ribonucleotides in the spacer sequence, or they may not base pair in the RNA molecule, or some of each of them may not base pair. In one embodiment, the dsRNA region is linked to a terminal loop. In one embodiment, the dsRNA region is flanked by terminal loops.

In one embodiment, when the dsRNA region of the RNA molecule of the invention has at least 3 non-canonical base pairs in any sub-region of 5 consecutive base pairs, the non-canonical base pairs are not contiguous, but are separated by one or more canonical base pairs, i.e. the dsRNA region does not have 3 or more consecutive non-canonical base pairs. In one embodiment, the dsRNA region does not have 4 or more contiguous non-canonical base pairs. For example, in one embodiment, a dsRNA region comprises at least 3 non-canonical base pairs in a 10 base pair sub-region, wherein each non-canonical base pair is separated by 4 canonical base pairs.

In one embodiment, the RNA molecule of the invention comprises more than one dsRNA region. For example, the RNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dsRNA regions. In this embodiment, one or more or all of the dsRNA regions may comprise the properties exemplified above, such as non-canonical base pairing and/or several antisense regulatory elements.

Silencing Activity

The RNA molecules of the invention have antisense activity because they comprise a sense ribonucleotide sequence that is substantially complementary to a region of the target RNA molecule. For example, the ribonucleotide sequence is substantially complementary to a region of a target RNA molecule in a eukaryotic cell. In one embodiment, the target RNA molecule can be in a bacterial cell, a fungal cell, a plant cell, an insect cell, or an animal cell. Such components of the RNA molecules defined herein may be referred to as "antisense regulatory elements". By "substantially complementary" is meant that the sense ribonucleotide sequence can have an insertion, a deletion, and a single point mutation as compared to the complement of the target RNA molecule in the eukaryotic cell. Preferably, the homology between the sense ribonucleotide sequence having antisense activity and the target RNA molecule is at least 80%, preferably at least 90%, preferably at least 95%, most preferably 100%. For example, the sense ribonucleotide sequence can comprise about 15, about 16, about 17, about 18, about 19 or more consecutive nucleotides with a sequence that is identical to the first region of the target RNA molecule in a eukaryotic cell. In another embodiment, the sense ribonucleotide sequence can comprise about 20 consecutive nucleotides that have the same sequence as the first region of the target RNA molecule in a eukaryotic cell.

"antisense activity" is used in the context of the present invention to refer to antisense regulatory elements from an RNA molecule as defined herein which modulate (increase or decrease) the expression of a target RNA molecule.

In various embodiments, an antisense regulatory element according to the invention can comprise a plurality of monomeric subunits linked together by a linker. Examples include primers, probes, antisense compounds, antisense oligonucleotides, External Guide Sequence (EGS) oligonucleotides, alternative splicers, gapmers, siRNA and microRNA. Thus, an RNA molecule according to the invention may comprise an antisense regulatory element having a single-stranded, double-stranded, circular, branched or hairpin structure. In one embodiment, the antisense sequence may contain structural elements, such as internal or terminal ridges or loops.

In one embodiment, the RNA molecules of the invention comprise a chimeric oligomeric component, such as a chimeric oligonucleotide. For example, the RNA molecule can comprise differently modified nucleotides, mixed backbone antisense oligonucleotides, or a combination thereof. In one embodiment, the chimeric oligomeric compound may comprise at least one modified region to confer increased resistance to nuclease degradation, increased cellular uptake and/or increased binding affinity for a target RNA molecule.

Antisense regulatory elements can be of various lengths. In various embodiments, the invention provides antisense regulatory elements consisting of X-Y linked bases, wherein X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 (provided X < Y). For example, in certain embodiments, the invention provides antisense regulatory elements comprising 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 11-23, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-28, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26, 22-27, 22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked bases.

The RNA molecule according to the invention may comprise a plurality of antisense regulatory elements. For example, an RNA molecule can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 antisense regulatory elements. In one embodiment, the antisense regulatory elements are identical. In this embodiment, the RNA molecule may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 copies of the antisense regulatory element. In another embodiment, the RNA molecule according to the invention may comprise different antisense regulatory elements. For example, antisense regulatory elements may be provided to target multiple genes in a pathway such as lipid biosynthesis. In this embodiment, the RNA molecule may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 different antisense regulatory elements.

Antisense regulatory elements according to the invention can modulate (increase or decrease) the expression or amount of various target RNA molecules. In one embodiment, the target RNA molecule is a fatty acid biosynthesis gene. Examples of such genes include genes encoding: acetyl transferase; acyl transporters (e.g., "acyl carrier proteins"); desaturases, such as stearoyl desaturase or microsomal D12-desaturase, in particular the Fad2-1 gene; malonyl transacylase; -a ketoacyl-ACP synthase; 3-keto-ACP reductase; enoyl-ACP hydratase; thioesterases, such as acyl-ACP thioesterases; enoyl-ACP reductase. In one embodiment, the target RNA molecule is the FAD2 gene (e.g., those described by Genbank accession number: Genbank). And (3) unpublished: AF124360 (Brassica carinata), AF042841 (Brassica rapa), L26296 (Arabidopsis thaliana), A65102 (Brassica napus). For example, the target RNA molecule can be a FAD2.1 gene. In another embodiment, the target RNA molecule can be a FAD2.2 gene. In another embodiment, the target RNA molecules can be FAD2.1 and FAD2.2 genes. Examples of other genes involved in modifying the lipid composition, which may be a target RNA molecule, are known in the art (see, e.g., Shure et al, 1983; Preiss et al, 1987; Gupta et al, 1988; Olive et al, 1989; Bhattacharyya et al, 1990; Dunwell, 2000; Brar et al, 1996; Kishore and Somerville, 1993; US5,530,192 and WO 94/18337).

In another embodiment, the target RNA molecule is an arthropod gene, such as an insect gene transcript. Examples of such genes include chitin synthase genes, such as CHS1 and/or CHS2 or other genes that control insect activity, behavior, reproduction, growth and/or development. Various essential genes for various pathogens are known to those skilled in the art (e.g., nematode resistance genes are summarized in WO 93/10251, WO 94/17194).

In another embodiment, the target RNA molecule is associated with a disease. For example, the target RNA molecule can be an oncogene or tumor suppressor gene transcript. Exemplary oncogenes include ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA, ERBB, EBRB2, FGR, FOS, FYN, HRAS, JUN, LCK, LYN, MYB, MYC, NRAS, RET, or SRC. Exemplary tumor suppressor genes include BRCA1 or BRCA 2; an adhesion molecule; cyclin kinases and inhibitors thereof.

In another embodiment, the target RNA molecule is associated with a delay in fruit ripening. Delaying fruit ripening can be achieved, for example, by reducing expression of a gene selected from the group consisting of: polygalacturonase, pectinesterase, beta- (1-4) -glucanase (cellulase), galactosidase (beta-galactosidase); ethylene biosynthetic genes such as 1-aminocyclopropane-1-carboxylic acid synthase; genes for carotenoid biosynthesis, such as, for example, the biosynthesis of pre-phytoene or phytoene.

In another embodiment, the target RNA molecule is associated with a delay in the symptoms of aging. Suitable target RNA molecules include cinnamoyl-CoA: NADPH reductase or cinnamoyl alcohol dehydrogenase. (in WO 1995/07993) further describe target RNA molecules.

In another embodiment, the target RNA molecule is associated with a modification of the fiber content in a food product, preferably a seed. For example, the RNA molecule may reduce the expression of caffeic acid O-methyltransferase or cinnamoyl alcohol dehydrogenase.

ledRNA molecules

In certain embodiments, the RNA molecules of the invention comprise a first RNA component covalently linked to a second RNA component. In a preferred embodiment, the RNA molecule hybridizes or folds upon itself to form a "dumbbell" or ledRNA structure, see, e.g., FIG. 1. In one embodiment, the molecule further comprises one or more of:

-a linking ribonucleotide sequence, which covalently links the first and second RNA components;

-a 5' leader sequence; and the combination of (a) and (b),

-a 3' trailer sequence.

In one embodiment, the first RNA component consists of, in 5 'to 3' order, a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide, wherein said first 5 'and 3' ribonucleotides in the RNA molecule are base-paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 consecutive ribonucleotides, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence of at least 20 consecutive ribonucleotides, wherein the first antisense ribonucleotide sequence is hybridized to the first sense ribonucleotide sequence in the RNA molecule, wherein the first antisense ribonucleotide sequence is capable of hybridizing to a first region of a target RNA molecule.

In another embodiment, the first RNA component consists of, in 5 'to 3' order, a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide, wherein the first 5 'and 3' ribonucleotides in the RNA molecule are base-paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 consecutive ribonucleotides, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence of at least 20 consecutive ribonucleotides, wherein the first antisense ribonucleotide sequence in the RNA molecule is fully base-paired with the first sense ribonucleotide sequence, wherein the first antisense ribonucleotide sequence is identical to the sequence of the complement of the first region of the target RNA molecule. Examples of the first RNA component of both embodiments are schematically shown in the left half of fig. 1A or the right half of fig. 1B.

In another embodiment, the first RNA component consists of a first 5 'ribonucleotide, a first RNA sequence and a first 3' ribonucleotide, wherein the first 5 'and 3' ribonucleotides in the first RNA component base pair with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence, a first loop sequence of at least 4 ribonucleotides and a first antisense ribonucleotide sequence, wherein the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence each have at least 20 consecutive ribonucleotides, whereby at least 20 consecutive ribonucleotides of said first sense ribonucleotide sequence are fully base paired with at least 20 consecutive ribonucleotides of said first antisense ribonucleotide sequence, wherein at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are substantially identical in sequence to the first region of the target RNA molecule.

In these embodiments, the base pair formed between the first 5 'ribonucleotide and the first 3' ribonucleotide is considered to be the terminal base pair of the dsRNA region formed by self-hybridization of the first RNA component, i.e. it defines the end of the dsRNA region.

In one embodiment, the first sense sequence has substantial sequence identity to a region of the target RNA, which identity can be for a sequence less than 20 nucleotides in length. In one embodiment, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive ribonucleotides, preferably at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical in sequence to the first region of the target RNA molecule. In another embodiment, at least 15, at least 16, at least 17, at least 18, at least 19 consecutive ribonucleotides of the first sense ribonucleotide sequence have 100% identity with the first region of the target RNA molecule. In one embodiment, the first 3, 4, 5, 6, or 7 ribonucleotides from the 5' end of the first sense ribonucleotide sequence are 100% identical to a region of the target RNA molecule and the remaining ribonucleotides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the target RNA molecule.

In one embodiment, at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the first region of the target RNA molecule. Also, in this embodiment, the first 3, 4, 5, 6, or 7 ribonucleotides can be 100% identical to a region of the target RNA molecule, with the remaining ribonucleotides being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the target RNA molecule. In another embodiment, at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence have 100% identity with the first region of the target RNA molecule.

In one embodiment, the first antisense sequence has substantial sequence identity to the complement of the region of the target RNA, which identity can be a sequence that is less than 20 nucleotides in length for the complement. In one embodiment, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive ribonucleotides, preferably at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical in sequence to the complement of the first region of the target RNA molecule. In another embodiment, at least 15, at least 16, at least 17, at least 18, at least 19 contiguous ribonucleotides of the first antisense ribonucleotide sequence have 100% identity with the complement of the first region of the target RNA molecule. In one embodiment, the first 3, 4, 5, 6, or 7 ribonucleotides from the 5' end of the first antisense ribonucleotide sequence are 100% identical to the complement of a region of the target RNA molecule, and the remaining ribonucleotides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the complement of the target RNA molecule.

In one embodiment, at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the complement of the first region of the target RNA molecule. Also, in this embodiment, the first 3, 4, 5, 6, or 7 ribonucleotides are 100% identical to the complement of a region of the target RNA molecule, and the remaining ribonucleotides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the complement of the target RNA molecule. In another embodiment, at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence have 100% identity with the first region of the target RNA molecule.

In another embodiment, the second RNA component consists of, in 5 'to 3' order, a second 5 'ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide, wherein the second 5 'and 3' ribonucleotides are base paired, wherein the second RNA sequence comprises a second sense ribonucleotide sequence, a second loop sequence of at least 4 ribonucleotides, and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence is base paired with the second antisense ribonucleotide sequence. In this embodiment, the base pair formed between the second 5 'ribonucleotide and the second 3' ribonucleotide is considered to be the terminal base pair of the dsRNA region formed by self-hybridization of the second RNA component.

In one embodiment, the RNA molecule comprises a 5 'leader sequence or a 5' extension sequence, which may be produced as a result of transcription from a promoter in the genetic construct, from the start site of transcription to the start of the polynucleotide encoding the remainder of the RNA molecule. Preferably, the 5 'leader sequence or 5' extension sequence is relatively short compared to the remainder of the molecule and can be removed from the RNA molecule post-transcriptionally, for example by rnase treatment. The 5 'leader sequence or 5' extension sequence may be largely non-base-paired, or may contain one or more stem-loop structures. In this embodiment, the 5' leader sequence may consist of a ribonucleotide sequence that is covalently linked to either the first 5' ribonucleotide if the second RNA component is linked to the first 3 ' ribonucleotide or to the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide. In one embodiment, the 5' leader sequence is at least 10, at least 20, at least 30, at least 100, at least 200 ribonucleotides long, preferably to a maximum length of 250 ribonucleotides. In another embodiment, the 5' leader sequence is at least 50 ribonucleotides long. In one embodiment, the 5' leader sequence may be used as an extension sequence for amplification of the RNA molecule by a suitable amplification reaction. For embodiments, the extension sequence may facilitate amplification by a polymerase.

In another embodiment, the RNA molecule comprises a 3 'trailer sequence or 3' extension sequence, which may result from continued transcription up to the transcription termination or polyadenylation signal in the construct encoding the RNA molecule. The 3 'trailer sequence or 3' extension sequence may comprise a poly A (polyA) tail. Preferably, the 3 'trailer sequence or 3' extension sequence is relatively short compared to the remainder of the molecule and can be removed from the RNA molecule following transcription, for example by RNase treatment. The 3 'trailer sequence or 3' extension sequence may be largely non-base-paired, or may contain one or more stem-loop structures. In this embodiment, the 3' tail sequence may consist of a ribonucleotide sequence, which is covalently linked to the second 3' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or to the first 3' ribonucleotide if the second RNA component is linked to the first 5 ' ribonucleotide. In one embodiment, the 3' leader sequence is at least 10, at least 20, at least 30, at least 100, at least 200 ribonucleotides long, preferably to a maximum length of 250 ribonucleotides. In another embodiment, the 3' leader sequence is at least 50 ribonucleotides long. In one embodiment, the 3' trailer sequence may be used as an extension sequence for amplification of an RNA molecule by a suitable amplification reaction. For embodiments, the extension sequence may facilitate amplification by a polymerase.

In one embodiment, all nucleotides except two ribonucleotides are covalently linked to two other nucleotides, i.e. the RNA molecule consists of only one RNA strand with a self-complementary region and thus has only one 5 'terminal nucleotide and one 3' terminal nucleotide. In another embodiment, all nucleotides except 4 ribonucleotides are covalently linked to two other nucleotides, i.e. the RNA molecule consists of 2 RNA strands with hybridized complementary regions and thus has only two 5 'terminal nucleotides and two 3' terminal nucleotides. In another embodiment, each ribonucleotide is covalently linked to two other nucleotides, i.e., the RNA molecule is circular and has a self-complementary region, and thus no 5 'terminal nucleotide and no 3' terminal nucleotide.

In one embodiment, the double-stranded region of the RNA molecule can comprise one or more bulges created by unpaired nucleotides in the sense RNA sequence or the antisense RNA sequence, or both. In one embodiment, the RNA molecule comprises a series of ridges. For embodiments, the double-stranded region of the RNA molecule can have 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bulges. Each protuberance can be independently one, two, or more unpaired nucleotides, up to 10 nucleotides. The longer sequence may loop out of the sense or antisense sequence in the dsRNA region, which may be internally base-paired or remain unpaired. In another embodiment, the double-stranded region of the RNA molecule does not comprise a bulge, i.e., complete base pairing along the entire length of the dsRNA region.

In another embodiment, the first sense ribonucleotide sequence is covalently linked to the first 5 'ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is covalently linked to the first 3' ribonucleotide without any intervening nucleotides, or both. In another embodiment, there are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 intervening nucleotides. It is understood that such intervening nucleotides are not related in sequence to the target RNA molecule, but may help stabilize base pairing of adjacent sense and antisense sequences.

In another embodiment, 20 consecutive nucleotides of the first sense ribonucleotide sequence are covalently linked to the first 5 'ribonucleotide without any intervening nucleotides, and 20 consecutive nucleotides of the first antisense ribonucleotide sequence are covalently linked to the first 3' ribonucleotide without any intervening nucleotides. In another embodiment, there are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 intervening nucleotides. The intervening nucleotides may base pair as part of the double-stranded region of the RNA molecule, but are not related in sequence to the target RNA. They may help provide increased stability to the double-stranded region or to bind the two ends of the RNA molecule together without leaving the 5 'or 3' ends, or both, that are not base-paired.

In one embodiment, the first and second RNA components comprise a linked ribonucleotide sequence. In one embodiment, the linking ribonucleotide sequence serves as a spacer between a first sense ribonucleotide sequence and the other components of the molecule, said first sense ribonucleotide sequence being essentially identical in sequence to the first region of the target RNA molecule. For example, the linking ribonucleotide sequence can serve as a spacer between the region and the loop. In another embodiment, the RNA molecule comprises a plurality of sense ribonucleotide sequences that are substantially identical in sequence to the first region of the target RNA molecule and a linking ribonucleotide sequence that serves as a spacer between these sequences. In one embodiment, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 ribonucleotide sequences are provided in the RNA molecule that are essentially identical in sequence to the first region of the target RNA molecule, each ribonucleotide sequence being separated from each other by a linking ribonucleotide sequence.

In one embodiment, the RNA molecule comprises a 5' leader sequence. In one embodiment, the 5' leader sequence consists of a ribonucleotide sequence that is covalently linked to either the first 5' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide. In one embodiment, the RNA molecule has a modified 5 'or 3' end, for example by attachment of a lipid group (such as cholesterol), or a vitamin (such as biotin), or a polypeptide. Such modifications can help to incorporate the RNA molecule into the eukaryotic cell in which the RNA will function.

In one embodiment, the length of the linked ribonucleotide sequence is less than 100 ribonucleotides. In one embodiment, the length of the linked ribonucleotide sequence is less than 50 ribonucleotides. In one embodiment, the length of the linked ribonucleotide sequence is less than 20 ribonucleotides. In one embodiment, the length of the linked ribonucleotide sequence is less than 10 ribonucleotides. In one embodiment, the length of the linked ribonucleotide sequence is less than 5 ribonucleotides. In one embodiment, the linking ribonucleotide sequence is between 1 and 100 ribonucleotides in length. In one embodiment, the linking ribonucleotide sequence is between 1 and 50 ribonucleotides in length. In one embodiment, the linking ribonucleotide sequence is between 1 and 20 ribonucleotides in length. In one embodiment, the linking ribonucleotide sequence is between 1 and 10 ribonucleotides in length. In one embodiment, the linking ribonucleotide sequence is 1 to 5 ribonucleotides in length. In one embodiment, the ribonucleotides that are linked to the ribonucleotide sequence are not base paired. In a preferred embodiment, the ribonucleotides of the linked ribonucleotide sequence are all base paired, or all but 1, 2 or 3 ribonucleotides are base paired.

In one embodiment, the first or second RNA component comprises a hairpin structure. In a preferred embodiment, the first and second RNA components each comprise a hairpin structure. In these embodiments, the hairpin structure may be a stem-loop. Thus, in one embodiment, an RNA molecule can comprise first and second RNA components that each comprise a hairpin structure, wherein the hairpins are covalently bound by a linker sequence. See, for example, fig. 1. In one embodiment, the linker sequence is one or more unpaired ribonucleic acids (RNAs). In one embodiment, the linker sequence is 1 to 10 unpaired ribonucleotides.

In one embodiment, the RNA molecule has a double hairpin structure, i.e. a "ledRNA structure" or a "dumbbell structure". In this embodiment, the first hairpin is a first RNA component and the second hairpin is a second RNA component. In these embodiments, the first 3 'ribonucleotide and the second 5' ribonucleotide, or the second 3 'ribonucleotide and the first 5' ribonucleotide, but not both, are covalently linked. In this embodiment, the other 5'/3' ribonucleotides can be separated by a cleft (i.e., a discontinuity in the dsRNA molecule in which there is no phosphodiester linkage between the 5'/3' ribonucleotides). Fig. 1B shows an embodiment of this type of arrangement. In another embodiment, the respective 5'/3' ribonucleotides can be separated by a loop. The 5 'leader sequence and the 3' trailer sequence may be the same or different in length. For embodiments, the 5 'leader may be about 5, 10, 15, 20, 25, 50, 100, 200, 500 ribonucleotides longer than the 3' trailer, and vice versa.

In embodiments where the RNA molecule has a double hairpin structure, the second hairpin (in addition to the first hairpin structure) comprises a sense RNA sequence and an antisense RNA sequence, which are substantially identical in sequence to the region of the target RNA molecule or its complement, respectively. In one embodiment, each hairpin has a series of ribonucleotides that are substantially identical in sequence to a region of the same target RNA molecule. In one embodiment, each hairpin has a series of ribonucleotides that are substantially identical in sequence to different regions of the same target RNA molecule. In one embodiment, each hairpin has a series of ribonucleotides that are substantially identical in sequence to regions of different target RNA molecules, i.e., an RNA molecule can be used to reduce the expression and/or activity of two target RNA molecules that may not be related in sequence.

In each hairpin of the double hairpin structure of the RNA molecule, the order in the sense and antisense RNA sequences in each hairpin can independently be sense followed by antisense, or antisense followed by sense, in 5 'to 3' order. In a preferred embodiment, the order of sense and antisense sequences in a double hairpin structure of an RNA molecule is where both sense sequences are contiguous antisense-sense-antisense (FIG. 1A), or where both antisense sequences are contiguous sense-antisense-sense (FIG. 1B).

In one embodiment, the RNA molecule can comprise, in 5 'to 3' order, a 5 'leader sequence, a first loop, a sense RNA sequence, a second loop, and a 3' trailer sequence, wherein the 5 'and 3' leader sequences are covalently bound to the sense strand to form a dsRNA sequence. In one embodiment, the 5 'leader sequence and the 3' trailer sequence are not covalently bound to each other. In one embodiment, the 5 'leader sequence and the 3' trailer sequence are separated by a cleft. In one embodiment, the 5 'leader sequence and the 3' trailer sequence are joined together to provide an RNA molecule having a closed structure. In another embodiment, the 5 'leader sequence and the 3' trailer sequence are separated by a loop.

The term "loop" as used in the context of the present invention refers to a loop structure formed by a series of non-complementary ribonucleotides in an RNA molecule as disclosed herein. The loop typically follows a series of base pairs between the first and second RNA components or joins the sense RNA sequence and the antisense RNA sequence in one or both of the first and second RNA components. In one embodiment, typically for a shorter loop of 4-10 ribonucleotides, all the cyclic ribonucleotides are non-complementary. In other embodiments, some ribonucleotides in one or more loops are complementary and are capable of base pairing within the loop sequence, provided that these base pairings are capable of forming a loop structure. For example, at least 5%, at least 10%, or at least 15% of the cyclic ribonucleotides are complementary. Embodiments of loops include stem loops or hairpins, pseudoknots (pseudokinot), and tetracyclic (tetracyoop).

In one embodiment, the RNA molecule comprises only two loops. In another embodiment, the RNA molecule comprises at least two, at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 loops, preferably at most 10 loops. For example, an RNA molecule can comprise 4 loops.

Various sizes of rings are contemplated by the present invention. For example, a loop may comprise 4, 5, 6, 7, 8, 9, 10, 11, or 12 ribonucleotides. In other embodiments, the loop comprises 15, 20, 25, or 30 nucleotides. In one embodiment, one or all of the loop sequences are longer than 20 nucleotides. In other embodiments, the loop is larger, e.g., comprises 50, 100, 150, 200, or 300 ribonucleotides. In one embodiment, the loop comprises 160 ribonucleotides. In another less preferred embodiment, the loop comprises 200, 500, 700 or 1000 ribonucleotides, provided that the loop does not interfere with the hybridization of the sense and antisense RNA sequences. In one embodiment, each loop has the same number of ribonucleotides. For example, the loop may have a length of 100 to 1000 ribonucleotides. For example, the loop may have a length of 600 to 1000 ribonucleotides. For example, a loop can have 4 to 1000 ribonucleotides. For example, the loop preferably has 4 to 50 ribonucleotides. In another embodiment, the loops comprise a different number of ribonucleotides.

In another embodiment, one or more of the loops comprises an intron that can be spliced out of the RNA molecule. In one embodiment, the intron is from a plant gene. Exemplary introns include intron 3 of zearalanol dehydrogenase 1(Adhl) (GenBank: AF044293), intron 4 of soybean β -conglycinin α subunit (GenBank: AB 051865); one of the introns of the pea rbcS-3A gene (GenBank: X04333) of the small subunit of ribulose-1, 5-bisphosphate carboxylase (RBC). Other embodiments of suitable introns are discussed in (McCullough and Schuler, 1997; Smith et al, 2000).

In various embodiments, a loop may be at the end of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 consecutive base pairs, which may be canonical base pairs or may include one or more non-canonical base pairs. In other embodiments, particularly vertebrate cells, less preferred, the loops may be at the end of at least 20, 30, 50, 100, 200, 500 or more consecutive base pairs.

In another embodiment, the RNA molecule comprises two or more sense ribonucleotide sequences and an antisense ribonucleotide sequence fully base-paired therewith, each of which is identical in sequence to a region of the target RNA molecule. For example, an RNA molecule can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more sense ribonucleotide sequences that are each independently identical in sequence to a region of the target RNA molecule, and an antisense ribonucleotide sequence that is fully base-paired therewith. In this embodiment, any one or more or all of the sequences may be separated by a linking ribonucleotide sequence. In this embodiment, any one or more or all of the sequences may be separated by loops.

In one embodiment, the two or more sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule. For example, the sequence may be identical to at least 2, at least 3, at least 4, at least 5, at least 6 regions of the same target molecule. In another embodiment, the two or more sense ribonucleotide sequences are identical in sequence. In one embodiment, the two or more sense ribonucleotide sequences are identical in sequence to the same region of the same target RNA molecule. In another embodiment, the two or more sense ribonucleotide sequences are identical in sequence to different target RNA molecules. For embodiments, the sequence may be identical to at least 2, at least 3, at least 4, at least 5, at least 6 regions of different target molecules.

In another embodiment, the two or more sense ribonucleotide sequences are devoid of an intervening loop (spacer) sequence.

In one embodiment, the RNA molecule can comprise a single strand of ribonucleotides having a 5 'end, at least one sense ribonucleotide sequence of at least 21 nucleotides in length, an antisense ribonucleotide sequence that is fully base paired with each sense ribonucleotide sequence over at least 21 consecutive nucleotides, at least two loop sequences and a 3' end. In this embodiment, the 5 '-terminal ribonucleotide and the 3' -terminal ribonucleotide are not directly covalently bound, but are adjacent to each base-pairing position.

In another embodiment, consecutive base pairs of the RNA component are separated by at least one gap. In one embodiment, a "gap" is provided by an unpaired ribonucleotide. In another embodiment, a "gap" is provided by an unligated 5 'leader sequence and/or 3' trailer sequence. In this embodiment, the gap may be referred to as an "unconnected gap". Mismatches and unligated gaps may be located at different positions in the RNA molecule. For embodiments, an unlinked gap may immediately follow the antisense sequence. In another embodiment, the unligated nick may be proximal to the loop of the RNA molecule. In another embodiment, the unconnected notches are positioned approximately equidistant between the at least two rings.

In one embodiment, the RNA molecule is produced from a single strand of RNA. In one embodiment, the single strand is not circularly closed, e.g., comprises an unlinked nick. In another embodiment, the RNA molecule is a circular closed molecule. The closed molecule can be generated by ligating the RNA molecules described above (e.g., with an RNA ligase) that contain unligated nicks.

In another embodiment, the RNA molecule comprises a 5 '-extended sequence or a 3' -extended sequence or both. For example, the RNA molecule can comprise a 5 'extension sequence covalently linked to a first 5' ribonucleotide. In another embodiment, the RNA molecule comprises a 3 'extension sequence covalently linked to a second 3' ribonucleotide. In another embodiment, the RNA molecule comprises a 5 'extension sequence covalently linked to a first 5' ribonucleotide and a 3 'extension sequence covalently linked to a second 3' ribonucleotide.

In another embodiment, the RNA molecule comprises a 5 'extension sequence covalently linked to a second 5' ribonucleotide. In another embodiment, the RNA molecule comprises a 3 'extension sequence covalently linked to a first 3' ribonucleotide. In another embodiment, the RNA molecule comprises a 5 'extension sequence covalently linked to a second 5' ribonucleotide and a 3 'extension sequence covalently linked to a first 3' ribonucleotide.

In another embodiment, the RNA molecule may comprise one or more of:

-a 5 'extension sequence covalently linked to a first 5' ribonucleotide;

-a 3 'extension sequence covalently linked to a second 3' ribonucleotide;

-a 5 'extension sequence covalently linked to a first 5' ribonucleotide and a 3 'extension sequence covalently linked to a second 3' ribonucleotide;

-a 5 'extension sequence covalently linked to a second 5' ribonucleotide;

-a 3 'extension sequence covalently linked to a first 3' ribonucleotide;

-a 5 'extension sequence covalently linked to the second 5' ribonucleotide and a 3 'extension sequence covalently linked to the first 3' ribonucleotide.

EncodingNucleic acid of RNA molecule

One skilled in the art will appreciate from the foregoing description that the present invention also provides isolated nucleic acids and components thereof encoding the RNA molecules disclosed herein. For example, a polypeptide comprising SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9, or a nucleic acid of any one or more of the sequences set forth in seq id no. The nucleic acid may be partially purified after expression in a host cell. The term "partially purified" is used to refer to RNA molecules that have been typically separated from lipids, nucleic acids, other peptides, and other contaminating molecules with which they are associated in a host cell. Preferably, the partially purified polynucleotide is at least 60% free, more preferably at least 75% free, more preferably at least 90% free of other components with which it is associated.

In another embodiment, the polynucleotide according to the invention is a heterologous polynucleotide. The term "heterologous polynucleotide" is well known in the art and refers to a polynucleotide that is not endogenous to a cell, or a native polynucleotide whose native sequence has been altered, or a native polypeptide whose expression has been quantitatively altered as a result of manipulation of the cell by recombinant DNA techniques.

In another embodiment, the polynucleotide according to the invention is a synthetic polynucleotide. For example, polynucleotides can be produced using techniques that do not require pre-existing nucleic acid sequences, such as DNA printing and oligonucleotide synthesis. In another embodiment, the polynucleotide is produced from a heterologous nucleic acid.

In one embodiment, the polynucleotide disclosed herein encodes an RNA precursor molecule comprising an intron, preferably in a 5' extension sequence or in at least one loop sequence, wherein the intron is capable of being spliced out during transcription of the polynucleotide in a host cell or in vitro. In another embodiment, the loop sequence comprises two, three, four, five or more introns. The invention also provides expression constructs, such as DNA constructs comprising the isolated nucleic acids of the invention operably linked to a promoter. In one embodiment, such isolated nucleic acids and/or expression constructs are provided in a cell or non-human organism. In one embodiment, the isolated nucleic acid is stably integrated into the genome of the cell or non-human organism. Various examples of suitable expression constructs, promoters, and cells comprising the same are discussed below.

The synthesis of RNA molecules according to the invention can be achieved using various methods known in the art. Examples of in vitro synthesis are provided in the examples section. In this example, constructs comprising the RNA molecules disclosed herein were subjected to restriction, precipitation, purification and quantification at the 3' end. After transformation of HT115 electrocompetent cells and induction of RNA synthesis using the T7, IPTG system, RNA synthesis can be achieved in bacterial culture.

Recombinant vector

One embodiment of the invention comprises a recombinant vector comprising at least one RNA molecule as defined herein and capable of delivering said RNA molecule into a host cell. Recombinant vectors include expression vectors. The recombinant vector contains a heterologous polynucleotide sequence, i.e., a polynucleotide sequence that is not naturally adjacent to an RNA molecule as defined herein, which is preferably derived from a different species. The vector may be RNA or DNA, and is typically a viral vector or plasmid derived from a virus.

Various viral vectors are useful for delivering and mediating expression of RNA molecules according to the invention. The choice of viral vector will generally depend on various parameters, e.g., the cell or tissue used for delivery, the transduction efficiency of the vector, and the pathogenicity. In one embodiment, the viral vector is integrated into host cell chromatin (e.g., lentivirus). In another embodiment, the viral vector is retained primarily in the nucleus as an extrachromosomal episome (e.g., adenovirus). Examples of these types of viral vectors include tumor retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpes viruses, and retroviruses.

Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, amplification and transformation of the expression cassette in prokaryotic cells, such as pUC-derived vectors, pGEM-derived vectors, or binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include origins of replication providing autonomous replication of the vector, preferably a selectable marker gene encoding antibiotic or herbicide resistance, a unique multiple cloning site providing multiple sites for insertion of nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences enhancing transformation of prokaryotic and eukaryotic (particularly plant) cells.

As used herein, "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) fragments. Generally, it refers to the functional relationship of a transcriptional regulatory element (promoter) to a transcribed sequence. For example, a promoter is operably linked to a coding sequence of an RNA molecule as defined herein if the promoter stimulates or regulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements, which are operably linked to a transcribed sequence, are physically contiguous with the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located near the coding sequence whose transcription is enhanced.

When multiple promoters are present, each promoter may be independently the same or different.

To facilitate the identification of transformants, the recombinant vector desirably contains a selectable or screenable marker gene. "marker gene" refers to a gene that confers a distinct phenotype on cells expressing the marker gene, thus allowing such transformed cells to be distinguished from cells not having the marker. Selectable marker genes confer a trait that one can "select" based on resistance to a selection agent (e.g., herbicide, antibiotic, etc.). The screenable marker gene (or reporter gene) confers a trait that can be identified by observation or testing, i.e., by "screening" (e.g., β -glucuronidase, luciferase, GFP, or other enzyme activities not present in the untransformed cells). Exemplary selectable markers for selection of plant transformants include, but are not limited to, the hyg gene encoding hygromycin B resistance; neomycin phosphotransferase (nptII) gene conferring resistance to kanamycin, paromomycin; glutathione-S-transferase gene from rat liver, which confers resistance to glutathione-derived herbicides, as described for example in EP 256223; a glutamine synthase gene which, when overexpressed, confers resistance to glutamine synthase inhibitors such as phosphinothricin, as described in WO 87/05327; acetyltransferase genes from Streptomyces viridodopromogenes (Streptomyces viridogenes) which confer resistance to the selective agent phosphinothricin, as described, for example, in EP 275957; a gene encoding 5-enol isothiocyanate-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine, as described, for example, in Hinche et al (1988); the bar gene, which confers resistance to bialaphos, as described, for example, in WO 91/02071; nitrilase genes, such as bxn from Klebsiella ozaenae (Klebsiella ozaenae), which confer resistance to bromobenzonitrile (Stalker et al, 1988); the dihydrofolate reductase (DHFR) gene, which confers resistance to methotrexate (Thillet et al, 1988); mutant acetolactate synthase (ALS) genes that confer resistance to imidazolinones, sulfonylureas, or other ALS-inhibiting chemicals (see EP154,204); a mutant anthranilate synthase gene that confers resistance to 5-methyltryptophan; or dalapon dehalogenase gene, which confers herbicide resistance.

Preferably, the recombinant vector is stably integrated into the genome of a cell, such as a plant cell. Thus, a recombinant vector may comprise appropriate elements that allow the vector to be incorporated into a genome or a chromosome of a cell.

Expression vector

As used herein, an "expression vector" is a DNA vector capable of transforming a host cell and effecting the expression of an RNA molecule as defined herein. The expression vectors of the invention comprise regulatory sequences, such as transcriptional control sequences, translational control sequences, origins of replication, and other regulatory sequences which are compatible with the host cell and which control the expression of the RNA molecules according to the invention. In particular, the expression vectors of the invention include transcriptional control sequences. Transcriptional control sequences are sequences that control the initiation, extension, and termination of transcription. Particularly important transcriptional control sequences are those that control the initiation of transcription, such as promoter, enhancer, operator and repressor sequences. The choice of the regulatory sequence used depends on the target organism, such as the plant and/or the target organ or tissue of interest. Such regulatory sequences may be obtained from any eukaryote, such as a plant or plant virus, or may be chemically synthesized.

Exemplary Vectors suitable for stably transfecting Plant cells or creating transgenic plants are described, for example, in Pouwels et al, Cloning Vectors, A Laboratory Manual (A Laboratory Manual), 1985, Supp.1987, Weissbach and Weissbach, Methods for Plant Molecular Biology (Methods of Plant Molecular Biology), Academic Press (U.S. Academic Press, 1989, and Gelvin et al, Plant Molecular Biology Manual (Plant Molecular Biology Manual), Kluwer Academic Publishers (Kluyverville Academic Press), 1990. Typically, plant expression vectors include one or more cloned plant genes, for example, under the transcriptional control of 5 'and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors can also comprise promoter regulatory regions (e.g., regulatory regions that regulate inducible or constitutive, environmental or developmental regulation, or cell or tissue specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or polyadenylation signals.

The vectors of the invention may also be used to produce the RNA molecules defined herein in cell-free expression systems, such systems being well known in the art.

In one embodiment, the polynucleotide encoding an RNA molecule according to the invention is operably linked to a promoter capable of directing expression of the RNA molecule in a host cell. In one embodiment, the promoter functions in vitro. In one embodiment, the promoter is an RNA polymerase promoter. For example, the promoter may be an RNA polymerase III promoter. In another embodiment, the promoter may be an RNA polymerase II promoter. However, the choice of promoter may depend on the target organism, such as the plant, insect, and/or tissue of interest. Exemplary mammalian promoters include CMV, EF1 α, SV40, PGK1, Ubc, human β actin, CAG, TRE, UAS, CaMKIIa, CAL1, 10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, and U6. Exemplary insect promoters include Ac5 and polyhedrin. Many constitutive promoters active in plant cells have been described. Suitable promoters for constitutive expression in plants include, but are not limited to, cauliflower mosaic virus (CaMV)35S promoter, Figwort Mosaic Virus (FMV)35S, light-inducible promoters from the small subunit of ribulose-1, 5-bisphosphate carboxylase (SSU), rice cytoplasmic trisaccharide phosphate isomerase promoter, Arabidopsis (Arabidopsis) adenine phosphoribosyltransferase promoter, rice actin 1 gene promoter, mannopine synthase and octopine synthase promoters, Adh promoter, sucrose synthase promoter, R gene complex promoter, chlorophyll α/α binding protein gene promoter. These promoters have been used to generate DNA vectors which have been expressed in plants, see for example WO 84/02913. All of these promoters have been used to generate various types of plant-expressible recombinant DNA vectors.

For expression in the source tissue of a plant such as leaf, seed, root or stem, it is preferable that the promoter used in the present invention has relatively high expression in these specific tissues. For this purpose, genes with tissue or cell specificity or enhanced expression can be selected from a number of promoters. Examples of such promoters reported in the literature include the chloroplast glutamine synthase GS2 promoter from pea, the chloroplast fructose-1, 6-bisphosphatase promoter from wheat, the nuclear photosynthetic ST-LSI promoter from potato, the serine/threonine kinase promoter from Arabidopsis thaliana (Arabidopsis thaliana), and the glucoamylase (CHS) promoter. Also reported are the ribulose-1, 5-bisphosphate carboxylase promoter from larch orientalis (laricina), the Cab gene from pine, the promoter of Cab6, the promoter of Cab-1 gene from wheat, the promoter of Cab-1 gene from spinach, the promoter of Cab 1R gene from rice, the promoter of Pyruvate Phosphate Dikinase (PPDK) from maize (Zea mays), the promoter of tobacco Lhcb1 x 2 gene, Arabidopsis thaliana (Arabidopsis thaliana) Suc2 sucrose-H³⁰The homeotropic transporter promoter, the promoter of the thylakoid membrane protein genes of spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS). Other promoters of chlorophyll alpha/beta-binding proteins may also be used in the present invention, such as those from the LhcB gene and PsbP gene of white mustard (Sinapis alba) A promoter.

Various plant gene promoters regulated in response to environmental, hormonal, chemical and/or developmental signals may also be used to express RNA binding protein genes in plant cells, including by (1) heat; (2) light (e.g., pea RbcS-3A promoter, corn RbcS promoter); (3) hormones, such as abscisic acid; (4) lesions (e.g., WunI); or (5) chemicals such as methyl jasmonate, salicylic acid, steroid hormones, alcohols, Safeners (WO 97/06269), or it may also be advantageous to use (6) organ-specific promoters.

The term "plant storage organ specific promoter" as used herein refers to a promoter that preferentially directs transcription of genes in a plant storage organ when compared to other plant tissues. For expression in sink tissues of plants, such as tubers of potato plants, fruits of tomato, or seeds of soybean, rape, cotton, maize, wheat, rice and barley, it is preferred that the promoter used in the present invention has relatively high expression in these specific tissues. The β -conglycinin promoter, or other seed-specific promoters, such as the rapeseed, zein, lin, and phaseolin promoters, can be used. Root-specific promoters may also be used. An example of such a promoter is the promoter of the acid chitinase gene. Expression in root tissue can also be achieved by using the root-specific subdomain of the CaMV 35S promoter which has been identified.

In a particularly preferred embodiment, the promoter directs expression in tissues and organs where lipid biosynthesis occurs. Such promoters may function at the appropriate time in seed development to modify lipid composition in seeds. Preferred promoters for seed-specific expression include: 1) a promoter derived from a gene encoding an enzyme involved in lipid biosynthesis and accumulation in seeds (such as desaturase and elongase), 2) a promoter derived from a gene encoding a seed storage protein, and 3) a promoter derived from a gene encoding an enzyme involved in carbohydrate biosynthesis and accumulation in seeds. Suitable seed-specific promoters are the rapeseed protein gene promoter of oilseed rape (US 5,608,152), the Vicia faba USP promoter (Baumlein et al, 1991), the Arabidopsis oleosin promoter (WO 98/45461), the phaseolin promoter of kidney bean (WO 98/45461) (US 5,504,200), the Brassica (Brassica) Bce4 promoter (WO 91/13980) or the legumin B4 promoter (Baumlein et al, 1992) and promoters which lead to the specific expression of seeds in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Suitable noteworthy promoters are the barley lpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230), or the promoters described in WO 99/16890 (promoters from the hordein gene, rice gluten gene, rice oryzin gene, rice prolamin gene, wheat gluten gene, zein gene, oat gluten gene, sorghum kasirin gene, rye triticale (secalin) gene). Other promoters include those described in Broun et al, (1998), Potenza et al, (2004), US20070192902 and US 20030159173. In one embodiment, the seed-specific promoter is preferably expressed in a defined part of the seed, such as the cotyledon or endosperm. Examples of cotyledon-specific promoters include, but are not limited to, the FPI promoter (Ellerstrom et al, 1996), the pea legumin promoter (Perrin et al, 2000); and the phaseolus vulgaris lectin promoter (Perrin et al, 2000). Examples of endosperm-specific promoters include, but are not limited to, the maize zein-1 promoter (Chikwamba et al, 2003), the rice gluten-1 promoter (Yang et al, 2003); the barley D-hordein promoter (Horvath et al, 2000); and the wheat HMW glutenin promoter (Alvarez et al, 2000). In another embodiment, the seed-specific promoter is not expressed or is expressed only at low levels after germination of the embryo and/or seed.

In another embodiment, the plant storage organ specific promoter is a fruit specific promoter. Examples include, but are not limited to, the tomato polygalacturonase E8 and Pds promoters, and the apple ACC oxidase promoter (for review see Potenza et al, 2004). In a preferred embodiment, the promoter preferentially directs expression in the edible part of the fruit, e.g. the pith of the fruit, relative to the fruit pericarp or the seed within the fruit.

In one embodiment, the inducible promoter is the Aspergillus nidulans (Aspergillus nidulans) alc system. Examples of inducible expression systems that can be used to replace the aspergillus nidulans alc system are described in reviews by Padidam (2003) and corado and Karali (2009). In another embodiment, the inducible promoter is a safener inducible promoter, such as the maize ln2-1 or ln2-2 promoter (Hershey and Stoner, 1991), the safener inducible promoter is the maize GST-27 promoter (Jepson et al, 1994) or the soybean GH2/4 promoter (Ulmarsov et al, 1995).

In another embodiment, the inducible promoter is a senescence-inducible promoter, such as the senescence-inducible promoters SAG (senescence-associated gene) 12 and SAG13(Gan, 1995; Gan and Amasino, 1995) from Arabidopsis thaliana (Arabidopsis), and LSC54(Buchanan-Wollaston, 1994) from Brassica napus (Brassica napus). Such promoters show increased expression at about the beginning of senescence in plant tissues, particularly leaves.

For expression in vegetative tissues, leaf-specific promoters may be used, such as the Rubisco (RBCS) promoter. For example, the tomato RBCS1, RBCS2, and RBCS3A genes are expressed in leaves and in light-grown seedlings (Meier et al, 1997). The rubisco promoter described by Matsuoka et al (1994) can be used, which is expressed at a high level almost exclusively in mesophyll cells of leaves and leaf sheaths. Another leaf-specific promoter is the photoplethysma/b binding protein gene promoter (see Shiina et al, 1997). The Arabidopsis (Arabidopsis thaliana) myb-related gene promoter (Atmby5), described by Li et al (1996), is leaf-specific. The Atmyb5 promoter was expressed in leaf hair, leaf-supporting and epidermal cells developing at the edges of young rosette and cauliflower leaves, as well as immature seeds. The leaf promoter identified in maize by Busk et al (1997) can also be used.

In some cases, for example when LEC2 or BBM is recombinantly expressed, it may be desirable that the transgene is not expressed at high levels. An example of a promoter that can be used in this case is a truncated napin a promoter that retains the seed-specific expression pattern but has reduced expression levels (Tan et al, 2011).

The 5' untranslated leader sequence may be derived from the promoter of a heterologous gene sequence selected to express the RNA molecule of the invention, or may be heterologous with respect to the coding region of the enzyme to be produced, and may be specifically modified to increase translation of mRNA, if desired. For a review on optimizing transgene expression, see Koziel et al, (1996). The 5' untranslated region may also be obtained from a suitable eukaryotic gene, plant gene (wheat and maize chlorophyll a/b binding protein gene leader sequence), or plant viral RNA (tobacco mosaic virus, tobacco etch virus, maize dwarf mosaic virus, alfalfa mosaic virus, etc.) of synthetic gene sequences. The present invention is not limited to constructs in which the untranslated region is derived from a 5' untranslated sequence that accompanies a promoter sequence. Leader sequences may also be derived from unrelated promoters or coding sequences. Leader sequences useful in the context of the present invention include the maize Hsp70 leader sequence (US 5,362,865 and US 5,859,347) and the TMV ω element.

Termination of transcription is achieved by a 3' untranslated DNA sequence operably linked to an RNA molecule of interest in an expression vector. The 3 'untranslated region of the recombinant DNA molecule contains a polyadenylation signal, which functions in plants to cause the addition of adenosine nucleotides to the 3' end of RNA. The 3' untranslated region can be obtained from various genes expressed in plant cells. The 3' untranslated region of nopaline synthase, the 3' untranslated region of the pea small subunit Rubisco gene, and the 3' untranslated region of the soybean 7S seed storage protein gene are commonly used in this capacity. Also suitable are 3' transcribed, untranslated regions containing the polyadenylation signal of the Agrobacterium tumor inducing (Ti) plasmid gene.

Transferring nucleic acids

The transfer nucleic acid can be used to deliver an exogenous polynucleotide to a cell and comprises one, preferably two border sequences and one or more RNA molecules of interest. The transfer nucleic acid may or may not encode a selectable marker. Preferably, the transfer nucleic acid forms part of a binary vector in a bacterium, wherein the binary vector further comprises elements that allow the vector to replicate in the bacterium, select for, or maintain a bacterial cell containing the binary vector. The transfer nucleic acid component of the binary vector can be integrated into the genome of the eukaryotic cell following transfer into the eukaryotic cell, or can only be expressed in the cell for transient expression experiments.

As used herein, the term "extrachromosomal transfer nucleic acid" refers to a nucleic acid molecule that is capable of being transferred from a bacterium, such as an Agrobacterium species, to a eukaryotic cell, such as a plant leaf cell. Extrachromosomal transfer of nucleic acids is a genetic element known as an element capable of being transferred, followed by integration of the nucleotide sequence contained within its borders into the genome of the recipient cell. In this regard, the transfer nucleic acid is typically flanked by two "border" sequences, although in some cases a single border at one end may be used and the second end of the transfer nucleic acid is randomly generated during the transfer process. The RNA molecule of interest is typically located between the left and right border-like sequences of the transferred nucleic acid. The RNA molecule contained within the transfer nucleic acid may be operably linked to a variety of different promoter and terminator regulatory elements that facilitate its expression, i.e., transcription of the RNA molecule and/or translation of the RNA molecule. The transfer of DNA (T-DNAs) from Agrobacterium species, such as Agrobacterium tumefaciens (Agrobacterium tumefaciens) or Agrobacterium rhizogenes (Agrobacterium rhizogenes) and artificial variants/mutants thereof, may be the best characterizing examples for the transfer of nucleic acids. Another example is P-DNA ("plant-DNA") comprising T-DNA border-like sequences from plants.

As used herein, "T-DNA" refers to T-DNA of an Agrobacterium tumefaciens (Ti) plasmid or an Agrobacterium rhizogenes (Ri) plasmid, or a variant thereof for transferring DNA into plant cells. The T-DNA may comprise the entire T-DNA, including the right and left border sequences, but need only comprise the minimal sequence required for cis-transfer, i.e., the right T-DNA border sequence. The T-DNA of the invention has been inserted into them, anywhere between the right and left border sequences (if present), into the RNA molecule of interest. Sequences encoding trans-factors (such as the vir genes) required for transfer of the T-DNA into a plant cell may be inserted into the T-DNA, or may be present on the same replicon as the T-DNA, or preferably are trans-form on a compatible replicon in an Agrobacterium host. Such "binary vector systems" are well known in the art. As used herein, "P-DNA" refers to a transfer nucleic acid isolated from a plant genome or an artificial variant/mutant thereof and comprising a T-DNA border-like sequence at each end or only at one end.

As used herein, the "border" sequence of a transfer nucleic acid may be isolated from a selected organism, such as a plant or bacterium, or be a man-made variant/mutant thereof. The border sequence facilitates and facilitates the transfer of the RNA molecule to which it is linked and may facilitate its integration in the genome of the recipient cell. In one embodiment, the border sequence is 10-80bp in length. Agrobacterium species T-DNA border sequences are well known in the art and include those described in Lacriox et al (2008).

Whereas traditionally only Agrobacterium (Agrobacterium) species have been used for gene transfer into plant cells, a number of systems have now been identified/developed which function in a similar manner to Agrobacterium (Agrobacterium) species. Several non-Agrobacterium species have recently been genetically modified to have the ability to transfer genes (Chung et al, 2006; Broothaerts et al, 2005). These include Rhizobium (Rhizobium) species NGR234, Sinorhizobium meliloti (Sinorhizobium meliloti) and Mezorhizobium loti (Mezohizobium loti).

Direct transfer of eukaryotic expression plasmids from bacteria to eukaryotic hosts was first achieved decades ago by fusion of protoplasts of mammalian cells and plasmid-carrying E.coli (Schaffner, 1980). Since then, the number of bacteria capable of delivering genes into mammalian cells has steadily increased (Weiss, 2003), and was independently discovered from four groups (Sizemore et al, 1995; Courvalin et al, 1995; Powell et al, 1996; Darji et al, 1997).

As used herein, the terms "transfection", "transformation" and variations thereof are generally used interchangeably. A "transfected" or "transformed" cell may have been manipulated to introduce an RNA molecule of interest, or may be a progeny cell derived therefrom.

Recombinant cell

The invention also provides a recombinant cell, e.g., a recombinant bacterial cell, fungal cell, plant cell, insect cell or animal cell, which is a host cell transformed with one or more RNA molecules or vectors as defined herein, or a combination thereof. Suitable cells of the invention include any cell that can be transformed with an RNA molecule or a recombinant vector according to the invention. In one embodiment, the transformed host cell is dead.

The recombinant cell may be a cell in culture, an in vitro cell, or a cell in an organism such as a plant, or a cell in an organ such as a seed or leaf. Preferably, the cell is in a plant, more preferably in a seed of a plant. In one embodiment, the recombinant cell is a non-human cell. Thus, in one embodiment, the invention relates to a non-human organism comprising one or more or all of the RNA molecules disclosed herein.

In one embodiment, the cell is an insect cell. In one embodiment, the insect cell is derived from Trichoplusia ni (Trichoplusia).

Another example of a suitable host cell is an electrocompetent HT115 cell.

The host cell into which the RNA molecule is introduced may be an untransformed cell or a cell which has been transformed with at least one nucleic acid. Such nucleic acids may or may not be associated with lipid synthesis. The host cell of the invention may be capable of endogenously (i.e. naturally) expressing an RNA molecule as defined herein, in which case the recombinant cell derived therefrom has an enhanced ability to produce an RNA molecule or is capable of producing said RNA molecule only after transformation with at least one RNA molecule as defined herein. In one embodiment, the cell is a cell that can be used to produce lipids. In one embodiment, the recombinant cells of the invention have an enhanced ability to produce non-polar lipids such as TAG.

The host cell of the invention can be any cell capable of expressing at least one RNA molecule described herein, and includes bacterial, fungal (including yeast), parasitic, arthropod, animal, and plant cells. Examples of host cells include Salmonella (Salmonella), Escherichia (Escherichia), Bacillus (Bacillus), Listeria (Listeria), Saccharomyces (Saccharomyces), Spodoptera (Spodoptera), Mycobacterium (Mycobacterium), Trichoplusia (Trichoplusia), Agrobacterium (Agrobacterium), BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells, and Vero cells. Further examples of host cells are E.coli, including E.coli K-12 derivatives; salmonella typhi (Salmonella typhi); salmonella typhimurium (Salmonella typhimurium), including attenuated strains; spodoptera frugiperda (Spodoptera frugiperda); cabbage loopers (Spodoptera frugiperda); and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Other suitable mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine, or chicken embryo fibroblast cell lines), myeloma cell lines, chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells, and/or HeLa cells.

In a preferred embodiment, the plant cell is a seed cell, in particular a cell in the cotyledon or endosperm of a seed. In one embodiment, the cell is an animal cell. The animal cell may be any type of animal, such as, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammalian cell, or a cell of an aquatic animal such as a fish or crustacean, an invertebrate, an insect, or the like. Examples of algal cells useful as host cells for the present invention include, for example, Chlamydomonas species (e.g., Chlamydomonas reinhardtii), Dunaliella species (Dunaliella), Haematococcus species (Haematococcus), Chlorella species (Chlorella), Thraustochytrium species (Thraustochytrium), Schizochytrium species (Schizochytrium), and Volvox species.

Transgenic plants

The present invention also provides a plant, a cell according to the present invention, a vector according to the present invention or a combination thereof comprising one or more exogenous RNA molecules as defined herein. When used as a noun, the term "plant" refers to a whole plant, while the term "part thereof" refers to a plant organ (e.g., leaf, stem, root, flower, fruit, seed); single cells (e.g., pollen); seeds; seed parts, such as the embryo, endosperm, blastoderm or seed coat; plant tissue, such as vascular tissue; plant cells and progeny thereof. As used herein, a plant part comprises a plant cell.

As used herein, the terms "in a plant" and "in the plant" in the context of making modifications to the plant mean that the modification has occurred in at least a portion of the plant, including where the modification has occurred throughout the plant, and does not preclude the case where the modification has occurred only in one or more, but not all, portions of the plant. For example, a tissue-specific promoter is said to be expressed "in a plant", even though it may be expressed only in certain parts of the plant. Similarly, "a transcription factor polypeptide that increases expression of one or more glycolytic and/or fatty acid biosynthesis genes in a plant" means that increased expression occurs in at least a portion of the plant.

As used herein, the term "plant" is used in its broadest sense, including any organism in the plant kingdom. Also comprises red algae, brown algae and green algae. Including, but not limited to, flowering plants of any kind, grasses, crops or cereals (e.g., oilseeds, corn, soybeans), feed or forage, fruit or vegetable plants, herbs, woody plants or trees. This is not meant to limit the plant to any particular structure. It also refers to unicellular plants (e.g., microalgae). The term "part thereof" in reference to a plant refers to a plant cell and its progeny, a plurality of plant cells, a structure present at any stage of plant development, or a plant tissue. Such structures include, but are not limited to, leaves, stems, flowers, fruits, nuts, roots, seeds, seed coats, embryos. The term "plant tissue" includes differentiated and undifferentiated tissues of a plant, including those present in leaves, stems, flowers, fruits, nuts, roots, seeds, such as embryonic tissue, endosperm, dermal tissue (e.g., epidermis, pericarp), vascular tissue (e.g., xylem, phloem), or primary tissue (including parenchyma, horny, and/or mesenchymal cells), as well as cells in culture (e.g., single cells, protoplasts, callus, embryos, etc.). The plant tissue may be an in situ transformant (in planta), an organ culture, a tissue culture or a cell culture.

Different amounts of 18:3 and 16:3 fatty acids are found in glycolipids of different plants. This is used to distinguish fatty acids with 3 double bonds and is usually always C₁₈18:3 plants of atomic length and comprising both C₁₆And C₁₈16:3 plants of fatty acids. In the 18:3 chloroplast, the enzymatic activity catalyzing the conversion of phosphatidic acid to diacylglycerol and diacylglycerol to Monogalactosyldiacylglycerol (MGD) is significantly lower than in the 16:3 chloroplast. In the 18:3 leaf, chloroplasts synthesize stearoyl ACP2 in the stroma, introduce a first double bond in the saturated hydrocarbon chain, and then hydrolyze the thioester. The released oleate is exported across the chloroplast envelope into the membrane of the eukaryotic portion of the cell, probably the endoplasmic reticulum, where it is incorporated into the PC. The PC-linked oleoyl groups are desaturated in these membranes and subsequently moved back into the chloroplasts. The MGD-linked acyl group is the substrate for the introduction of a third double bond to produce an MGD with two linolenic acid residues. The galactolipids are characteristic of 18:3 plants such as Asteraceae and Leguminosae (Fabaceae). In photosynthetically active cells of, for example, 16:3 plants represented by members of the Umbelliferae (Apiaceae) and Brassicaceae (Brassicaceae), two pathways operate in parallel to provide thylakoids with MGD. Synergistic "eukaryotic" sequences are supplemented to varying degrees by the "prokaryotic" pathway. Their response is restricted to chloroplasts and results in the typical arrangement of acyl groups and their complete desaturation once esterified to MGD. The prokaryotic DAG backbone carries C16:0 and its desaturation product at the C18: fatty acid excluded C-2 position. The C-1 position is occupied by a C18 fatty acid and to a small extent by a C16 group. The similarity of the cyanobacterial lipid DAG backbone in 16:3 plants and the DAG backbone synthesized by the chloroplast transit pathway illustrates this phylogenetic relationship and demonstrates the presence of prokaryotic cells.

As used herein, the term "vegetative tissue" or "vegetative plant part" is any plant tissue, organ or part other than an organ used for sexual reproduction of a plant. The organ of sexual reproduction of the plant is specifically a seed organ, flower, pollen, fruit and seed. Vegetative tissues and parts include at least plant leaves, stems (including corms and tillers, but not head ends), tubers and roots, but not flowers, pollen, seeds including seed coats, embryos and endosperm, fruits including mesocarp tissue, pods with seeds and head ends with seeds. In one embodiment, the vegetative part of the plant is an above ground plant part. In another or further embodiment, the vegetative plant part is a green part, such as a leaf or a stem.

"transgenic plant" or variants thereof refers to a plant that contains a transgene not found in wild-type plants of the same species, variety or cultivar. Transgenic plants as defined in the context of the present invention include plants and progeny thereof which have been genetically modified using recombinant techniques to produce at least one polypeptide as defined herein in a desired plant or part thereof. Transgenic plant parts have corresponding meanings.

The terms "seed" and "grain" are used interchangeably herein. "grain" refers to mature grain, e.g., harvested grain or grain still on a plant but ready for harvest, but may also refer to bloated or germinated grain, depending on the context. Mature grains typically have a moisture content of less than about 18%. In a preferred embodiment, the moisture content of the grain is at a level which is generally considered safe for storage, preferably 5% -15%, 6% -8%, 8% -10%, or 10% -15%. As used herein, "developing seed" refers to pre-mature seed, typically found in the reproductive structure of a plant after fertilization or flowering, but may also refer to such pre-mature seed isolated from a plant. Mature seeds typically have a moisture content of less than about 12%.

As used herein, the term "plant storage organ" refers to a part of a plant that stores energy exclusively in the form of, for example, proteins, carbohydrates, lipids. Examples of plant storage organs are seeds, fruits, tuber roots and tubers. Preferred plant storage organs of the invention are seeds.

As used herein, the term "phenotypically normal" refers to a genetically modified plant or part thereof, e.g., a transgenic plant, or a storage organ of the invention, such as a seed, tuber, or fruit, that does not have significantly reduced growth and reproductive capacity as compared to an unmodified plant or part thereof. Preferably, the biomass, growth rate, germination rate, storage organ size, seed size and/or number of viable seeds produced is no less than 90% of the biomass, growth rate, germination rate, storage organ size, seed size and/or number of viable seeds of a plant lacking the recombinant polynucleotide when grown under the same conditions. The term does not include plant characteristics that may differ from wild-type plants but do not affect the usefulness of the plant for commercial purposes, such as the inland-free (billerin) phenotype of seedling leaves. In one embodiment, said genetically modified plant or part thereof with a normal phenotype comprises a recombinant polynucleotide encoding a silencing suppressor operably linked to a plant storage organ specific promoter and having substantially the same growth or reproductive capacity as a corresponding plant or part thereof which does not comprise said polynucleotide.

Plants provided by or contemplated for use in the practice of the present invention include monocots and dicots. In a preferred embodiment, the plant of the invention is a crop (e.g., cereals and dried beans, maize, wheat, potatoes, rice, sorghum, millet, cassava, barley) or a legume such as soybean, kidney bean or pea. The plants may be grown for the production of edible roots, tubers, leaves, stems, flowers or fruits. The plant may be a vegetable plant whose vegetative parts are used as food. The plants of the invention may be: makeba palm (Acrocomia acuminata), Arabidopsis thaliana (Arabidopsis thaliana), Arachis hypogaea (Aracanis hypogaea), palm butter fat (Astrocaryum murumuru), Astrocaryum palmatum (Astrocaryum vulgare), Pelargonium graveolens (Attaleia geraesensis), Elaea americana (Attaleia humilis), Camellia oleifera (Attaleia oleifera) (andrani), Attalea phalerata (uricaria), Brazilian palm (Attaleia specosa), Avena sativa (EnAva sativa), sugar beet (Beta vulgaria), Brassica (Brassica sp), such as Isaria italica (Brassica carinata), Brassica juncea (Brassica juncea), Brassica napus (Brassica napus), Brassica oleracea (Brassica oleracea), Canarium sativum (L) and Brassica oleracea (Brassica), Canarium sativum (Carpesium), Canarium sativum (Carpesium), Canarium (Brassica) and Brassica sativum (Brassica napus), such as sunflower (Helianthus annuus), barley (Hordeum vulgare), Jatropha curcas (Jatropha curcas), Anda trees (Joannesia princeps), Lemna species (Lemna) such as Lemna aequinoctialis (Lemna aequinoctialis), Lemna disperma, Lemna eculariensis, Lemna swell (Lemna gibba), Lemna japonica (Lemna japonica), Lemna minor (Lemna minor), Lemna monoraphica (Lemna minor), Lemna obscura, Lemna paucicosta, Lemna paucicostata (Lemna paucicosta), Lemna minor (Lemna pusilla), Lemna tenia, Lemna minor (Lemna minor), Lemna tena tenua, Lemna minor (Lemna sultica), and Nicornia species (Lingnaria), such as Niacinosa (Niacina), Niacinosa (Niacinia), or Niacinia species (Lingnanus), Niacinia (Niacinia) such as, Niacinula (Niacinosa), Missilia), Missiia (Niacinia) and Niacinia (Niacinia) species (Lingna), Missiia) such as (Micanadensis), oenocarpus bacabaa, Bauhua (Oenocarpus bataua), Oenocarpus distichus, Oryza (Oryza), such as Oryza sativa (Oryza sativa) and Oryza sativa (Oryza glaberrima), switchgrass (Panicum virgatum), Maria (Paraquea paraensis), avocado (Persea americana), Populus tomentosa (Populus trichocarpa), Ricinus communis (Ricinus communis), Saccharum sinensis (Saccharum sinensis) species, sesame (Sesamum indicum), potato (Solanum turberosa), Sorghum (Sorgum medium) species, such as Sorghum bicolor (Sorgum Sorghum vulgare), Sorghum vulgare, Therobacilus (Therobacillus), Sorghum medium (Sorghum vulgare), Triticum sativum), Triticum (Triticum sativa), Triticum (Triticum) species, Triticum sativa (Triticum) species, Triticum (Triticum) such as Triticum sativa), Triticum (Zeyla), Triticum sativa (corn), Triticum) species (Zeyla), citrus (Citrus) species, cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa) species, avocado (Persea americana) fig (Ficus Carica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew nut (Anacardium occidentale), Macadamia nut (Macadamia intergrifolia) and almond (Prunus amygdalus). For example, the plant of the present invention may be Nicotiana benthamiana (Nicotiana benthamiana).

Other preferred plants include C4 grasses, for example, miscanthus (Andropogon gerardia), tassel (Boutelloua), tall gastrodia tuber (B. gracilis), Buchloe dactyloides (Buchloe dactyloides), Schizophyllum commune (Schizochytrium scoparium), sorghum halepense (Sorghastum montanus), Sargassum niveus (Sporobolocarpus crepidus), in addition to those described above; c3 grasses, such as Elymus canadensis (Elymus canadensis), Lespedeza capitata (Lespedeza capitata) and Scutellaria barbata (Petalostumvillosum), Heteropappus (Aster azureus); and woody plants such as Quercus ellipsoidea (Quercus ellipsoidea) and Quercus macrocarpa (q. macrocarpa). Other preferred plants include C3 grass.

In a preferred embodiment, the plant is an angiosperm.

In one embodiment, the plant is an oilseed plant, preferably an oilseed crop plant. As used herein, an "oilseed plant" is a plant species used for the commercial production of lipids from the seeds of the plant. The oilseed plant may be, for example, oilseed rape (e.g. canola), maize, sunflower, safflower, soybean, sorghum, flax (linseed) or sugar beet. In addition, the oilseed plant can be other brassicas, cotton, peanut, poppy, brassicas, mustard, castor, sesame, safflower, Jatropha curcas (Jatropha curcas) or nut producing plants. The plants can produce high levels of lipids in their fruits applied, for example, to olives, oil palms or coconut. Horticultural plants to which the invention may be applied are lettuce, cabbage or vegetable brassicas including cabbage, broccoli or cauliflower. The invention can be applied to tobacco, melons, carrots, strawberries, tomatoes or peppers.

In a preferred embodiment, the transgenic plant is homozygous for each gene (transgene) that has been introduced, so that its progeny do not segregate for the desired phenotype. The transgenic plant may also be heterozygous for the introduced transgene, preferably consistently heterozygous for the transgene, for example in the F1 progeny that have been grown from hybrid seed. Such plants may provide advantages well known in the art, such as heterosis.

Transformation of

The RNA molecules disclosed herein can be stably introduced into the above-described host cells and/or non-human organisms, such as plants. For the avoidance of doubt, embodiments of the present invention encompass the above-mentioned plants stably transformed with the RNA molecules disclosed herein. As used herein, the terms "stably transformed", "stably transformed" and variants thereof refer to the integration of an RNA molecule or a nucleic acid encoding an RNA molecule into the genome of a cell such that they are transferred to progeny cells during cell division without the need for positive selection for their presence. Stable transformants or progeny thereof may be identified by any method known in the art, such as Southern blotting on chromosomal DNA, or in situ hybridization of genomic DNA, so that they can be selected.

Transgenic Plants can be produced using techniques known in The art, such as those generally described in Slater et al, Plant Biotechnology-Genetic Manipulation of Plants (Plant Biotechnology-The Genetic management of Plants), Oxford university Press (2003), and Christou and Klee, Handbook of Plant Biotechnology (Handbook of Plant Biotechnology), John Wiley and Sons (2004).

In one embodiment, a plant may be transformed by topically applying an RNA molecule according to the invention to the plant or part thereof. For example, the RNA molecule can be provided as a formulation with a suitable carrier and sprayed, dusted, or otherwise applied to the surface of the plant or portion thereof. Thus, in one embodiment, the methods of the invention encompass introducing an RNA molecule disclosed herein into a plant, the method comprising topically applying a composition comprising the RNA molecule to the plant or portion thereof.

Agrobacterium-mediated transfer is a widely used system for introducing genes into plant cells, since DNA can be introduced into cells throughout plant tissues, plant organs or explants in tissue culture, for transient expression or for stable integration of DNA into the plant cell genome. For example, the in situ conversion (in planta) method may be used. The use of Agrobacterium-mediated plant integration vectors to introduce DNA into plant cells is well known in the art. The DNA region to be transferred is defined by border sequences, and intervening DNA (T-DNA) is usually inserted into the plant genome. This method was chosen because of the ease and clear nature of gene transfer.

Acceleration methods that may be used include, for example, particle bombardment and the like. One example of a method for delivering a transforming nucleic acid molecule to a plant cell is microprojectile bombardment. This method has been reviewed in Yang et al, Particle Bombardment technique for Gene Transfer (Particle Bombardment Technology for Gene Transfer), Oxford Press, Oxford, UK (1994). Non-biological particles (microparticles) that can be coated with nucleic acids and delivered by propulsive force into cells such as immature embryos. Exemplary particles include those composed of tungsten, gold, platinum, and the like.

In another method, the plasmid can be stably transformed. The disclosed methods for plasmid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of DNA to the plasmid genome by homologous recombination (US5,451,513, US5,545,818, US5,877,402, US5,932479, and WO 99/05265). Other methods of cell transformation may also be used, including but not limited to introducing DNA into a plant by direct transfer of the DNA into pollen, by direct injection of the DNA into the reproductive organs of a plant, or by direct injection of the DNA into cells of immature embryos followed by rehydration of the dried embryos.

Regeneration, development and culture of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al, in: methods of plant molecular biology (methods of plant molecular biology), academic Press, san Diego, Calif. (1988)). This regeneration and growth process typically includes the following steps: transformed cells are selected and those individualized cells are cultured from the plantlet stage of rooting to the usual stage of embryonic development. Transgenic embryos and seeds were regenerated similarly. The resulting transgenic rooted shoots are then planted in a suitable plant growth medium, such as soil.

The development or regeneration of plants containing exogenous genes is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plant is crossed with a plant grown from seed of an agronomically important line. Instead, pollen from plants of these important lines is used to pollinate regenerated plants. The transgenic plants of the invention containing the desired polynucleotide are grown using methods well known to those skilled in the art.

To confirm the presence of the transgene in the transgenic cells and plants, Polymerase Chain Reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. The expression product of the transgene can be detected in any of a variety of ways, depending on the nature of the product, and including Northern blot hybridization, western blot, and enzymatic assays. Once the transgenic plants are obtained, they can be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant part may be harvested, and/or seeds collected. Seeds can be used as a source for growing additional plants having tissues or parts with desired characteristics. Preferably, the vegetative plant parts are harvested at the time of highest yield of non-polar lipids. In one embodiment, the vegetative plant parts are harvested at about the time of or after the start of flowering. Preferably, the plant parts are harvested at about the beginning of senescence, usually manifested as leaf yellowing and dryness.

Transgenic plants formed using Agrobacterium or other transformation methods typically contain a single locus on one chromosome. Such transgenic plants can be considered hemizygous for the added gene. More preferred are transgenic plants that are homozygous for the added gene, i.e., transgenic plants containing two added genes, one at the same locus on each chromosome of the chromosome pair. Homozygous transgenic plants can be obtained by self-fertilizing a hemizygous transgenic plant, germinating some of the seeds produced and analyzing the resulting plant for the gene of interest.

It is also understood that two different transgenic plants containing two independently segregating exogenous genes or loci can also be crossed (mated) to produce progeny containing both sets of genes or loci. Selfing of the appropriate F1 progeny may produce plants that are homozygous for the exogenous gene or locus. Backcrossing with parental plants and outcrossing with non-transgenic plants, as well as vegetative propagation, are also contemplated. Similarly, a transgenic plant may be crossed with a second plant comprising a genetic modification, such as a mutant gene, and progeny containing both the identified transgene and genetic modification. Other breeding methods commonly used for different traits and crops are described In Fehr, In: breeding Methods for Cultivar Development (Breeding Methods for solvar Development), WilcoxJ. The American society for agriculture (Breeding Methods for Cultivar Development), Madison division, Wis.A. (1987).

Preparation

The RNA molecule according to the invention can be provided as various agents. For example, the RNA molecule may be in the form of a solid, ointment, gel, cream, powder, paste, suspension, colloid, foam, or aerosol. Solid forms may include powders, dusts, granules, microspheres, pills, lozenges, tablets, filled films (including seed coatings), and the like, which may be water dispersible. In one embodiment, the composition is in the form of a concentrate.

In one embodiment, the RNA molecule can be provided as a topical formulation. In one embodiment, the formulation stabilizes the RNA molecule in the formulation and/or in vivo. For example, the RNA molecule may be provided in a lipid formulation. For example, the RNA molecule may be provided in a liposome. In one embodiment, the formulation comprises a transfection facilitating agent.

As used herein, the term "transfection facilitating agent" refers to a composition that is added to an RNA molecule to enhance uptake into a cell, including but not limited to a plant cell, an insect cell, or a fungal cell. Any transfection facilitating agent known in the art to be suitable for transfecting cells may be used. Examples include cationic lipids, such as one or more of the following: DOTMA (N- [1- (2.3-dioleoyloxy) -propyl ] -N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-)3- (trimethylammonium) propane), DMRIE (1, 2-dimyristoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide), DDAB (dimethyloctacosylammonium bromide). Lipspermine, particularly DOSPA (2, 3-Diglycoloxy-N- [2 (spermicarboxamido) ethyl ] -N, N-dimethyl-1-propanaminium-trifluoroacetate) and DOSPER (1, 3-Diglycoloxy-2- (6-Carbospermidine) -propyl-amide and dialkyl-and tetraalkyl-tetramethylspermidine, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermidine), TMTOS (tetramethyltetraoleylspermidine), TMTLS (tetramethyltetralauryl spermidine), TMTMTMTMTMTMDS (tetramethyltetradecyl spermidine) and TMDS (tetramethyldioleoyl spermine). cationic lipids are optionally combined with non-cationic lipids, particularly neutral lipids, such as lipids, e.g., DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol Life Technologies) and Lipofectamine 2000(Life Technologies).

In one embodiment, the RNA molecule can be incorporated into a formulation suitable for in situ administration. In one embodiment, the field comprises plants. Suitable plants include agricultural crops (e.g., cereals and legumes, corn, wheat, potato, tapioca, rice, sorghum, soybean, cassava, barley or pea), or legumes). The plants may be grown for the production of edible roots, tubers, leaves, stems, flowers or fruits. In one embodiment, the crop plant is a cereal plant. Examples of cereals include, but are not limited to, wheat, barley, sorghum oats and rye. In these embodiments, the RNA molecule can be formulated for application to the plant or any part of the plant in any suitable manner. For example, the composition may be formulated for application to the leaves, stems, roots, fruits, vegetables, grains, and/or shoots of a plant. In one embodiment, the RNA molecule is formulated for application to the leaves of a plant, and may be sprayed onto the leaves of the plant.

The RNA molecules described herein can be formulated with a variety of other agents depending on the desired formulation. Exemplary agents include one or more of suspending agents, coalescing agents, bases, buffers, bittering agents, fragrances, preservatives, propellants, thixotropic agents, antifreeze agents, and coloring agents.

In other embodiments, the RNA molecule formulation can comprise an insecticide, pesticide, fungicide, antibiotic, anthelmintic, antiparasitic, antiviral, or nematicide.

In another embodiment, the RNA molecule can be incorporated into a pharmaceutical composition. Such compositions generally comprise an RNA molecule as described herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, oral, and rectal administration.

In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. For example, liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811.

The RNA molecule according to the invention may be provided in a kit or package. For example, the RNA molecules disclosed herein can be packaged in a suitable container with written instructions for producing the above-mentioned cells or organisms or treating a condition.

Method for controlling non-human organisms

In one embodiment, the RNA molecules according to the invention can be used for controlling non-human organisms, such as insects. These uses involve the administration of RNA molecules according to the invention using various methods. In one embodiment, the RNA molecule according to the invention may be provided as an insect decoy for ingestion by an insect. In another example, the RNA molecules can be sprayed on the insects as desired. In another example, the RNA molecule can be sprayed on a plant or crop to protect the plant or crop from insects. Exemplary crops include cotton, corn, tomato, chickpea, pigeon pea, alfalfa, rice, sorghum, and cowpea.

In one embodiment, RNA molecules may be provided to modify insect behavior. In another embodiment, RNA molecules may be provided to kill insects. In another embodiment, RNA molecules can be provided to reduce insect fertility. Exemplary insect targets include household insects. Other exemplary insect targets include sap-sucking insects such as aphids (e.g., Myzus persicae (Myzus persicae), aphids reticulum (metropolium dirhodum), aphids Rhopalosiphum padi (Rhopalosiphum padi), bean aphids (Aphis glycines) further exemplary insect targets include arachnids, mosquitoes, ectoparasites, flies, spider mites, thrips, ticks, red bird mites, ants, cockroaches, termites, crickets including domesticated crickets, silverfish, booklice, beetles, midges, mosquitoes and fleas.

In one embodiment, the insect is a sap-sucking insect. In this example, the RNA molecule may have antisense activity to MpC002 and/or MpRack-1. In one embodiment, the sap-sucking insect is an aphid. In another embodiment, the aphid is a green peach aphid (Myzus persicae).

In one embodiment, the insect target is an ant (e.g., argentina (Linepithama humile)), cotton bollworm or corn earworm (Helicoverpa armigera) or fly (e.g., Lucilia cuprina). In one embodiment, the target insect is cotton bollworm (Helicoverpa armigera) and the RNA molecule has antisense activity to the ABC transporter gene (ABCwhite). In another embodiment, the target insect is an argentina ant (Linepihema humile) and the RNA molecule has antisense activity to a pheromone biosynthesis-activating neuropeptide (PBAN). In another embodiment, the target insect is Lucilia cuprina (Lucilia cuprina) and the RNA molecule has antisense activity against one or more genes encoding a protein selected from the group consisting of type V proton atpase catalytic subunit A, RNA enzyme 1/2, chitin synthase, ecdysone receptor, and gamma-tubulin 1/1-like.

In the above embodiments, the compositions and RNA molecules disclosed herein can be provided in a dispenser. In one embodiment, the dispenser is a trap or bait. In one embodiment, the trap and/or bait comprises a bait comprising one or more RNA molecules disclosed herein.

In one embodiment, the invention encompasses a method of controlling the behavior of insects, comprising spraying, dusting, or otherwise applying to the insects an RNA molecule disclosed herein. In this embodiment, the RNA molecule can be sprayed, dusted, or otherwise applied directly to the insect. In another embodiment, the RNA molecule can be sprayed, dusted, or otherwise applied to the plant or crop prior to insect infestation.

In one embodiment of the invention, the insects or arachnids may belong to the following orders: acarina (Acari), Arachnida (Arachnida), louse (Anoplura), blattaria (Blattodea), Coleoptera (Coleoptera), colletotrichum (colleoptera), collectiforme (Collembola), Dermaptera (Dermaptera), Dictyoptera (Dictyoptera), Dipura, embotera, medetoda (Ephemeroptera), desmoteda (griylobadodea), hemiptera (Hem iptera), Heteroptera (heterptera), Homoptera (Homoptera), hymenoptera (hymenoptera), Isoptera (Isoptera), Lepidoptera (Lepidoptera), trichoderma (Mallophaga), longoptera (melanchopera), Neuroptera (neoptera), dragonfly (edoptera), Orthoptera (Orthoptera), trichloptera (phytoptera), trichloptera (cephaloptera), trichtera (trichtera), trichtera (trichtera), trichtera (.

In a preferred but non-limiting embodiment of the invention, the insect or arachnid is selected from the group consisting of: (1) acarina (Acari): mites, including acarina (Ixodida) (2) arachnids (arachnids): spider (Araneae) and whitefly (opioies), examples include: black oligogynes (Latrodectus macrants) and brown squitoes (Loxosceles recise) (3) aphids (anoplora): lice, such as human body lice (Pediculus humanus) (4) blattaria (Blattodea): cockroaches, including german cockroach (Blatella germanica), Periplaneta (Periplaneta), including american cockroach (Periplaneta americana) and australian cockroach (Periplaneta australianae), Blatta (Blatta), including Blatta orientalis and Periplaneta palmaris (Blatta longipalea). The most preferred target is German cockroach (Blatella germanica). (5) Coleoptera order: beetles, examples include: the family of the Powderpost beetles (family of the family Bostrichholidae); bark beetle (Dendroctonus) species (black pine beetle, southern pine beetle, IPS beetle); carpet beetles (bark beetles, pissodes (Attagenus)); family of domestic longicorn (family of longidae (cerambycidae) longicorn (hylotrupus bajunus), family of furniture beetles (Anobium puncatum), genus Tribolium (Tribolium) gracilium (troglodorma granarium), valley beetles (oryzaephius sarinaensis) and the like (book worms) (6) Dermaptera (Dermaptera) family of wigwighidaceae family (7) Diptera (Diptera) mosquitos (culidae) and flies (Brachycera) such as Anopheles (Anopheles), for example Anopheles (apophylline), for example Anopheles (Anopheles) and moseriidae (culidae) such as yellow spot mosquitoes (Aedes fulvestris), mulberculidae (mange) such as yellow spot flies (aedius), gadidae (tabulariaceae), for example malacia (tarkipedigree), family of cerales (sartoriae), family of cephalosporidae (mangrove), such as trichoderma (leucotrichineae), common muscales (leucotrichia) and the like (leucotrichia (leucotrichinea) such as leucotrichinella (leucotrichinea) and common muscoideae) such as leucotrichia (leucotrichinea) and common muscoideae (leucotrichinea) such as leucotrichinea (leucotrichinella) and common trichoderma and common muscoideae) such as leucotrichia (trichoderma mange (leucotrichia) such as leucotrichia (trichoderma mange (trichopteroides (trichoderma mange) and common trichoderma mange (trichoderma mange), phanerochaete (Monomorium pharaonis), Achnotis (Camponotus) species, Formica fusca (Iasius niger), Dermaptera (Tetramorium caespitum), Anthemis ruber (Myrmica rubera), Formica (Formica) species, Dermacentella (Crematographelta) species, Argentina (Iridomyremax leis), Formica (Pheidole) species, Anseria (Dasytilla occidentalis), etc. (10) Isoptera (Isoptera): termites, embodiments include: florida black wing subterranean termites (Amitermes floridensis), oriental subterranean termites (reciltermes flavipes), western subterranean termites (r. hesperus), termopsis formosanus (coptottermes formosanus), western wood termites (inc.) minor, forest termites (Neotermes conneus) and termitaceae (tertidae) (11) Lepidoptera (Lepidoptera): moths, examples include: the families of the glutamineidae (tinedae) and the folliculoperidae (Oecophoridae), such as the common chlamydomonas (teneola bisseliella), and the family of the cartialidae (Pyrolidae), such as the rice moth (Pyrolisfarinalis) and the like (12) rodentia (rodentia): booklice (Psocids) (13) fleas (Siphonaptera): fleas, for example, human fleas (Pulex irritans) (14) of the order Fumithida (Sternorhyncha): aphididae (Aphididae) (15) order Pernychiformes (Zygentoma): whitebait, examples are: family clothes fish (Thermobia domestica) and silverfish (Lepisma sacchara);

Other target insects or arachnids include household insects, ectoparasites and insects related to public health and wellness and/or arachnids such as, but not limited to, flies, spider mites, thrips, ticks, red poultry mites, ants (e.g., by targeting PBANs), cockroaches, termites, crickets including house-crickets, silverfish, booklice, beetles, earwigs, mosquitoes and fleas. More preferred targets are cockroaches (Blattodea), such as, but not limited to, species of the genus Blatta (blattolla) (e.g., Blattodea (blattolla germanica)), species of the genus Periplaneta (Periplaneta) (e.g., Periplaneta americana (Periplaneta americana), Periplaneta australis (Periplaneta australianae), species of the genus Blatta (blattola) such as species of the genus oriental cockroach (Blatta orientalis) and the genus of the genus palmoplatta (superella) (e.g., the genus palmoplanta (supralongipalata), species of the general family of ants (formicoiae), such as, but not limited to, the genus leophagis (Solenopsis) such as the genus red ant (Solenopsis invicta), species of the genus fagopyrum (monaria) (e.g., the genus of the genus malacophyta), species of the genus fagopyrum (Solenopsis) (e.g., the genus of the genus malarial (chlamydia)), species of the genus fagopyrum (e) (e.g., the genus malarial (chlamydia)), species such as the genus), species of the genus) such as (chlamydia) (e.g., the genus), species of the genus) such as (chlamydia) (e.g., malarial (chlamydia) (e), species of the genus, termites (e.g., bellying termites (creatogen lineolara)), argentina (creatogen) (e.g., argentina (iridoxymex humilis)), macroterus (Pheidole) species, and velvet termites (dasymulus) (e.g., swan-teres (dasymus occidentalis)) termites (Isoptera) and/or termitaceae (tergitidae)), such as, but not limited to, termites (tertidae) (e.g., florida black subterranean termites (amitomes florides), termites (e.g., oriental subterranean termites (rotiitermes flavipes)), western subterranean termites (rotissereus sperus), termites (coptermes sperus) (e.g., coptermes formosanus (coptermes formosanus)), termites (e.g., coptermes alba (inco)), western termites (e.g., termites (termite), termites (e.g., coptermes alba (termite), termites (e) (e.g., coptermes alba (neotermites) (e.g., termites (inco) s (e.g., termites (comentrophores)).

In one embodiment, the target RNA encodes an insect lactate synthase.

When delivered and/or expressed in a plant, the RNA molecules of the invention can have a wide range of desirable properties that affect, for example, agronomic traits, insect resistance (e.g., by targeting genes such as MpC002, MpRack-1 and ABC transporter genes), disease resistance (e.g., by targeting genes such as LanR or MLO), herbicide resistance, sterility, grain characteristics, and the like. The target RNA molecule may be involved in the metabolism of oil, starch, carbohydrates, nutrients, etc., or may be involved in the synthesis of proteins, peptides, fatty acids, lipids, recombination frequencies (by targeting genes such as DDMl and FANCM), waxes, oils (by targeting genes such as TOR), starches, sugars, carbohydrates, fragrances, odors, toxins, carotenoids, hormones (by targeting genes such as EIN2, NCEDl and NCED2), polymers, flavonoids (by targeting genes such as chalcone synthase), storage proteins, phenolic acids, alkaloids, lignin, tannins, cellulose, glycoproteins, glycolipids, etc.

In particular embodiments, the plant produces: increased levels of enzymes for oil production in plants, such as brassica (Brassicas) plants, e.g. oilseed rape or sunflower, safflower, flax, cotton, soybean or corn; enzymes, involved in starch synthesis in plants such as potato, corn and cereals, e.g. wheat, barley or rice; synthetic enzymes, or proteins themselves, natural drugs, such as pharmaceuticals or veterinary products.

In another embodiment, the RNA molecule of the invention is intended for prophylactic or therapeutic treatment of an infection by a fungal pathogen selected from the group consisting of: alternaria (Altemaria) species; armillaria mellea (Armillaria mellea); armillaria oligospora (Armillaria mellae); powdery mildew (Blumeria graminis) of the family poaceae (targeted to the Mlo gene by using the RNA molecule as described in example 17), Boletus (Boletus grandis); botrytis cinerea (Botritis cinerea); botrytis fabae (botrytis fabae); candida albicans (Candida albicans); ergot (Claviceps purpurea); rust of bullous (Cronartium ribicola); rhodococcus (Epicoccum purpurens); epidermophyton floccosum (Epidermophyton floccosum); fomes angularis (Fomes innosus); fusarium oxysporum (Fusarium oxysporum); wheat holomyces graminis var.tritici; pleurotus circinelloides (Glomerella cingulata); ruscus alvarezii (Gymnosphaora juniperi-virginianae); microsporidia canis (Microsporum canis); monilinia fructicola (Monilinia fructicola); alfalfa rust (physioderma alfalfalfalfa); potato late blight (phytophthora infestans); malaytrophus furfur (pityriosporum orbicular); polyporus sulphureus (Polyporus sulphureus); puccinia spp (Puccinia spp.); saccharomyces cerevisiae (Saccharomyces cerevisiae); septoria apiacea (Septoria apiicola); trichophyton rubrum (Trichophyton rubrum); trichophyton mentagrophytes (t.mentagrophytes); smut (Ustilago spp.); venturia inaequalis (Venturia inaegulis); and Verticillium wilt (Venturia inaqualis).

Exemplary conditions to be treated

The RNA molecules according to the invention can be used in methods of various conditions. In some embodiments, the invention relates to a method of treating cancer comprising administering an RNA molecule disclosed herein. The term "cancer" refers to or describes a physiological condition in mammals that is generally characterized by dysregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer), non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, lentigo acrival melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (including low grade/follicular non-hodgkin lymphoma (NHL)); mantle cell lymphoma; AIDS-related lymphomas; and waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphocytic Leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD) and abnormal vascular proliferation associated with scarring nevus, edema (e.g., edema associated with brain tumors), megger syndrome, brain and head and neck cancer, and associated metastases. Thus, in one embodiment, the invention relates to a method of treating breast, ovarian, colon, prostate, lung, brain, skin, liver, stomach, pancreas or blood based cancers.

In other embodiments, the methods described herein are used to treat cancer associated with mutations in BRCA1, BRCA2, PALB2, OR RAD51B, RAD51C, RAD51D, OR related genes. In other embodiments, the methods described herein are used to treat cancers associated with mutations in DNA mismatch repair-related genes, such as MSH2, MLH1, PMS2, and related genes. In other embodiments, the methods described herein are used to treat cancers with silenced DNA repair genes, such as BRCA1, MLH1, OR RAD51B, RAD51C, OR RAD 51D.

In other embodiments of the invention, the methods described herein are used to kill cells that have impaired DNA repair processes. For example, cells with impaired DNA repair may abnormally express genes involved in DNA repair, DNA synthesis, or homologous recombination. Exemplary genes include XRCC, ADPRT (PARP-1), ADPRTL, (PARP-2), POLYMERASE BETA, CTPS, MLH, MSH, FACCD, PMS, p, PTEN, RPA, RPAl, RPA, XPD, ERCC, XPF, MMS, RAD51, DMC, XRCCR, XRCC, BRCA, PALB, RAD, MREU, NB, WRN, BLM, KU, ATM, ATR CPIK, HK, FACCA, FACCB, FACCC, FACCD, FACCE, FACCF, FACCG, FACCC, FACCD, FACCE, FACCF, FACSC, FACCD, FACCE, FACCF, and NCG. In one embodiment, the methods described herein are used to kill cells having a mutant tumor suppressor gene. For example, the cell may have one or more mutations in BRCA1 or BRCA 2.

In other embodiments of the invention, the methods described herein are used to treat virally transformed cells. In other embodiments of the invention, the methods described herein are used to kill cells transformed with a latent virus. Exemplary latent viruses include CMV, EBV, herpes simplex virus (type I and type II) and varicella zoster virus. In other embodiments of the invention, the methods described herein are used to treat active viral infections caused by a virus that causes cancer, immunodeficiency, hepatitis, encephalitis, pneumonia, or respiratory disease. Exemplary viruses include parvovirus, poxvirus, herpesvirus.

In other embodiments of the invention, the methods described herein are used to treat Zika (Zika) virus, Colorado tick fever (caused by the RNA virus Coltivrus), West Nile fever (encephalitis, caused by flaviviruses that occur primarily in the middle east and Africa), yellow fever, rabies (caused by different strains of the Rhabdoviridae), viral hepatitis, infections caused by norwalk and norwalk-like viruses, rotaviruses, gastroenteritis caused by caliciviruses and astroviruses (viral) -acute viral gastroenteritis, polio, influenza caused by orthomyxoviruses able to undergo frequent antigenic variation (Flu), measles (rubella), paramyxoviridae, mumps, respiratory syndromes, including pneumonia and acute respiratory syndrome, including laryngitis caused by various viruses collectively known as acute respiratory virus, and respiratory diseases caused by respiratory syncytial virus.

Examples

Example 1: materials and methods

Synthesis of genetic constructs

To design a typical ledRNA construct, a region of target RNA of about 100-1000 nucleotides in length, typically 400-600 nucleotides in length, is identified. In one embodiment, the 5 'half of the sequence and about 130nt of the flanking region and similarly the 3' half of the flanking region and 130nt are oriented in the antisense direction relative to the promoter. These sequences were interrupted by a 400-nucleotide sense target sequence (FIG. 1A). The 5 'end of the resulting construct is preceded by a promoter, such as the T7 or SP6 RNA polymerase promoter, and the 3' end is engineered to include a restriction enzyme cleavage site to allow in vitro transcription termination.

For transcription in cells such as bacterial cells, inducible promoters are used, for example, to introduce promoter and terminator sequences to facilitate expression as a transgene. The duplex and loop sequence lengths may vary. Constructs were prepared using standard cloning methods or ordered from commercial service providers.

RNA Synthesis

After digestion with restriction enzymes to linearize the DNA at the 3' end, transcription with RNA polymerase produces the 5' and 3' arms of the ledna RNA transcript that anneal to a central target sequence, which molecule comprises a central stem or double-stranded region with a single nick and terminal loop. The central sequence may be oriented in sense or antisense orientation relative to the promoter (FIG. 1A, FIG. 1B, respectively).

For in vitro synthesis, the DNA of the construct was digested at the 3' restriction site using appropriate restriction enzymes, precipitated, purified and quantified. RNA synthesis was achieved using RNA polymerase according to the manufacturer's instructions. The ledRNA was resuspended in annealing buffer (25mM Tris-HCl, pH 8.0, 10mM MgCl) using DEPC treated water₂) To inactivate any trace of rnase. The yield and integrity of RNA produced by this method was determined by nano-droplet analysis and gel electrophoresis (see figure 2, respectively).

The synthesis of ledRNA in bacterial cells was achieved by introducing the construct into E.coli strain HT 115. The transformed cell culture was induced with IPTG (0.4mM) to express T7 RNA polymerase, providing transcription of the ledRNA construct. RNA extraction from bacterial cells and purification was essentially as described by Timmons et al (2001).

For transcription of Cy 3-labeled RNA, the ribonucleotide (rNTP) mix contained 10mM each of ATP, GTP, CTP, 1.62mM UTP and 8.74mM Cy 3-UTP. The transcription reaction was incubated at 37 ℃ for 2.5 hours. The transcription reaction (160. mu.1) was transferred to an Eppendorf (Eppendorf) tube, 17.7. mu.1 turbo DNase buffer and 1. mu.1 turbo DNA were added, and incubated at 37 ℃ for 10 minutes to digest the DNA. Then 17.7. mu.1 Turbo DNAse inactivation solution was added, mixed and incubated at room temperature for 5 min. The mixture was centrifuged for 2 minutes and the supernatant was transferred to a new rnase-free Eppendorf tube. 1.5. mu.l of each transcription reaction sample was run on a gel to test the quality of the RNA product. Typically, depending on the construct, one RNA band ranging in size from 500bp to 1000bp is observed. RNA was precipitated by adding 88.5. mu.l of 7.5M ammonium acetate and 665. mu.l of cold 100% ethanol to each tube. The tubes were cooled to-20 ℃ for several hours or overnight and then centrifuged at 4 ℃ for 30 minutes. The supernatant was carefully removed and the RNA pellet washed with 1ml 70% ethanol (prepared with nuclease-free water) at-20 ℃ and centrifuged. The pellet was dried and the purified RNA was resuspended in 50. mu.l of 1 × RNAi annealing buffer. RNA concentration was measured using the nano-drop method and stored at-80 ℃ until use.

Example 2: design of ledRNA

As schematically shown in fig. 1A, a typical ledRNA molecule comprises a sense sequence of two adjacent sense sequences that can be considered to be covalently linked and have identity to a target RNA, an antisense sequence complementary to the sense sequence and divided into two regions, and two loops separating the sense sequence from the antisense sequence. Thus, a DNA construct encoding such a form of ledRNA comprises, in 5 'to 3' order, a promoter for transcription of the ledRNA coding region, a first antisense region complementary to the region toward the 5 'end of the target RNA, a first loop sequence, a sense sequence, a second loop sequence, then a second antisense region complementary to the 3' end region of the target RNA, and finally a means for terminating transcription. In this arrangement, the two antisense sequences flank the sense and loop sequences. When transcribed, two regions of the antisense sequence anneal to the sense sequence, forming a dsRNA stem with two flanking loops.

In another but related form of ledRNA, the sense region is divided into two regions, while the two antisense regions remain as a single sequence (FIG. 1B). Thus, a DNA construct encoding this second form of ledRNA comprises, in 5 'to 3' order, a promoter for transcription of the ledRNA coding region, a first sense region, first loop sequence, antisense sequence, second loop sequence, identical to the region toward the 3 'end of the target RNA, then a second sense region, identical to the 5' end of the target RNA, and finally means for terminating transcription. In this arrangement, the two sense sequences flank the antisense and loop sequences.

Without wishing to be bound by theory, because of the closed loop at each end, these ledRNA structures will be more resistant to exonuclease than open-ended dsRNA formed between single-stranded sense and antisense RNA and without loops, and also more resistant to exonuclease than hairpin RNA with only a single loop. Furthermore, the inventors contemplate that loops at both ends of the dsRNA stem will allow efficient Dicer access to both ends, thereby enhancing dsRNA processing into sRNA and silencing efficiency.

As a first example, genetic constructs for in vitro transcription were prepared using T7 or SP6 RNA polymerase to form LedRNA targeting genes encoding GFP or GUS. The ledGFP construct comprises the following regions in order: the first half of the antisense sequence corresponding to nucleotide 358-131 of the GFP coding sequence (CDS) (SEQ ID NO: 7), the first antisense loop corresponding to nucleotide 130-1 of the GFP CDS, the sense sequence corresponding to nucleotide 131-591 of the GFP CDS, the second antisense loop corresponding to nucleotide 731-592 of the GFP CDS, and the second half of the antisense sequence corresponding to nucleotide 591-359 of the GFP CDS.

The ledGUS construct comprises the following regions in order: the first half of the antisense sequence corresponding to nucleotide 609-357 of the GUS CDS (SEQ ID NO: 8); and a first antisense loop corresponding to nucleotides 356 to 197 of the GUS CDS, a sense sequence corresponding to nucleotide 357-860 of the GUS CDS, a second antisense loop corresponding to nucleotide 1029-861 of the GUS CDS; the second half of the antisense sequence corresponding to nucleotide 861-610 of the GUS CDS.

To prepare a single-stranded sense/antisense GUS dsRNA (conventional dsRNA), the same target sequence corresponding to nucleotides 357 and 860 of the GUS CDS was ligated between T7 and SP6 promoter in the pGEM-TEAsy vector. The sense and antisense strands are transcribed with T7 or SP6 polymerase, respectively, the transcripts are mixed and the mixture is heated to denature the RNA strand and then annealed in annealing buffer.

Example 3: stability of ledRNA

The ability of ledRNA to form dsRNA structures was compared to open-ended dsRNA (i.e., no loops, annealed by separate single-stranded sense and antisense RNAs) and long hpRNA. The mixture of ledRNA, long hpRNA and sense and antisense RNA was denatured by boiling and allowed to anneal in annealing buffer (250mM Tris-HCl, pH 8.0 and 100mM MgCl)₂Buffer) and then run in a 1.0% agarose gel under non-denaturing conditions.

As shown in FIG. 2, both GUS ledRNA and GFP ledRNA gave a dominant RNA band of the expected mobility of the double-stranded molecule, indicating the predicted formation of ledRNA structure. This is in contrast to the mixture of sense and antisense RNA which shows only a weak band of dsRNA, indicating that most of the sense and antisense RNA do not readily anneal to each other to form dsRNA. The hairpin RNA sample gave two prominent bands, indicating that only a portion of the transcript formed the predicted hairpin RNA structure. Thus, ledRNA is most effective in forming predicted dsRNA structures.

The ability of ledRNA to stay and diffuse on the leaf surface was also compared to dsRNA. When applied to the lower part of the tobacco leaf surface, GUS ledRNA (ledGUS) could be easily detected in the upper part of untreated tobacco leaves after 24 hours (fig. 3). However, no single strand GUS dsRNA (dsGUS) could be detected in the upper part of the untreated leaves (fig. 3). This result indicates that ledRNA is more resistant to degradation than dsRNA and therefore capable of spreading within plant leaf tissue.

Example 4: testing ledRNA by local delivery

The ability of ledRNA to induce RNAi after local delivery was tested in Nicotiana benthamiana (Nicotiana benthamiana) and Nicotiana tabacum (Nicotiana tabacum) plants expressing GFP or GUS reporter genes, respectively. The GFP and GUS target sequences and the sequence of the ledRNA encoding construct are shown in SEQ ID NO: 7. 8, 4 and 5. The ribonucleotide sequence of the encoded RNA molecule is set forth in SEQ ID NO: 1(GFPLedRNAGFP ledRNA) and 2(GUS ledRNA).

To facilitate reproducible and uniform application of ledRNA to the leaf surface, 10mM MgCl at 25mM Tris-HCl, pH8.0, using a soft paint brush₂And ledRNA at a concentration of 75-100. mu.g/ml in Silwet 77 (0.05%) was applied to the sub-axial surface of the leaf. Leaf samples were taken for analysis of targeted gene silencing 6 hours and 3 days after application of ledRNA.

Application of GFP to leaves of Nicotiana benthamiana (n.benthamiana) and ledRNA to GUS, tabacum (Nicotiana tabacum) resulted in a significant reduction of 20-40% and 40-50% of the activity of the corresponding target gene at the mrna (GFP) or protein activity (GUS) level 6 hours after treatment. However, in this experiment, the reduction did not persist for 3 days after treatment. The inventors believe that the observation at 3 days may be due to some non-specific response of the transgene to dsRNA treatment or the amount of ledRNA dissipated. However, in a separate experiment, GUS silencing was detected in both treated and distal untreated leaf regions 24 hours after ledRNA treatment (figure 4).

Example 5 LedRNA-induced silencing of endogenous target genes

In another example, ledRNA is designed to target mRNA encoded by the endogenous gene, i.e., the FAD2.1 gene of Nicotiana benthamiana (N.benthamiana). The sequences of the target FAD2.1 mRNA and ledfad2.1 encoding constructs are shown in SEQ ID NO: 9 and 6. The ribonucleotide sequence of the encoded RNA molecule is provided as SEQ ID NO: 3 (Nicotiana benthamiana (N.benthamiana) FADD 2.1ledRNA).

FAD2.1ledRNA construct consists of: the first half of the antisense sequence corresponding to nucleotide 678-flyash 379 of FAD2.1 CDS (Niben101Scf09417g 01008.1); a first antisense loop corresponding to nt378-242 of FAD2.1 CDS; a sense sequence corresponding to nt 379-979; a second antisense loop corresponding to nt 1115-980; the second half of the antisense sequence corresponding to nt979-nt679 of FAD2.1 CDS.

ledGUS RNA from the previous example was used in parallel as a negative control. In the first experiment, FAD2.1mRNA levels and accumulated C18:1 fatty acids were determined for target gene silencing (fig. 5). The level of activity of the related gene FAD2.2 was also determined. For each sample, approximately 3 μ g of total RNA was treated with DNase and reverse transcribed for 50 minutes at 50 ℃ using oligo dT primer. The reaction was terminated at 85 ℃ for 5 minutes and diluted to 120. mu.l with water. Relative expression of FAD2.1 and FAD2.2 mRNA was analyzed in triplicate for 5 μ Ι of each sample using a rotor gene PCR instrument using gene specific primers for housekeeping gene actin. In subsequent experiments, Northern blot hybridization was also used to confirm silencing of the FAD2.1 gene by topically applied ledfad2.1 RNA (fig. 6).

FAD2.1mRNA decreased significantly to levels barely detectable in leaf tissue treated with ledRNA at the 2, 4 and 10 hour time points (fig. 5). In this experiment, it is not clear why fad2.1mrna levels did not decrease as much at the 6 hour time point. In the repeated experiments shown in fig. 6, strong FAD2.1 down-regulation occurred at 6 and 24 hours, in particular at the 24 hour time point. The related FAD2.2 gene with sequence homology to FAD2.1 was also shown to be down-regulated by ledRNA at the 2 and 4 hour time points (fig. 5).

Since FAD2.1 and FAD2.2 encode fatty acid Δ 12 desaturases, which desaturate oleic acid to linoleic acid, the levels of these fatty acids were determined in leaf tissue treated with ledRNA. At time points of 2, 4 and 6 hours, there was a significant increase in oleic acid (18:1) accumulation in ledRNA-treated leaf tissue, indicating a decrease in the amount of FAD2 enzyme (FIG. 5). Therefore, qRT-PCR and fatty acid composition analysis showed ledRNA-induced FAD2.1 gene silencing.

Example 6: design and testing of hairpin RNAs comprising G: U base pairs or mismatched nucleotides

Modified hairpin RNA targeting GUS RNA

Reporter genes such as the gene encoding β -Glucuronidase (GUS) provide a simple and convenient assay system that can be used to measure the efficiency of gene silencing in eukaryotic cells, including plant cells (Jefferson et al, 1987). Accordingly, the present inventors designed, produced and tested some modified hairpin RNAs for the ability to reduce GUS gene expression as a target gene, provided hairpin RNAs to cells using a gene delivery method, and compared the modified hairpin RNAs with conventional hairpin RNAs. The conventional hairpin RNA used as a control in the experiment had a double-stranded region of 200 consecutive base pairs in length, in which all base pairs were canonical base pairs, i.e., G: C and A: U base pairs, without any G: U base pairs in the double-stranded region and without any non-base-pairing nucleotides (mismatches), targeting the same 200nt region of the GUS mRNA molecule as the modified hairpin RNA. The sense and antisense sequences forming the double-stranded region are covalently linked by a spacer sequence comprising the intron of PDK (Helliwell et al, 2005; Smith et al, 2000), which upon splicing of the intron from the primary transcript provides an RNA loop of 39 or 45 nucleotides in length (depending on the cloning strategy used). The DNA fragment for the antisense sequence was flanked at the 5 'end by the XhoI-BamHI restriction site and at the 3' end by the HindIII-KpnI restriction site for ease of cloning into the expression cassette, each sense sequence being flanked by the XhoI and KpnI restriction sites, respectively. For the control hairpin and the modified hairpin, the 200bp dsRNA region of each hairpin RNA included an antisense sequence of 200 nucleotides that was fully complementary to the wild-type GUS sequence from within the protein coding region. The antisense sequence, corresponding to SEQ ID NO: 10, nucleotides 13-212, are the complement of nucleotides 804-1003 of the GUS Open Reading Frame (ORF) (the cDNA sequence is provided as SEQ ID NO: 8). The GUS target mRNA is therefore greater than 1900nt in length. The length of 200 nucleotides for the sense and antisense sequences was selected to be small enough to reasonably facilitate synthesis of DNA fragments using synthetic oligonucleotides, but also long enough to provide multiple sRNA molecules after Dicer processing. As part of the ORF, the sequence is unlikely to contain a cryptic splice site or transcription termination site.

Preparation of genetic constructs

PCR amplification of a 200bp GUS ORF sequence was performed using oligonucleotide primers containing XhoI and BamHI sites or HindIII and KpnI for GUS-WT-F (SEQ ID NO: 52) and GUS-WT-R (SEQ ID NO: 53), respectively, to introduce these restriction enzyme sites 5 'and 3' of the GUS sequence. The amplified fragment was inserted into the vector pGEM-T Easy and the correct nucleotide sequence was verified by sequencing. The GUS sequence was inserted in the antisense orientation relative to the operably linked CaMV e35S promoter (Grave, 1992), and the Ocs gene polyadenylation/transcription terminator (Ocs-T) by excising the GUS fragment by digestion with BamHI and HindIII and inserting it into the BamHI/HindIII site of pKannibal (Helliwell and Waterhouse, 2005). The resulting vector was designated pMBW606 and contained 35S:: PDK intron:: antisense GUS:: Ocs-T expression cassette in 5 'to 3' order. This vector is an intermediate vector that serves as the basic vector for the assembly of the four hpRNA constructs.

Construct hpGUS with canonical base pairs only [ wt ]

To prepare a vector with canonical base pairs only, encoding a hairpin RNA molecule used as a control, named hpGUS [ wt ], a 200bp GUS PCR fragment with XhoI and KpnI was excised from the pGEM-T Easy plasmid and inserted into the XhoI/KpnI site between the 35S promoter and PDK intron in pMBW 606. This resulted in a vector named pMBW607 containing 35S:: sense GUS [ wt ]: PDK intron:: antisense GUS:: OCS-T expression cassette. The cassette was excised by digestion with NotI and inserted into the NotI site of pART27 (Gleave, 1992) to give a vector named hpGUS [ wt ] encoding a canonical base-paired hairpin RNA targeting the GUS mRNA.

When self-annealed by hybridization of 200nt sense and antisense sequences, the hairpin had a double-stranded region corresponding to 200 consecutive base pairs of the GUS sequence. The sense and antisense sequences in the expression cassette were flanked by BamHI and HindIII restriction sites at the 5 'and 3' ends, respectively, relative to the GUS sense sequence. When transcribed, the nucleotides corresponding to these sites are also capable of hybridizing, extending the double-stranded region by 6bp at each end. Following transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure is predicted to have a 39 nucleotide loop structure prior to any processing by Dicer or other rnases. The nucleotide sequence of the hairpin RNA structure including its loop is represented by SEQ ID NO: 15 and its free energy of folding is predicted to be-471.73 kcal/mol. This is therefore an energy stable hairpin structure. Free energy was calculated based on the nucleotide sequence after splicing of the PDK intron sequence using "RNAfold" (http:// rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi).

When transcribed from an expression cassette with a 35S promoter and OCS-T terminator, the resulting hairpin RNA is embedded in a larger RNA molecule with 8 nucleotides added at the 5' end and approximately 178 nucleotides added at the 3' end, without regard to any poly A tail added at the 3' end. Since the modified hairpin RNAs use the same promoter-terminator design, these molecules also have these extensions at the 5 'and 3' ends. Thus, the hairpin RNA molecule after splicing of the PDH intron is about 630 nucleotides in length.

Construct hpGUS comprising G: U base pairs [ G: U ]

A DNA fragment comprising the same 200 nucleotide sense sequence but with all 52 cytosine nucleotides (C) of the corresponding wild-type GUS region replaced by thymine nucleotides (T) was assembled by annealing overlapping oligonucleotides GUS-GU-F (SEQ ID NO: 54) and GUS-GU-R (SEQ ID NO: 55) and PCR extension of the 3' end using high fidelity LongAmp Taq polymerase (New England Biolabs, Cat. No. M0323). The amplified DNA fragment was inserted into pGEM-T Easy vector and the correct nucleotide sequence was verified by sequencing. (SEQ ID NO: 11). The DNA fragment containing the modification sequence was then excised by digestion with XhoI and KpnI and inserted into the XhoI/KpnI site of the basic vector pMBW 606. This resulted in a construct designated pMBW608 comprising the expression cassette 35S:, sense GUS [ G: U ]: PDK intron:, antisense GUS:: OCS-T. This expression cassette was excised by NotI digestion and inserted into the NotI site of pART27, resulting in a vector designated hpGUS [ G: U ] that encodes a G: U base-paired hairpin RNA molecule.

This cassette encodes a hairpin RNA targeting GUS mRNA which when self-annealed by hybridization of 200nt sense and antisense sequences has 52G: U base pairs (instead of the G: C base pairs in hpGUS [ wt ]) and 148 canonical base pairs, i.e., 26% of the nucleotides in the duplex region are involved in G: U base pairs. The 148 canonical base pairs in hpGUS [ G: U ] are identical to those in the control hairpin RNA and include 49U: A base pairs, 45A: U base pairs and 54G: C base pairs at corresponding positions. The longest extension of the double-stranded region of contiguous canonical base pairing is 9 base pairs. Thus, the antisense nucleotide sequence of hpGUS [ G: U ] is identical in length (200nt) and sequence to the antisense sequence of the control hairpin RNAhpGUS [ wt ]. Following transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure is predicted to have a 45 nucleotide loop structure prior to any processing by Dicer or other rnases. The nucleotide sequence of the hairpin structure including its loop is represented by SEQ ID NO: 16 and its folding free energy is predicted to be-331.73 kcal/mol. For hpGUS [ wt ], although the 52G: U base pairs are much weaker than the G: C base pairs in hpGUS [ wt ], respectively, this is therefore an energetically stable hairpin structure.

FIG. 7 shows the alignment of the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 11) with the corresponding region of the GUS target gene (SEQ ID NO: 14).

Construct hpGUS comprising nucleotides with every fourth nucleotide mismatch [1:4]

A DNA fragment comprising the same 200bp sense sequence but in which every 4 th nucleotide of the corresponding wild-type GUS sequence was replaced was designed and assembled. By changing C 'to G', G 'to C', a 'to T' and T 'to a', every 4 th nucleotide of each of the 4 nucleotide groupings (nucleotides at positions 4, 8, 12, 16, 20, etc.) is replaced, leaving the other nucleotides unchanged. These substitutions are all translocation substitutions, which are expected to destabilize the resulting hairpin RNA structure more than transition substitutions. The DNA fragments were assembled by annealing overlapping oligonucleotides GUS-4M-F (SEQ ID NO: 56) and GUS-4M-R (SEQ ID NO: 57) and PCR extension of the 3' end using LongAmp Taq polymerase. The amplified DNA fragment was inserted into pGEM-T Easy vector and the correct nucleotide sequence was verified by sequencing. (SEQ ID NO: 12). The DNA fragment containing the modification sequence was then excised by digestion with XhoI and KpnI and inserted into the XhoI/KpnI site of the basic vector pMBW 606. This resulted in a construct designated pMBW609 comprising the expression cassette 35S:, sense GUS [1:4]: PDK intron:, antisense GUS:: OCS-T. This expression cassette was excised by NotI digestion and inserted into the NotI site of pART27, resulting in a vector designated hpGUS [1:4] encoding a 1:4 mismatched hairpin RNA molecule.

The cassette encodes a hairpin RNA targeting the GUS mRNA and which, when self-annealed by hybridization of the sense and antisense sequences, has a mismatch to 50 nucleotides of the 200nt antisense sequence, including a mismatch to the nucleotide at position 200. In addition to position 200, the double-stranded region of the hairpin RNA has 150 canonical base pairs and 49 mismatched nucleotide pairs in the sense and antisense sequences of 199nt in length, i.e. 24.6% of the nucleotides of the predicted double-stranded region are mismatched (base pairs not involved). Following transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure is predicted to have a 45 nucleotide loop structure prior to any processing by Dicer or other rnases. The nucleotide sequence of the hairpin structure including its loop is represented by SEQ ID NO: 17 and its free energy of folding is predicted to be-214.05 kcal/mol. For hpGUS [ wt ], this is therefore an energetically stable hairpin structure, except for mismatched nucleotides.

FIG. 8 shows the alignment of the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 12) with the corresponding region of the GUS target gene (SEQ ID NO: 14).

Construct hpGUS [2:10], in which 9 and 10 of the 10 nucleotides are mismatched

A DNA fragment comprising the same 200bp sense sequence but in which every ninth and tenth nucleotide of the corresponding wild-type GUS sequence was replaced was designed and assembled. By changing C 'to G', G 'to C', a 'to T' and T 'to a', every 9 th and 10 th nucleotide (nucleotides at positions 9, 10, 19, 20, 29, 30, etc.) of each of the 10 nucleotide groupings was replaced, leaving the other nucleotides unchanged. The DNA fragments were assembled by annealing overlapping oligonucleotides GUS-10M-F (SEQ ID NO: 58) and GUS-10M-R (SEQ ID NO: 59) and PCR extension of the 3' end using LongAmp Taq polymerase. The amplified DNA fragment was inserted into pGEM-T Easy and the correct nucleotide sequence was verified by sequencing. (SEQ ID NO: 13). The DNA fragment containing the modification sequence was then excised by digestion with XhoI and KpnI and inserted into the XhoI/KpnI site of the basic vector pMBW 606. This resulted in a construct designated pMBW610 comprising the expression cassette 35S:, sense GUS [2:10]: PDK intron:, antisense GUS:: OCS-T. The expression cassette was excised by NotI digestion and inserted into the NotI site of pART27, resulting in a vector designated hpGUS [2:10] that encodes a 2:10 mismatched hairpin RNA molecule.

The cassette encodes a hairpin RNA targeting the GUS mRNA which, when self-annealed by hybridization of the sense and antisense sequences, has mismatches for 50 nucleotides of the 200nt antisense sequence, including mismatches for the nucleotides at positions 199 and 200. In addition to position 199 and position 200, the double-stranded region of the hairpin RNA had 160 canonical base pairs and 19 dinucleotide mismatches in the sense and antisense sequences of length 198nt, i.e., 19.2% of the nucleotides of the predicted double-stranded region were mismatched (base pairs not involved). The 160 base pairs in hpGUS [2:10] are identical to those in the control hairpin RNA and include 41U: A base pairs, 34A: U base pairs and 42G: C and 43G: C base pairs at corresponding positions. Following transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure is predicted to have a 45 nucleotide loop structure prior to any processing by Dicer or other rnases. The nucleotide sequence of the hairpin structure including its loop is represented by SEQ ID NO: 18 and its free energy of folding is predicted to be-302.78 kcal/mol. For hpGUS [ wt ], this is therefore an energetically stable hairpin structure, except for mismatched nucleotides that are expected to bulge out of the stem of the hairpin structure.

FIG. 9 shows the alignment of the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 13) with the corresponding region of the GUS target gene (SEQ ID NO: 14).

FIG. 10 schematically shows four genetic constructs for expression of control and modified hairpin RNAs.

Example 7: detection of modified hairpin RNA in transgenic plants

Plants of the Nicotiana tabacum (Nicotiana tabacum) species transformed with the GUS target gene were used to test the efficacy of the four hairpin RNA constructs described above. Specifically, the target plants were from two homozygous independent transgenic lines PPGH11 and PPGH24, each containing a single copy insertion of the GUS transgene from the vector pwbpgh, as schematically shown in fig. 11. The GUS gene in the T-DNA of pWBPPGH has the GUS coding region (nucleotides 7-1812 of SEQ ID NO: 8) operably linked to a 1.3kb long promoter from the phloretin 2(PP2) gene of Cucurbita pepo (Cucurbita pepo L. cv. Autumn Gold) (Wang et al, 1994; Wang, 1994). The construct pwbpgh was constructed by excision of the PP2 promoter plus the 5' UTR from the lambda genome clone CPP1.3(Wang, 1994) and 54 nucleotides of the coding region of the PP2 protein (the first 18 amino acids encoding PP2), and fusion of the fragment with the GUS coding sequence starting with the nucleotide encoding the 3 rd amino acid of GUS to produce an N-terminal fusion polypeptide with GUS activity. pPP2 GUS: Nos-T cassette was inserted into pWBVec2a (Wang et al, 1998) to produce pWBPPGH, which was used to transform plants of Nicotiana tabacum cv. Wisconsin 38 selects for resistance to hygromycin using Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediated leaf disc transformation (Ellis et al, 1987). GUS activity was similar in homozygous progeny plants of both transgenic lines PPGH11 and PPGH 24. GUS expression in both transgenic plants is not limited to phloem, but is present in most tissues of the plants. Thus, GUS expression from the PP2 promoter appears to be constitutive in these plants. The PP2-GUS plant was selected as the test plant for two reasons: i) they gave approximately the same constitutive high level of GUS expression as the 35S-GUS plants; ii) the PP2 promoter is an endogenous PP2 gene promoter derived from Cucurbita pepo (Cucurbita pepo) having a sequence different from the 35S promoter used to drive expression of the hpRNA transgene, and therefore it is not co-repressed by transcription of the imported 35S promoter.

All 4 hairpin RNA constructs (example 6) were used to transform PPGH11 and PPGH24 plants using the Agrobacterium-mediated leaf disc method (Ellis et al, 1987) using 50mg/L kanamycin as selection agent. This selection system using kanamycin as a reagent different from the previously used hygromycin for the introduction of the T-DNA of pwbpgh was observed to produce only transformed plants, and no untransformed plants were regenerated. Regenerated transgenic plants containing T-DNA from the hpGUS construct were transferred to soil for growth in the greenhouse and maintained for about 4 weeks prior to determination of GUS activity. When assayed, the transgenic plants were healthy and actively growing and were identical in appearance to the untransformed control plants and the parental PPGHII and PPGH24 plants. In general, 59 transgenic plants transformed with T-DNA encoding hpGUS wt, 74 plants transformed with T-DNA encoding hpGUS G: U, 33 plants transformed with T-DNA encoding hpGUS 1:4, and 41 plants transformed with T-DNA encoding hpGUS 2:10 were obtained.

GUS expression levels were determined using the fluorescent 4-methylumbelliferyl β -D-glucuronide (MUG) assay (Jefferson et al, 1987) according to the modified kinetic method described by Chen et al (2005). Plants were determined by taking leaf samples of approximately 1cm in diameter from 3 different leaves of each plant, selecting well-swelled, healthy and green leaves. Note that the test plants were at the same stage of growth and development as the control plants. Each assay used 5. mu.g of protein extracted from each leaf sample and the cleavage rate of MUG was measured as described by Chen et al (2005).

Representative data are shown in fig. 12, showing GUS activity (MUG units in the assay) for each individual transgenic plant. Since the data for the hpGUS [ wt ] constructs show that some plants show strong silencing with at least 90% reduction in activity, while others are less silent, herein, 10% GUS activity relative to control plants was chosen as a means for classifying plants into two classes and comparing the activity levels of the different constructs.

Using the 10% activity level as a baseline for strong silencing, the genetic construct encoding canonical base-paired hpGUS [ wt ] induced strong GUS silencing (54.2%) in 32 of the 59 transgenic plants tested. The other 27 plants all showed reduced GUS activity but retained more than 10% of the enzyme activity relative to the control plant and were therefore considered herein to show weak silencing. Transgenic plants with this construct showed a wide range of degrees of GUS gene silencing (fig. 12), from less than 1% to about 80% activity retention, which is typical for conventional hairpin constructs (Smith et al, 2000).

In clear contrast, the hpGUS [ G: U ] construct induced consistent and uniform silencing across independent transgenic lines, with 71 (95.9%) of the 74 plants tested exhibiting strong GUS silencing. Again, all 33 hpGUS [1:4] plants tested showed reduced levels of GUS activity, yielding < 10% GUS activity relative to only 8 (24%) of the control plants, and the other 25 were classified as having poor silencing. These results indicate that the construct induces weaker but more uniform levels of GUS downregulation across transgenic lines. The hpGUS [2:10] construct performed more like the hpGUS [ wt ] construct, inducing a good level of silencing in some lines (28 out of 41, or 68.3%) and little or no GUS silencing in the remaining 13 plants.

When silent lines alone (residual activity < 10%) were used for comparison and the average GUS activity was calculated, hpGUS [ wt ] plants showed the highest average degree of silencing, followed by hpGUS [ G: U ] plants and hpGUS [2:10] plants (FIG. 13). The average reduction in GUS activity was minimal in HPGUS [1:4] plants. The degree of GUS silencing showed a good correlation with the thermodynamic stability of the predicted hpRNA structure derived from four different hpRNA constructs (example 6).

To test whether these differences persist in progeny plants, representative transgenic plants containing the target GUS gene (homozygous) and hpGUS transgene (hemizygous) were self-pollinated. Kanamycin resistant progeny plants from the hpGUS line were selected, thus discarding any null segregants lacking the hpGUS transgene. This ensures that the hpGUS transgene is present in all progeny in either homozygous or heterozygous state. Progeny plants were assayed for GUS activity and representative data are shown in figure 14. Progeny containing the hpGUS [ wt ] transgene are clearly divided into two categories, i.e., progeny with strong GUS silencing and progeny that show weak or no silencing. These types correlated well with the previous generation phenotype, indicating that the degree of target gene silencing is heritable. All plants in the hpGUS [ G: U ] lines tested consistently showed strong silencing, while plants in the hpGUS [1:4] lines consistently showed weaker silencing. The inventors concluded that the phenotype observed in the parent is generally retained in the progeny plant.

Southern blot hybridization experiments on transformed plants

In use with hpGUS [ G: U ]]The uniformity of strong gene silencing observed in a large number of independent transgenic plants produced by the constructs is surprising and unexpected. The inventors sought to determine other than by hpGUS [ G: U]Any explanation beyond the role of RNA induction is whether it causes uniformity of silencing. To test whether multiple transgenic plants resulted from expected independent transformation events, plants isolated from 18 plants contained hpGUS [ G: U ]]DNA from representative transgenic plants of the constructs was subjected to Southern blot hybridization experiments. DNA was isolated from leaf tissue using the thermal phenol method described by Wang et al (2008). For Southern blot hybridization, approximately 10. mu.g of DNA from each plant sample was digested with HindIII enzyme, separated by gel electrophoresis in a 1% agarose gel in TBE buffer, and blotted onto Hybond-N + membrane using a capillary method (Sambrook et al, 1989). The film is contacted with a solution from the OCS-T terminator region at 42 DEG C³²The P-labeled DNA fragment was hybridized overnight. This probe was chosen because it interacts with hpGUS [ G: U ]]The transgene was crossed but not to the GUS target gene without the OCS-T terminator sequence. The membrane was washed at high stringency and the retained probes were visualized with a phosphoric acid imager.

An autoradiogram of the hybridization blot is shown in FIG. 15. Each lane shows 1-5 or 1-6 hybridizing bands. No two lanes show the same pattern, i.e., the autoradiogram showed that each of the 16 representative hpGUS [ G: U ] plants had a different pattern of post-cross HindIII fragments and thus were from different transgene insertions. The inventors concluded that the uniform GUS silencing observed for hpGUS [ G: U ] lines was not due to a similar transgene insertion pattern in plants, and that the uniformity of silencing was due to the structure of hpGUS [ G: U ] RNA. The inventors also concluded that: multiple copies of the hpGUS [ G: U ] transgene are not required for strong gene silencing; a single copy of the transgene is sufficient.

Northern blot hybridization experiments on transformed plants

To determine whether hpGUS [ G: U ] RNA was processed in the same manner as control hairpin RNA in transgenic plants, Northern blot hybridization experiments were performed on RNA isolated from leaves of transgenic plants. Northern blot experiments were performed to detect shorter RNAs (sRNA, approximately 21-24 nucleotides in length) produced by Dicer-processing of hairpin RNA. Experiments were performed on small RNAs isolated from transgenic HPGUS [ wt ] and HPGUS [ G: U ] plants that also contained the GUS target gene expressed as a (sense) mRNA. 9 plants per construct were selected for sRNA analysis. For the hpGUS [ wt ] transgenic population, plants showing weak GUS silencing as well as some showing strong GUS silencing were included. Small RNA samples were isolated using the pyrogallol method (Wang et al, 2008) and subjected to Northern blot hybridization as described in Wang et al (2008), and gel electrophoresis of RNA samples was performed under denaturing conditions. The probe used was a probe corresponding to SEQ ID NO: 8 nucleotides 804 and 1003 corresponding to the sense or antisense sequence.

FIG. 16 shows autoradiographs of Northern blots hybridized to antisense probes (top panel) to detect sense sRNA molecules derived from hairpin RNA, or to sense probes to detect antisense sRNA (bottom panel). At the bottom, the figure shows the qualitative score of GUS expression level relative to control plants lacking hpGUS constructs. In other experiments, hybridization to small RNAs of about 20-25 nucleotides was observed based on the mobility of srnas compared to RNAs of known length. The hpGUS [ wt ] line shows a range of variation in sRNA accumulation. This was observed for both sense and antisense sRNA, although the antisense sRNA bands are less distinct than the sense band. Since hpGUS [ wt ] plants contain both hpGUS transgene expressing sense and antisense sequences corresponding to the 200nt target region and GUS target gene expressing the full length sense gene, sense sRNA can be generated from hairpin RNA or target mRNA. sRNA levels in hpGUS [ wt ] were inversely correlated with the degree of GUS silencing. For example, both plants represented in lanes 4 and 5 accumulated relatively more sRNA, but showed only a moderate degree of GUS downregulation. In contrast, the two plants presented in lanes 7 and 8 had strong GUS silencing but accumulated relatively low levels of sRNA.

In contrast to hpGUS [ wt ] plants, which accumulate consistent amounts of antisense sRNA across lines, and consistent with the relatively consistent degree of silencing of hpGUS [ G: U ] constructs. Furthermore, the degree of GUS silencing appears to show a good correlation with the amount of antisense sRNA. Few sense srnas were detected in these plants. This is expected because the RNA probe used in the Northern blot hybridization was transcribed from the wild-type GUS sequence and therefore had a lower level of complementarity to sense sRNA from hpGUS [ G: U ], where all C nucleotides were replaced with U nucleotides, allowing only lower stringency hybridization. However, this experiment does not exclude the possibility of processing hpGUS [ G: U ] RNA to produce less sense sRNA or to degrade them more rapidly.

Repeat the Northern blot hybridization experiment, this time using only the sense probe to detect antisense sRNA; autoradiography is shown in figure 17. Again, the production of antisense sRNA from the hpGUS [ wt ] construct was negatively correlated with GUS activity (upper panel of FIG. 17). Strongly silenced plants produced high levels of antisense sRNA (lanes 1, 3, 5, 8 and 10), while plants showing only weak or no silencing produced no hybridization signal in this experiment (lanes 2, 4, 6, 7 and 9). In contrast, plants expressing hpGUS [ G: U ] produced much lower but consistent amounts of antisense sRNA. It is interesting to observe that strongly silenced plants expressing hpGUS [ G: U ] accumulate much lower sRNA levels than strongly silenced plants expressing hpGUS [ wt ] and suggest that the inventors hpGUS [ wt ] are processed in plants by different mechanisms, but still as effective as hpGUS [ wt ] constructs. Further observations in this experiment provide clues that the two relatively weak antisense bands for hpGUS [ G: U ] plants appear to have the same mobility as the second and fourth bands observed for the antisense sRNA band from hpGUS [ wt ]. This was confirmed in further experiments described below. The present inventors postulated that the four bands of sRNA from hpGUS [ wt ] represent 24-, 22-, 21-and 20-mers, and primarily processed hpGUS [ G: U ] RNA to produce 22-and 20-mer antisense sRNA.

An important clear conclusion from the above data is that hpGUS [ G: U ] RNA molecules are processed by one or more Dicer enzymes to produce sRNA, particularly antisense sRNA, which is thought to be a mediator of RNA interference in the presence of various proteins such as Argonaute. The observed production of antisense sRNA suggests that sense sRNA was also produced, but this experiment did not distinguish between degradation/instability of sense sRNA or that sense sRNA was not detected due to insufficient hybridization with the probe used. From these experiments, the inventors also concluded that: the hpGUS [ wt ] and hpGUS [ G: U ] RNA molecules differ significantly in their processing. This indicates that the molecule is recognized differently by one or more Dicers.

Example 8: transgenic plant sRNA analysis for expression of modified hairpin RNA

Another Northern blot hybridization experiment was performed to detect antisense sRNA from hpGUS [ G: U ] plants and compare its size to those produced by hpGUS [ wt ]. The autoradiogram is shown in figure 18. At this time, the size difference of the two antisense sRNA bands from hpGUS [ G: U ] was more pronounced than the two major bands from hpGUS [ wt ]. This can best be seen by comparing the mobility of the bands in adjacent lanes 9 and 10 of figure 18. This result demonstrates that the two hairpin RNAs are processed differently in plants by one or more Dicers.

To further investigate this, small RNA populations from hpGUS [ wt ] and hpGUS [ G: U ] were analyzed by deep sequencing of total, adaptor-amplifiable sRNA isolated from plants. The frequency of sRNA mapped to the hairpin RNA double-stranded region was determined. And the length distribution thereof was measured. The results indicate that the frequency of 22-mer antisense RNA is increased for hpGUS [ G: U ] constructs compared to hpGUS [ wt ] constructs. An increase in the sRNA ratio of 22nt in length indicates that Dicer-2 has altered processing of the hpGUS [ G: U ] hairpin relative to hpGUS [ wt ].

Example 9: transgenic plants DNA methylation assay

The observation of variability in the degree of GUS silencing conferred by hpGUS [ wt ] and the detection of antisense 24-mer sRNA in hpGUS [ wt ] plants, but apparently no antisense 24-mer sRNA in hpGUS [ G: U ] plants, led the inventors to question whether two plant populations differ in the level of DNA methylation of the target GUS gene. Sequence-specific 24-mer sRNA is thought to be involved in promoting DNA methylation of inverted repeats in plants (Dong et al, 2011). Thus, the inventors tested the DNA methylation level of the GUS transgene, in particular the 35S promoter region of the hairpin-encoding gene (silenced gene), in hpGUS plants.

For this purpose, the DNA methylation dependent endonuclease McrBC was used. McrBC is a commercially available endonuclease that cleaves DNA containing methylcytosine on one or both strands of double-stranded DNA ((R))^mC) DNA of bases (Stewart et al, 2000). McrBC recognizes two sites on DNA, these sites are defined by 5' (G or A)^mTwo half-sites of C3', preferably G^mC. These half-sites may be separated by several hundred base pairs, but the optimal spacing is 55 to about 100 bp. Double-stranded DNA with such linked GmC dinucleotides on both strands is the best substrate. McrBC activity is dependent on one or two methylated GC dinucleotides. Since plant DNA can be methylated at C in CG, CHG or CHH sequences, where H represents A, C or T (Zhang et al, 2018), digestion of DNA using McrBC and subsequent PCR amplification of gene-specific sequences can be used to detect specific DNA sequences in plant genomes^mThe presence or absence of C. In this assay, PCR amplification of methylated McrBC digested genomic DNA produces a reduced amount of amplification product compared to unmethylated DNA, but if the DNA is unmethylated, will produce the same amount of PCR product as untreated DNA.

From hpGUS containing the target except the GUS Gene [ wt ] by standard methods ]、hpGUS[G:U]Or hpGUS [1:4 ]]Genomic DNA was isolated from plants of the constructs (Draper and Scott, 1988). According to the manufacturer's instructions, including the presence of Mg required for endonuclease activity²⁺Ions and GTP, purified DNA samples were analyzed using McrBC (catalog No. M0272; Masa)New england bio laboratory, seikhou). In summary, approximately 1. mu.g of genomic DNA was digested overnight with McrBC in a 30. mu.l reaction volume. The digested DNA sample was diluted to 100. mu.l and PCR amplified of the region of interest was performed as follows.

The treated DNA sample was used for PCR reaction using the following primers. For the 35S-GUS linker sequence of hpGUS [ wt ]: forward primer (35S-F3), 5'-TGGCTCCTACAAATGCCATC-3' (SEQ ID NO: 60); reverse primers (GUST-R2, 5'-CARRAACTRTTCRCCCTTCAC-3' (SEQ ID NO: 61). 35S-GUS linker sequence for hpGUS [ G: U ]: forward primers (GUGUGU-R2), 5'-CAAAAACTATTCACCCTTCAC-3' (SEQ ID NO: 62); reverse primers (GUS4m-R2), CACRAARTRTACRCRCTTRAC (SEQ ID NO: 63). 35S promoter sequence for both constructs forward primer (35S-F2, reverse, 5'-GAGGATCTAACAGAACTCGC-3' (SEQ ID NO: 64); reverse primer (35S-R1), 5'-CTCTCCAAATGAAATGAACTTCC-3' (SEQ ID NO: 65). in each case, R or G, Y ═ C or T. PCR reactions were carried out under cycling conditions of 94 ℃ for 1 minute, 35 cycles of 94 ℃ for 30 seconds, 55 ℃ for 45 seconds, 68 ℃ for 1 minute, and finally extension at 68 ℃ for 5 minutes. The PCR amplification product was electrophoresed and the intensity of the band was quantified.

Representative results are shown in fig. 19 and 20. Most hpGUS [ wt ] plants show significant levels of DNA methylation for a 200bp 35S-GUS junction region that includes a 35S promoter sequence containing a transcription start site. Individual plants that retain higher levels of GUS activity, i.e., less silencing, within the hpGUS [ wt ] plant population appear to have more methylation of the promoter-GUS sense junction region. The results for the 35S promoter region were similar. In contrast, most of the hpGUS [ G: U ] and hpGUS [1:4] plants showed weaker DNA methylation at the 35S-GUS junction. The inventors believe that this proximal promoter sequence is important for expression of the transgene, and methylation at this region will likely reduce expression of the silencing construct through Transcriptional Gene Silencing (TGS) of the transgene. This is called "self-silencing".

General discussion of examples 6-9

Disruption of inverted repeat DNA structure in transgenes enhances their stability

Both the hpGUS [ wt ] and hpGUS [2:10] transgenic plant populations exhibit a broad degree of target gene silencing. In contrast, both populations containing hpGUS [ G: U ] and hpGUS [1:4] plants showed relatively uniform GUS silencing in many independent lines, with the former constructs observing strong silencing and the latter constructs observing a significant reduction in gene activity, albeit relatively weak. In hairpin RNAs from [ G: U ] and [1:4] constructs, about 25% of the nucleotides in the sense and antisense sequences are involved in G: U base pairs or in sequence mismatches distributed evenly over the 200 nucleotide sense/antisense sequences. Because of sequence differences between sense and antisense sequences, it is believed that a mismatch in the DNA construct between the sense and antisense "arms" or the inverted request structure significantly disrupts the inverted repeat DNA structure. The repetitive DNA structure can attract DNA methylation and silencing in various organisms (Hsieh and Fire, 2000). The hpGUS [2:10] construct also contains mismatches in the sense and antisense regions, but each of the 2bp mismatches between the sense and antisense sequences is flanked by 8-bp consecutive mismatches, so that the mismatches may not disrupt the inverted repeat DNA structure as in the [ G: U ] and [1:4] transgenes. Thus, the uniformity of GUS silencing induced by hpGUS [ G: U ] and hpRNA [1:4] may be due, at least in part, to the disruption of the inverted repeat DNA structure, which results in lower methylation and thus reduced self-silencing of both transgenes. Another benefit of mismatches between sense and antisense DNA regions is that they aid in cloning of inverted repeats in E.coli, since bacteria tend to delete or rearrange perfect inverted repeats.

The thermodynamic stability of hpRNA is an important factor affecting the degree of silencing of target genes

When compared to strongly silenced transgenic lines, the target gene of hpGUS wt plants is down-regulated to the greatest extent, followed by hpGUS [ G: U ], hpGUS [2:10] and hpGUS [1:4 ]. RNAFld analysis predicts that the hpGUS [ wt ] hairpin RNA structure has the lowest free energy, i.e., the greatest stability, followed by hpGUS [ G: U ], hpGUS [2:10] and hpGUS [1:4] hairpins. The inventors believe that the more stable the hairpin RNA structure, the greater the degree to which it can induce silencing of the target gene. This also favors longer double-stranded RNA structures over shorter double-stranded RNA structures. It is believed that stable double stranded RNA formation is required for efficient Dicer processing. The experimental results described herein demonstrate another important advantage of G: U base pairing constructs over constructs containing most simple mismatched nucleotides, such as hpGUS [1:4], although both types of constructs disrupt the inverted repeat DNA structure, which reduces self-silencing, but at the RNA level, hpGUS [ G: U ] RNA is more stable due to the ability of G and U to form base pairs. Combinations of both types of modifications are also considered beneficial, including G: U base pairs and some mismatched nucleotides in double-stranded RNA structures, but involving at least 2, 3, 4, or even 5 times more nucleotides in the G: U base pairs than in mismatches.

Dicer can efficiently process hpGU RNA

An important question answered in these experiments is whether U base-paired hpRNA can be processed by Dicer into small RNA (sRNA). Strong silencing in hpGUS [ G: U ] plants and 1:4 and 2:10 mismatched hpRNA plants suggests that these hairpin RNA structures are processed by Dicer. This was confirmed by sRNA Northern blot hybridization of [ G: U ] molecules, which readily detected antisense sRNA. Furthermore, the degree of GUS silencing in HPGUS [ G: U ] plants showed a good correlation with the amount of accumulated antisense sRNA. Deep sequencing analysis of small RNAs from two selected lines of each line (only one for hpGUS [ wt ]) confirmed that hpGUS [ G: U ] plants, such as hpGUS [ wt ] plants, produced large amounts of sRNA, whereas hpGUS [1:4] plants also produced sRNA, but with much lower abundance (FIG. 21). Lower levels of sRNA in hpGUS [1:4] plants are consistent with relatively low GUS silencing efficiency, indicating that the low thermodynamic stability of dsRNA stems in hpGUS [1:4] RNA decreases Dicer processing efficiency. It was noted that the degree of GUS silencing showed relatively poor correlation with sRNA levels of the hpGUS [ wt ] construct, and some strongly silenced lines contained relatively low amounts of sRNA. This indicates that GUS silencing in some hpGUS [ wt ] lines is due at least in part to transcriptional silencing rather than sRNA-directed PTGS. The present inventors have recognized that by using modified hairpin RNA constructs, particularly G: U constructs, self-silencing of hairpin-encoded genes involved in methylation of gene sequences, such as promoter regions, is reduced.

The G: U and 1:4hpRNA transgenes show a reduction in DNA methylation of the proximal 35S promoter region

McrBC restriction-PCR analysis showed that the DNA methylation levels in the 240bp 35S sequence near the Transcription Start Site (TSS) in hpGUS [ G: U ] and hpGUS [1:4] were reduced relative to the hpGUS [ wt ] population. This result indicates to the inventors that disruption of the perfect inverted repeat structure minimizes transcriptional self-silencing of the hpRNA transgene due to C to T modifications (in hpGUS [ G: U ]) or 25% nucleotide mismatches (in hpGUS [1:4 ]) in the sense sequence. This is consistent with the uniformity of GUS gene silencing observed in the hpGUS [ G: U ] and hpGUS [1:4] populations relative to the hpGUS [ wt ] population. The present inventors have recognized that hpGUS [ G: U ] constructs are more desirable than hpGUS [1:4] constructs in reducing promoter methylation, at least because they have a reduced number or even a deletion of cytosine nucleotides in the sense sequence, and thus do not attract DNA methylation that can diffuse to the promoter.

Example 10: design and testing of hairpin RNAs comprising G: U base pairs targeting endogenous genes

Modified hairpin RNA targeting EIN2 and CHS RNA

Since the G: U modified hairpin RNA appears to induce more consistent and uniform silencing of the target gene compared to conventional hairpin RNA as described above, the inventors wanted to test whether the improved design would also reduce the expression of the endogenous gene. Thus, the inventors designed, produced and tested several [ G: U ] modified hairpin RNA constructs targeting either the EIN2 or the CHS gene or both, which are endogenous genes in Arabidopsis (Arabidopsis thaliana), selected as exemplary target genes for attempting silencing. The EIN2 gene (SEQ ID NO: 19) encodes ethylene insensitive protein 2(EIN2), which is a central factor in the signaling pathway regulated by the plant signal molecule ethylene, i.e., a regulator protein, and the CHS gene (SEQ ID NO: 20) encodes chalcone synthase (CHS), which is involved in anthocyanin production in Arabidopsis thaliana (A.thaliana). Another G: U modified construct was generated that targets both the EIN2 and CHS genes, where the EIN2 and CHS sequences were transcriptionally fused to produce a single hairpin RNA. In addition, three additional constructs were made targeting either EIN2, CHS, or both EIN2 and CHS, in which the cytosine bases in both the sense and antisense sequences were replaced with thymine bases (referred to herein as G: U/U: G constructs), rather than just the sense sequence as was done for the modified hairpin targeting GUS. Modified hairpin RNA constructs were tested for their ability to reduce expression of endogenous EIN2 gene or EIN2 and CHS gene using gene delivery methods to provide hairpin RNA to cells. Conventional hairpin RNAs used as controls in this experiment had a double-stranded RNA region of 200 base pairs in length targeting either EIN2 or CHS mRNA alone, or a chimeric double-stranded RNA region comprising 200 base pairs from each of the EIN2 and CHS genes fused together as a single hairpin molecule. In the fused RNA, the double stranded portion of EIN2 is adjacent to the hairpin loop and the CHS region is distal to the hairpin loop. All base pairs in the double-stranded region of the control hairpin RNA are canonical base pairs.

Construct preparation

A DNA fragment of the 200bp region of wild type EIN2(SEQ ID NO: 19) and CHS cDNA (SEQ ID NO: 20) was PCR-amplified from Arabidopsis thaliana (Arabidopsis thaliana) Col-0 cDNA using oligonucleotide primer pairs EIN2wt-F (SEQ ID NO: 66) and EIN2wt-R (SEQ ID NO: 67) or CHSwt-F (SEQ ID NO: 68) and CHSwt-R (SEQ ID NO: 69), respectively. For the GUS hairpin construct, the fragment was inserted into pGEMT-Easy (example 6). DNA fragments comprising 200bp modified sense EIN2[ G: U ] (SEQ ID NO: 22) and CHS [ G: U ] (SEQ ID NO: 24) fragments or 200bp modified antisense EIN2[ G: U ] (SEQ ID NO: 25) and modified antisense CHS [ G: U ] (SEQ ID NO: 26) fragments, each flanked by restriction enzyme sites, were assembled by annealing the corresponding oligonucleotides to EIN2gu-F + EIN2gu-R, CHSgu-F + CHSgu-R, ASEIN2gu-F + ASEIN2gu-R and ASCHSgu-F + ASCHSgu-R (SEQ ID NO: 70-77) followed by PCR extension of the 3' end using LongAmp Taq polymerase. All G: U modified PCR fragments were cloned into pGEM-T Easy vector and the target nucleotide sequence was verified by sequencing. The CHS [ wt ]: EIN2[ wt ], CHS [ G: U ]: EIN2[ G: U ], asCHS [ G: U ]: aseIN2[ G: U ] fusion fragment was prepared by ligating the appropriate CHS and EIN2 DNA fragments at the common XbaI site of pGEM-T Easy plasmid.

35S sense fragment PDK intron antisense fragment OCS-T cassette was prepared in a similar manner to the hpGUS construct. Basically, antisense fragments were excised from the corresponding pGEM-T Easy plasmid by digestion with HindIII and BamHI and inserted into pKannibal between the BamHI and HindIII sites so that they were in antisense orientation relative to the 35S promoter. The sense fragment was then excised from the corresponding pGEM-T Easy plasmid using XhoI and KpnI and inserted into the appropriate same site containing the antisense clone. All cassettes in the pGEM-T Easy plasmid were then excised with noti and inserted into pART27 to form the final binary vector for plant transformation.

FIGS. 22-25 show an alignment of modified sense [ G: U ] and antisense [ G: U ] nucleotide sequences with corresponding wild type sequences, showing the positions of the substituted nucleotides. The design of the expression cassette for hairpin RNA is schematically shown in fig. 26.

The free energy of hairpin RNA formation was estimated using the FOLD program. These are calculated as (kcal/mol): hpEIN2[ wt ], -453.5; hpEIN2[ G: U ], -328.1; hpCHs [ wt ], -507.7; hpCHS [ G: U ] -328.5; hpEIN2[ G: U/U: G ], -173.5; hpCHs [ G: Y/U: G ], -186.0; hpCHS, EIN2 wt, -916.4; hpCHS, EIN2[ G: U ], -630.9; hpCHs (EIN 2 (G: U/U: G)) 333.8.

Plant transformation

All the EIN2, CHS and chimeric EIN2/CHS constructs were used to transform Col-0 plants of Arabidopsis (Arabidopsis thaliana) species using the floral dip method (Clough and Bent, 1998). For selection of transgenic plants, seeds collected from Agrobacterium-impregnated flowers were sterilized with chlorine and plated on MS medium containing 50mg/L kanamycin. Multiple transgenic lines were obtained for all 9 constructs (table 1). These primary transformants (passage T1) were transferred to soil, self-pollinated and grown to maturity. Seeds collected from these plants (T2 seeds) were used to create T2 plants and to screen lines homozygous for the transgene. These were used to analyze EIN2 and CHS silencing.

TABLE 1 summary of transgenic plants obtained in the Col-0 background

Construct	Number of transgenic lines obtained
		hpEIN2[wt]	46
hpCHS[wt]	34
		hpEIN2[G:U]	23
hpCHS[G:U]	32
		hpEIN2[G:U/U:G]	52
hpCHS[G:U/U:G]	13
		hpCHS::EIN2[wt]	28
hpCHS::EIN2[G:U]	26
		hpCHS::EIN2[G:U/U:G]	20

EIN2 analysis of silencing degree

EIN2 is a gene in arabidopsis thaliana (a. thaliana) that encodes a receptor protein involved in ethylene sensing. The gene is expressed in the seedling after the seed germination and also expressed in the plant growth and development process. EIN2 mutant seedlings showed hypocotyl elongation relative to isogenic wild type seedlings when germinated in the dark in the presence of 1-aminocyclopropane-1-carboxylic Acid (ACC), an intermediate of ethylene synthesis in plants. Thus, the expression and degree of silencing of the EIN2 gene in transgenic plants was determined by germinating seeds on MS medium containing 50 μ g/L ACC in total darkness and measuring their hypocotyl length compared to wild type seedlings. Hypocotyl length is an easily measurable phenotype and a good indicator of the degree of reduction in EIN2 gene expression, indicating different levels of EIN2 silencing. Depending on the level of EIN2 silencing, plants with silenced EIN2 gene expression are expected to have varying degrees of hypocotyl elongation, ranging between wild type seedlings (short hypocotyls) and null mutant seedlings (long hypocotyls). Seeds from 20 randomly selected independently transformed plants from each construct were assayed. Seeds from 20 plants containing the hpCHS: (EIN 2[ G: U ] construct) did not germinate. Data for hypocotyl length are shown in figure 27.

Lines hpEIN2[ wt ] showed a considerable range in the extent of EIN2 silencing, of which 7 lines (plant lines 2, 5, 9, 10, 12, 14, 16 in fig. 27) clearly showed low levels of silencing or the same hypocotyl length relative to wild type, and the other 13 lines had moderate to strong EIN2 silencing. Individual plants within each independent line tend to exhibit a range of degrees of EIN2 silencing, as indicated by differences in hypocotyl length. In contrast, two lines (plant lines 5, 18 in FIG. 27) containing only the hpEIN2[ G: U ] construct showed weak EIN2 silencing, the remaining 18 showed uniform strong EIN2 silencing. Furthermore, individual plants in each of the 18 lines appeared to have relatively uniform EIN2 silencing compared to plants transformed with the hpEIN2[ wt ] construct. The inventors conclude that U-modified hairpin RNA constructs are capable of conferring more consistent, less variable gene silencing to endogenous genes, which is more uniform and more predictable than conventional hairpin RNAs that target the same region of the endogenous RNA.

The EIN2 silencing levels of the transgenic hpEIN2[ wt ] and hpEIN2[ G: U ] populations also varied with respect to transgene copy number. Transgene copy number-resistance: susceptible seedling ratio of 3:1 indicates single locus insertion, while higher ratios indicate multi-locus transgene insertion, indicated by segregation ratio of kanamycin resistance marker gene in progeny plants. Several multicopy strains transformed with the hpEIN2[ wt ] construct showed low levels of EIN2 silencing, but this was not the case for hpEIN2[ G: U ] strains, where both single and multicopy loci showed strong EIN2 silencing.

The EIN2 gene was also silenced in CHS:, EIN2 fused hairpin RNA transformed seedlings. Similar to plants containing a single hpEIN2[ G: U ] construct, hpCHI: EIN2[ G: U ] seedlings clearly showed more uniform EIN2 silencing between independent lines compared to hpCHI: EIN2[ wt ] seedlings. Silencing between individual plants within independent lines also appeared to be more uniform for hpCHS:: EIN2[ G: U ] than for the hpCHS:: EIN2[ wt ] line. Also, the EIN2 silencing degree of the highly silenced hpCHs:: EIN2[ wt ] plants was slightly stronger than that of hpCHs:: EIN2[ G: U ] plants, similar to the comparison between plants transformed with hpGUS [ wt ] and hpGUS [ G: U ]. Comparison of the degree of silencing indicated that the fusion construct did not induce more potent EIN2 silencing than the single hpEIN2[ G: U ] construct, and indeed, the fusion G: U hairpin construct appeared to induce less potent EIN2 silencing than the single gene-targeted hpEIN2[ G: U ] construct.

When plants transformed with the G: U/U: G construct, in which cytosine (C) nucleotides of the sense and antisense sequences were modified to thymine (T) nucleotides, were examined, little increase in hypocotyl length was observed for all 20 independent lines analyzed, compared to wild-type plants. This was observed for both the hpEIN2[ G: U/U: G ] and hpCHS: (EIN 2[ G: U/U: G ] constructs. These results indicate that the inventors, hairpin RNA constructs with G: U/U: G base pairing of about 46% substitutions, were not able to efficiently induce target gene silencing, probably because the base pairing of the hairpin RNA was too unstable. The inventors believe that two possible causes may lead to inefficiency. First, the EIN2 double-stranded region of the hairpin RNA has 92G: U base pairs of 200 potential base pairs between the sense and antisense sequences. Second, alignment of the modified antisense sequence with the complement of the wild-type sense sequence showed that a 49C to T substitution in the antisense sequence might reduce the effectiveness of the antisense sequence in targeting EIN2 mRNA. The inventors concluded from this experiment that there is an upper limit on the number of nucleotide substitutions that can be tolerated in hairpin RNAs, at least for the EIN2 target gene, and still maintain a sufficient silencing effect. For example, substitution of 92/200 ═ 46% may be too high a percentage.

CHS silencing degree analysis

The CHS gene expression level of transgenic plants was determined by quantitative reverse transcription PCR (qRT-PCR) on RNA extracted from whole plants grown in vitro on tissue culture medium. Primers for CHS mRNA were: forward primer (CHS-200-F2), 5'-GACATGCCTGGTGCTGACTA-3' (SEQ ID NO: 78; reverse primer (CHS-200-R2) 5'-CCTTAGCGATACGGAGGACA-3' (SEQ ID NO: 79.) the primers used as the standard reference gene Actin (Actin)2 were forward primer (Actin2-For) 5'-TCCCTCAGCACATTCCAGCA-3' (SEQ ID NO: 80), and reverse primer (Actin2-Rev) 5'-GATCCCATTCATAAAACCCCAG-3' (SEQ ID NO: 81).

The data show that the level of accumulated CHS mRNA in plants is reduced in the range of 50-96% relative to the reference mRNA for the Actin2 gene (figure 28).

Arabidopsis thaliana (a. thaliana) seeds that completely lack CHS activity have a light seed coat color compared to the brown color of wild-type seeds. Thus, the seed coat color of the seeds of the transgenic plants was visually observed. A significant reduction in seed coat colour was observed in seeds from several plants, while no significant reduction in seed coat colour was observed in other plants, despite the reduction in CHS mRNA in the leaves of those plants. However, it is believed that the seed coat color phenotype is only exhibited when the CHS activity is almost completely eliminated in the developing seed coat during plant growth. In addition, the 35S promoter may not have sufficient activity in developing seed coats to provide reduced levels of CHS activity to provide the light seed phenotype seen in null mutants. The improvement of the visual seed coat color phenotype can be obtained by using a promoter that is more active in the seed coat of the seed.

Reduction of expression of PDS Gene in Arabidopsis thaliana (Arabidopsis thaliana)

Selection of another Arabidopsis(Arabidopsis)Genes as exemplaryThe target gene, Phytoene Desaturase (PDS) gene, encodes a phytoene desaturase, which catalyzes the desaturation of phytoene to ζ -carotene during carotenoid biosynthesis. PDS silencing is expected to result in photobleaching of arabidopsis plants, which is easily observed visually. Thus, G: U modified hpRNA constructs were made and tested in comparison to traditional hpRNA constructs targeting a 450 nucleotide PDS mRNA sequence. A450 nucleotide PDS sequence containing 82 cytosines (C) was substituted with thymidine (T) resulting in hpRNA hpPDS [ G: U]18.2% of the base pairs in the dsRNA region of (a) are G: U base pairs. Encoding hpPDS [ G: U ] using Agrobacterium-mediated transformation]And encoding hpPDS [ WT ]]The control genetic construct of (a) was introduced into the Arabidopsis thaliana Col-0 ecotype.

For the hpPDS [ WT ] and hpPDS [ G: U ] constructs, 100 and 172 transgenic lines were identified, respectively. Surprisingly, all of these lines showed photobleaching in the cotyledons of young T1 seedlings that appeared on kanamycin-resistant selective media, with no significant difference between the two transgenic populations at the early stages of plant growth. These indicate that both constructs are equally effective in inducing PDS silencing in the cotyledons. However, some of the developed true leaves of T1 plants were no longer photobleached, but appeared green or pale green, indicating that PDS silencing was released or attenuated in the true leaves. The hpPDS [ WT ] population showed a much higher proportion of green true leaf transgenic lines than the hpPDS [ G: U ] population. Transgenic plants were classified into three distinct classes based on strong PDS silencing (strong photobleaching throughout the plant), moderate PDS silencing (pale green or mottled leaves) and weak PDS silencing (fully green or weakly mottled leaves). The proportion of plants with weak PDS silencing in the hpPDS [ WT ] line was 43%, compared to 7% for the hpPDS [ G: U ] line. In fact, all hpPDS [ G: U ] lines of the weakly silenced group still showed mild mottle on true leaves, compared to that the weakly silenced hpPDS [ WT ] plants mostly had completely green leaves. These results indicate that the G: U modified hpRNA construct provides more uniform PDS silencing across independent transgenic populations than the conventional (fully canonical base-paired) hpPDS construct, consistent with the results of the GUS and EIN2 silencing assays described above. More importantly, PDS silencing results indicate that developmental variability in plants of hpRNA transgene-induced gene silencing has not been previously discovered and that hpRNA transgene silencing is more efficient and stable in cotyledons than in true leaves. Based on uniform gene silencing across independent lines, PDS silencing results indicate that G: U modified hpRNA transgenes are developmentally more stable, providing more stable and durable silencing than conventional hpRNA constructs.

Example 11: analysis of sRNA from hairpin RNA constructs

Northern blot hybridization of RNA samples to detect DNA from hpEIN2[ G: U]Antisense sRNA of plants, and the amount and size thereof were compared with hpEIN2[ wt ]]The generated srnas were compared. The probe was conjugated with hpEIN2[ wt ]]Corresponding to a sense sequence of 200 nucleotides in the construct³²P-labeled RNA probes, and hybridization is performed under low stringency conditions to allow detection of shorter (20-24 nucleotides) sequences. An autoradiogram from the probed Northern blot is shown in FIG. 29. This experiment showed hpEIN2[ G: U ]]Hairpin RNA was processed to sRNA and reacted with hpEIN2[ wt ]]In comparison to those of the lines, hpEIN2[ G: U ] was transformed in 9 independent transformants analyzed]The level of accumulation in plants is relatively uniform. Similar to a similar experiment for GUS hairpin RNA, from hpEIN2[ G: U]Two antisense sRNA bands of (2) and the DNA from hpEIN2[ wt ]]The difference in movement of the main two bands is quite apparent. This can best be seen by comparing the mobility of the bands in the adjacent lanes 10 and 11 of figure 28.

To further investigate this, small RNA populations from hpEIN2[ wt ] and hpEIN2[ G: U ] were analyzed by deep sequencing of the total sRNA population isolated from whole plants. The proportion of each population of double-stranded regions mapped to hpEIN2[ wt ] and hpEIN2[ G: U ] was determined. Of the approximately 16000000 reads in each population, approximately 50,000 sRNA mapped to the hpEIN2[ wt ] double-stranded region, while only approximately 700 mapped to hpEIN2[ G: U ]. This indicates that less sRNA was produced from the [ G: U ] hairpin. An increased proportion of EIN 2-specific 22-mers was also observed.

FIG. 29 shows that both traditional (fully canonical base pairing) and G: U modified hpRNA lines accumulate two major siRNA size fragments. Consistent with previous reports, the major sirnas in the traditional hpRNA lines migrated similarly to 21nt and 24nt sRNA size markers. However, the two major siRNA bands from the two G: U modified transgenes migrated slightly faster on the gel, indicating that they were smaller in size than the conventional hpRNA transgene, or that their terminal chemical modifications were different from the conventional hpRNA transgene.

To investigate whether the size distribution of siRNAs might be different between the two different types of constructs, small RNAs were isolated from one hpGUS [ WT ] line and two lines of hpGUS [ G: U ], respectively, hpEIN2[ WT ] and hpEIN2[ G: U ], and sequenced using the Illumina platform, yielding approximately 1600 million sRNA reads per sample. Samples from two highly silent hpGUS [1:4] lines were also sequenced. The number of srnas mapped to the double-stranded and intron spacers of the hairpin RNA was determined. siRNAs were also mapped to the upstream and downstream regions of the target GUS mRNA and ENI2 mRNA to detect the delivery siRNAs. Sequencing data confirmed that the hpGUS [ G: U ] line produced a large amount of siRNA similar to the hpGUS [ WT ] line, whereas the hpGUS [1:4] line also produced siRNA, but in much lower abundance. The lower levels of siRNA in the hpGUS [1:4] line are consistent with the relatively low efficiency of silencing GUS by hpGUS [1:4], and this suggests that the lower thermodynamic stability of the dsRNA stem in hpGUS [1:4] RNA reduces Dicer processing efficiency relative to traditional hairpins. Although the mobility of antisense siRNA was shown to vary significantly in Northern blots, there was no significant difference in the size distribution of siRNA between the classical and mismatched hpRNA lines, and all samples showed 21-nt sRNA as the major size class. There was a slight difference in the proportional abundance of 22nt antisense siRNA between the traditional and mismatched hpGUS lines: the hpGUS [ G: U ] and hpGUS [1:4] lines showed a higher proportion of 22nt size classes than the hpGUS [ WT ] line. A significant feature of the sequencing data for the conventional and mismatched hpRNA lines is that the 24-nt siRNA is much less abundant than the 21-nt siRNA in all samples, i.e., about 3-21 times less siRNA for the sense 24-nt and about 4-35 times less siRNA for the antisense 24-nt. This is significantly different from Northern blot results, which show that the amounts of the two major size classes are relatively equal. Interestingly, the hpEIN2[ WT ] -7 and hpEIN2[ G: U ] -14/15 samples showed similar antisense siRNA abundance on Northern blots, but in the sequencing data, the total number of 20-24nt antisense siRNAs (17290 and 29211) was much less for hpEIN2[ G: U ] line compared to hpEIN2[ WT ] -7 line (134112 reads).

For the hpGUS [ G: U ] and hpEIN2[ G: U ] lines, almost all sense siRNAs matched the G: U modified sense sequence of hpRNA, while most antisense siRNAs had wild-type antisense sequences. This indicates that most of these sense and antisense siRNAs are processed directly from the original hpRNA [ G: U ] transcript, rather than due to RDR-mediated amplification of the hpRNA or target RNA transcript, which would otherwise result in sense and antisense siRNAs of the same template sequence. Consistent with this, only a small number of 20-24nt sRNA reads (delivery sirnas) were detected from the loop region of the hpRNA transgene (PDK intron) or the non-targeted downstream region of GUS or EIN2 mRNA. However, both hpGUS [1:4] lines showed a relatively high proportion of wild-type sense siRNA, suggesting that strong GUS silencing in both lines (relatively rare for the hpGUS [1:4] population) may be involved in RDR amplification. Indeed, in hpGUS [1:4] lines, the amount of siRNA detected from the target gene sequence downstream of the hpRNA target region was higher than that detected from the dsRNA stem, indicating the presence of transmitted silencing in these lines.

Taken together, the sRNA sequencing data indicated that sirnas from the classical and mismatched hpRNA lines had similar size distributions, except for the 22-nt size class, indicating that differential migration detected by Northern blots was due to different 5 'or 3' chemical modifications. The relative sRNA abundance differences between Northern blot results and sequencing data (e.g., differences between the hpEIN2[ WT ] and hpEIN2[ G: U ] derived siRNAs and 21-nt and 24-nt) indicate that different siRNA populations and size classes may have different cloning efficiencies during sRNA library preparation.

Plant srnas are known to have a 2 '-O-methyl group at the 3' terminal nucleotide, which is thought to stabilize srnas. 3 'adaptor (adaptor) ligation previously shown that 3' methylation can inhibit but cannot prevent reduction in sRNA cloning efficiency (Ebhardt et al, 2005). Thus, hpRNA [ WT ] and hpRNA [ G: U ] derived siRNAs were used with sodium periodate in the beta-elimination assay. This treatment did not result in changes in gel mobility for hpRNA [ WT ] and hpRNA [ G: U ] derived siRNAs, indicating that both siRNA populations were methylated at the 3 'end and that there was no difference in 3' chemical modification between hpRNA [ WT ] and hpRNA [ G: U ] derived siRNAs.

Standard sequencing protocols for sRNA are based on sRNA with 5 'monophosphates (allowing for 5' adaptor ligation) (Lau et al, 2001). It is assumed that sRNA treated by Dicer has a 5' monophosphate, but in c.elegans, many sirnas are found to have diphosphates or triphosphates at the 5' end, which alters the gel mobility of sRNA and prevents 5' adaptor ligation of sRNA in standard sRNA cloning procedures (Pak and Fire 2007). It is not clear whether plant srnas also have differential 5' phosphorylation. hpRNA [ WT ]]And hpRNA [ G: U ]]The 5' phosphorylation status of the derived siRNAs was therefore examined by treating the total RNA with alkaline phosphatase followed by Northern blot hybridization. This treatment reduced the gel mobility of all hpRNA-derived srnas, indicating the presence of 5' phosphorylation. However, after phosphatase treatment, hpRNA [ G: U ] ]Derived siRNA showed specific hpRNA [ WT ]]The greater mobility change of the derivatized siRNA resulted in migration of both dephosphorylated sirnas at the same location on the gel. Using polynucleotide kinase reactions³²P radiolabels the 5 'end of 21 and 24-nt sRNA size markers and therefore should have a monophosphorylated 5' end. This indicates that hpRNA [ WT ] migrated at the same position as the size marker]The derived siRNA may be monophosphorylated siRNA, whereas hpRNA [ G: U]The derivatized siRNA migrated faster, with more than one phosphate at the 5' end. Thus, it was concluded that sirnas generated from the classical and G: U modified hpRNA transgenes have different phosphorylation in plant cells.

Example 12 DNA methylation analysis of EIN2 silenced plants

GUS and EIN2 silencing results indicate that hpRNA constructs with unmodified sense sequences induce highly variable levels of target gene silencing compared to constructs with modified sense sequences providing G: U base pairs. As noted above, the promoter region of the hpGUS [ G: U ] construct appears to have less methylation than the hpGUS [ wt ] construct. To test for DNA methylation and to compare hpEIN2[ wt ] and hpEIN2[ G: U ] transgenic plants, 12 plants from each population were analyzed for DNA methylation at the 35S promoter and 35S-promoter-sense EIN2 junction regions using the McrBC method. Primers for the 35S promoter region: forward primer (Top-35S-F2), 5' -AGAAAATYTTYGTYAAYATGGTGG-3 ' (SEQ ID NO: 82), reverse primer (Top-35S-RyGTYAAYAYATGGGG-3 ') (SEQ ID NO: 82), reverse primer (Top-35S-R2), 5' -TCARTRRARATRTCACATCAATCC-3 ' (SEQ ID NO: 83). Primers for the 35S promoter-sense EIN2 junction region: a forward primer (Link-35S-F2), 5 '-YYATYATTGYGATAAAGGAAAGG-3' (SEQ ID NO: 84), and a reverse primer (Link-EIN2-R2), 5 '-TAATTRCCACCAARTCATACCC-3' (SEQ ID NO: 85). In each of these primer sequences, Y ═ C or T and R ═ a or G.

Quantification of the degree of DNA methylation was determined by performing real-time PCR analysis. Calculate per plant: quotient of DNA fragment amplification rate after treatment of genomic DNA with McrBC/DNA fragment amplification rate after treatment of genomic DNA without McrBC.

Almost every hpEIN2[ wt ] plant showed significant levels of DNA methylation at the 35S promoter, particularly at the 35S-EIN2 linker, but some were higher than others. As shown in fig. 30 and 31, the plant lines represented in lanes 1, 4, 7, 9, 11 and 12 all showed strong EIN2 silencing as indicated by the longer hypocotyl length. In contrast, the other 6 lines represented in lanes 2, 3, 5, 6, 8 and 10 showed relatively weak EIN2 silencing, resulting in shorter hypocotyls. These less silent lines showed more DNA methylation at the promoter and linker as indicated by the much lower PCR band intensity when genomic DNA was pre-digested with McrBC. Quantitative real-time pcr (qpcr) assays confirmed these observations (fig. 31). All 12 tested lines had some degree of DNA methylation in both the 35S promoter region and the 35S-sense junction region. For hpEIN2[ wt ] lines 2, 3, 5, 6, 8, and 10, the maximal degree of methylation, the lowest quotient in the qPCR assay, was completely correlated with a reduction in silencing as measured by hypocotyl length. These results demonstrate that reduced EIN2 silencing in some hpEIN2[ wt ] strains is associated with increased promoter methylation. Even in the hpEIN2[ wt ] plant line in which EIN2 is silenced, the DNA methylation level is still quite high, especially in the 35S-sense EIN2 linked fragment region. When a promoter is methylated, this is thought to cause transcriptional silencing. In the case of silencing constructs, this is thus a form of "self-silencing".

In contrast to the hpEIN2[ wt ] line, the hpEIN2[ G: U ] line showed less DNA methylation at the 35S promoter and 35S-EIN2 junction. Indeed, 4 of these 12G: U lines, corresponding to lanes 1, 2, 3 and 7 in figure 30 (lanes 13, 14, 15 and 20 in figure 31), had no significant DNA methylation as indicated by PCR bands of equal intensity between the McrBC-treated and untreated samples. When these amplifications were quantified by qPCR, 6 of the 12 lines showed little to no reduction in fragments from McrBC treatment and therefore little to no DNA methylation-see lower panel of figure 31, lines 13, 14, 15, 18, 19 and 20. These results indicate that, at least in some strains, the relatively uniform EIN2 silencing of the hpEIN2[ G: U ] construct is due to significantly less promoter methylation and self-transcriptional silencing compared to hpEIN2[ wt ].

These conclusions were further confirmed by bisulfite sequencing analysis of genomic DNA of transgenic plant lines. This assay makes use of the fact that: treatment of DNA with bisulfite converts unmethylated cytosine bases in DNA to uracil (U) in excess, but leaves 5-methylcytosine bases ((U))^mC) Is not affected. After bisulfite treatment, the defined DNA fragment of interest is amplified in a PCR reaction in such a way that only the sense strand of the treated DNA is amplified. The PCR products were then subjected to batch sequencing, revealing the location and extent of methylation of individual cytosine bases in the DNA fragments. Thus, the assay yields single nucleotide resolution information about the methylation status of the DNA fragment.

Three plant lines showing the strongest levels of EIN2 silencing of each of hpEIN2[ wt ] and hpEIN2[ G: U ] by bisulfite sequencing analysis correspond to hpEIN2[ wt ] lines 1, 7 and 9 and hpEIN2[ G: U ] lines 13, 15 and 18 in FIG. 31. These plant lines showed the longest hypocotyl length, and therefore each construct was expected to have the lowest DNA methylation level of the 20 lines. The results for hpEIN2[ wt ] and hpEIN2[ G: U ] are shown in FIGS. 32 and 33, respectively. When compared, it is clear that many cytosines in the 35S promoter region and EIN2 sense region of hpEIN2[ wt ] plants are extensively methylated. In contrast, the 3 hpEIN2[ G: U ] plant lines showed much lower levels of cytosine methylation in the 35S promoter region.

Example 13: hpGUS [1:4]]DNA methylation levels in promoters of constructs

When DNA methylation of genomic DNA isolated from hpGUS [1:4] plants was analyzed using the McrBC and bisulfite methods described above, it was similarly observed that there was less methylation of cytosine bases in the 35S promoter and 35S promoter-GUS sense sequence regions relative to hpGUS [ wt ] plants.

General discussion of examples 10-13

Double-stranded RNA with G: U base pairs induces more uniform gene silencing than conventional dsRNA

Similar to the GUS construct, both hpEIIN 2[ G: U ] and hpCHN 2[ G: U ] induced more consistent and uniform EIN2 silencing than the corresponding hpRNA [ wt ] constructs encoding conventional hairpin RNA. This identity occurs not only between many independent transgenic lines, but also between siblings within transgenic lines each having the same transgene insertion. In addition to uniformity, the degree of EIN2 silencing induced by hpEIN2[ G: U ] approached that of the strongly silenced hpEIN2[ wt ] strain. Analysis of CHS gene silencing indicated that the hpCHS [ G: U ] construct was effective at reducing CHS mRNA levels by 50-97%, but few plants showed a significantly visible phenotype of reduced seed coat color. A possible explanation for the lack of more visible phenotype in the seed coat color is that even low levels of CHS activity may be sufficient to produce flavonoid pigments. Other possible explanations were that the 35S promoter was not active enough in developing seed coats to produce a phenotype, or that the hpCHS [ G: U ] construct sequence contained 65 cytosine substitutions (32.5%) compared to the EIN2 sequence only 43 (21.5%) and the GUS sequence only 52 (26%). In addition, many of these cytosine bases in the CHS sequence occur in groups of two or three consecutive cytosines, and thus not all cytosine bases need to be substituted. When all cytosines in the sense strand are substituted, this results in more, and possibly more than optimal, G: U base pairs in the hpCHS [ G: U ] RNA than in the hpEIN2[ G: U ] and hpGUS [ G: U ] RNAs. To verify this, another set of CHS constructs was prepared using sequences containing a range of cytosine substitutions ranging from about 5%, 10%, 15%, 20%, or 25% cytosine base substitutions. These constructs were tested and the optimal level was determined.

The hpEIN2[ G: U ] line expresses more uniform levels of siRNA

Consistent with more uniform EIN2 gene silencing, the hpEIN2[ G: U ] line accumulated sRNA in independent lines at a more uniform level. This confirms the conclusion with the hpGUS construct that [ G: U ] modified hpRNA is efficiently processed by Dicer and is capable of inducing efficient target gene silencing.

Fusion constructs also provide gene silencing

The purpose of including the CHS: EIN2 fusion construct in the experiment was to test whether two target genes could be silenced with a single hairpin-encoding construct. GUS experiments show that the free energy and the stability of hairpin structure RNA are positively correlated with the silencing degree of a target gene. The results indicate that the CHS EIN2 fusion construct results in silencing of at least two genes of the CHS at the mRNA level.

Two hpRNA constructs, hpEIN2[ G: U/U: G ] and hpCHEIN 2[ G: U/U: G ], in which both the sense and antisense sequences were modified from C to T such that 46% of the base pairs were converted from canonical to G: U base pairs, induced only weak or no EIN2 or EIN2 silencing in most transgenic plants. Possible explanations include i) too many G.U base pairs present, resulting in inefficient Dicer processing, and ii) sRNA binding to target mRNA that includes too many G.U base pairs does not induce efficient mRNA cleavage, or a combination of factors.

U base pairing constructs increased uniformity of target gene silencing associated with reduced promoter methylation

DNA methylation analysis using McrBC-digestion PCR and bisulfite sequencing showed that all hpEIN2[ wt ] plant lines showed DNA methylation in the promoter region, and that the degree of methylation was inversely correlated with the level of EIN2 silencing. Even as judged by McrBC-digestion PCR, DNA methylation levels of about 40% relative to all methylated cytosines were shown in the 35S promoter. Extensive promoter methylation is believed to be due to sRNA-directed DNA methylation at the EIN2 repeat, which diffuses to adjacent promoter regions. Many lines of hpEIN2[ G: U ] showed little to no promoter methylation compared to lines of hpRNA [ wt ] plants, and most plants analyzed showed less methylated cytosines. As discussed for hpGUS lines, a number of factors can lead to reduced methylation: i) the inverted repeat DNA structure is disrupted by changing C bases to T bases in the sense sequence, and ii) the sense EIN2 sequence lacks cytosine and therefore cannot be methylated by sRNA-directed DNA methylation, and iii) some Dicer recognition is altered due to a reduced level of 24-mer RNA production resulting from the altered structure of the dsRNA region with G: U base pairs, so Dicer3 and/or Dicer4 activity and Dicer2 activity are relatively high. Thus, the hpEIN2[ G: U ] transgene can behave like a normal, non-RNAi transgene (e.g., an overexpressing transgene), and the promoter methylation observed in some lines is due to the T-DNA insertion pattern rather than the inherent inverted repeat DNA structure of the hpRNA transgene.

Example 14: modified hairpins for reducing expression of another endogenous gene

Genetic constructs for the generation of modified silencing RNAs against hairpin RNAs or ledrnas targeted to other endogenous genes were designed and synthesized. These include the following.

FANCM genes in arabidopsis (a. thaliana) and Brassica napus (Brassica napus) encode fanconi anemia complementation group m (FANCM) protein, which is DEAD/DEAH box RNA helicase protein, accession nos. NM _001333162 and XM _ 018659358. The nucleotide sequence corresponding to the protein coding region of cDNA of the FANCM gene of arabidopsis thaliana (a. thaliana) is provided in SEQ ID NO: 31, and for Brassica napus (Brassica napus), the sequence provided in SEQ ID NO: 32 (c).

Genetic constructs were designed and prepared to express hairpin RNA with or without C to T substitutions and to target the FANCM gene in arabidopsis (a. thaliana) and Brassica napus (Brassica napus). Target region selection in arabidopsis (a. thaliana) SEQ ID NO: 31, nucleotide 675-1174(500 nucleotides). Target region selection in brassica napus (b.napus) SEQ ID NO: nucleotide 896-1395(500bp) of 32. Constructs encoding hairpin RNA using either wild-type sense sequence or modified (G: U) sense sequence were designed and assembled. The nucleotide sequences of the hpFANM-At [ wt ], hpFANM-At [ G: U ], hpFANM-Bn [ wt ], and hpFANM-Bn [ G: U ] constructs are provided in SEQ ID NO: 33-36. To make the G: U construct, all cytosine bases in the sense sequence were replaced with thymine bases — 102/500 (providing 20.4% G: U base pairs) in arabidopsis (a. thaliana) constructs, 109/500 (21.8% G: U base pairs) in brassica napus (b. napus). The longest stretch of consecutive canonical base pairs in the double-stranded region of the U-modified hairpin is 17 base pairs for Brassica napus (B.napus) G: 16 consecutive base pairs for the second length.

The Brassica napus (B.napus) DDM1 gene encodes a methyltransferase of methylated cytosine bases in DNA (Zhang et al, 2018). The nucleotide sequence corresponding to the protein coding region of the cDNA of the DDM1 gene of Brassica napus (Brassica napus) is provided in SEQ ID NO: 37, respectively.

Genetic constructs were designed and prepared to express hairpin RNA with or without C to T substitutions and to target the DDM1 gene in Brassica napus (Brassica napus). Two non-contiguous target regions of the brassica napus (b.napus) gene were selected: SEQ ID NO: nucleotides 504 and 815 and 1885 of 37, and directly joined to produce the chimeric sense sequence. The total length of the sense sequence is thus 502 nucleotides. Constructs encoding hairpin RNA using either wild-type sense sequence or modified (G: U) sense sequence were designed and assembled. The nucleotide sequences of the hpDDM1-Bn [ wt ] and hpDDM1-Bn [ G: U ] constructs are provided in SEQ ID NO: 38-39. To make the G: U construct, cytosine-106/502 (21.1% G: U base pair) in the sense sequence was replaced with thymine in a Brassica napus (B.napus) construct. The longest stretch of consecutive canonical base pairs in the double-stranded region of the U-modified hairpin is 20 base pairs and the second length is 15 consecutive base pairs.

For another construct targeting the endogenous gene, the genetic construct was designed to express hairpin RNA with 95C to T substitutions in the sense sequence, in 104C in the sense sequence of 350 nucleotides, providing 95/350 ═ 27.1% G: U base pairs in the double-stranded region of the hairpin RNA. That is, not all of the C's in the sense sequence are replaced by T'. In particular, when 3, 4 or 5 consecutive C's are present in the sense sequence, only 1 or 2 of the 3C's, or only 2 or 3 of the 4C's, or only 2, 3 or 4 of the 5 consecutive C's are replaced by T '. This provides a more uniform distribution of G: U base pairs in the double stranded RNA region. The longest stretch of the double-stranded region of contiguous canonical base pairing is 15 base pairs and the second length is 13 contiguous base pairs.

Another construct was designed in which one or two base pairs in each of the 4, 5, 6 or 7 nucleotide groupings were modified with C to T or a to G substitutions. Wherein the wild-type sense sequence has a stretch of 8 or more nucleotides consisting of T 'or G', one or more nucleotides being substituted in the sense strand to produce a mismatched nucleotide within the grouping, or C to T or a to G substitutions being made in the antisense strand, thereby avoiding double-stranded extension of 8 or more contiguous canonical base pairs in the double-stranded region of the resulting hairpin RNA transcribed from the construct.

Example 15: modified hairpins for reducing gene expression in animal cells

To test modified silencing RNA for G, U base pairing form, ledRNA form, or a combination of both modifications in animal cells, the gene encoding Enhanced Green Fluorescent Protein (EGFP) was used as a model target gene in the following experiments. The nucleotide sequence of the EGFP coding region is shown as SEQ ID NO: shown at 40. A target region of 460 nucleotides was selected, corresponding to SEQ ID NO: 40 nucleotides 131-.

Designing and preparing a genetic construct called hpeGFP [ wt ] expressing a hairpin RNA comprising, in 5 'to 3' order with respect to the promoter used for expression, an antisense EGFP sequence of 460 nucleotides which is fully complementary to the corresponding region of the EGFP coding region (nucleotides 131 and 590); a loop sequence of 312 nucleotides derived in part from the GUS coding region (corresponding to nucleotides 802-1042 of the GUS ORF); and a 460 nucleotide sense EGFP sequence which is identical in sequence to nucleotide 131-590 of the EGFP coding region. The DNA sequence (SEQ ID NO: 41) encoding hairpin RNA hpEGGFP [ wt ] included a NheI restriction enzyme site at the 5 'end and a SalI site at the 3' end for cloning into the vector pCI (Promega corporation). The vector is suitable for mammalian cell transfection experiments and provides expression of a strong CMV promoter/enhancer. The construct also has a T7 promoter sequence inserted between the NheI site and the start of the antisense sequence to provide in vitro transcription to produce hairpin RNA using T7RNA polymerase. The hairpin coding cassette is inserted into the expression vector pCI at the NheI to SalI site, thereby operably linking the RNA coding region to the CMV promoter and SV 40-late polyadenylation/transcription termination region.

Corresponding hairpin constructs, having 157C to T substitutions in the sense sequence and NO substitutions in the antisense sequence, were designed and prepared, and were designated as hpEGFP [ G: U ] (SEQ ID NO: 42). The target region of the EGFP coding region is nucleotide 131-590. In the stem of hairpin RNA, the percentage of C to T substitutions and thus G: U base pairs was 157/460-34.1%. The sense and antisense sequences are 460 nucleotides in length. In the field of gene silencing, long double-stranded RNA is generally avoided due to the potential to activate cellular responses, including interferon activation.

Designing and preparing a ledRNA construct called ledEGFP [ wt ] to express ledRNA comprising in 5 'to 3' order with respect to the promoter used for expression a 228 nucleotide antisense EGFP sequence fully complementary to nucleotide 131-358 of the EGFP coding sequence; a loop sequence of 150 nucleotides; a 460 nucleotide sense EGFP sequence, which has the same sequence as nucleotide 131-590 of the EGFP coding region (SEQ ID NO: 40); a loop sequence of 144 nucleotides; and an antisense sequence of 232 nucleotides which is fully complementary to nucleotide 359-590 of the EGFP coding sequence, flanked by NheI and SalI restriction sites (SEQ ID NO: 43). The codified ledRNA is thus of the type shown in FIG. 1A. When self-annealed by base pairing between one sense and two antisense sequences, the ledRNA structure has a 460 base pair double-stranded region corresponding to the EGFP target region, where the two antisense sequences are not directly covalently linked to each other, but rather have a "gap" or "cleft" between the ends corresponding to nucleotides 358 and 359. In the sequence of the CMV promoter and SV40 late polyadenylation/transcription termination region, the LedRNA construct is embedded in a larger RNA transcript that includes a 5 'upstream region and a 3' downstream region.

Corresponding ledRNA constructs having 162C to T substitutions in the sense sequence and NO substitutions in the antisense sequence were designed and prepared, designated ledEGFP [ G: U ] (SEQ ID NO: 44). In each case, the target region in the EGFP coding region was nucleotide 131-590(SEQ ID NO: 40) relative to the protein coding region that begins with the ATG start codon. In the stem of ledRNA, the percentage of C to T substitutions and thus G: U base pairs was 162/460-35.2%.

Testing of the encoded hpeGFP [ wt ] by transfection of the vector into cells]、hpEGFP[G:U]、ledEGFP[wt]And ledEGFP [ G: U]Gene silencing Activity of RNA silencing plasmids in CHO, HeLa and VERO cells. Assays were performed by co-transfection of the test plasmid with the GFP expression plasmid. All assays were performed in triplicate. CHO cells (Chinese hamster ovary cells) and VERO cells (African Green monkey kidney cells) were plated at 1X 10 per well⁵The density of individual cells was seeded in 24-well plates. CHO cells were grown in MEM alpha modification (Sigma) usa) and HeLa and VERO cells were grown in DMEM (Invitrogen usa). Both basal media were supplemented with 10% fetal bovine serum, 2mM glutamine, 10mM hepes, 1.5g/L sodium bicarbonate, 0.01% penicillin and 0.01% streptomycin. Cells were incubated at 37 ℃ with 5% CO ₂And (4) growing. Cells were then transfected with plasmid DNA or siRNA as EGFP silencing controls at 1 μ g/well using Lipofectamine 2000 (liposomes). Briefly, the test siRNA or plasmid was combined with a GFP reporter plasmid (pGFP N1) and then mixed with 1. mu.l of Lipofectamine 2000, both diluted in 50. mu.l of OPTI-MEM (Invitrogen, USA), and incubated at room temperature for 20 minutes. The complex was then added to the cells and incubated for 4 hours. The cell culture medium was replaced and the cells were cultured for 72 hours. Cells were then flow cytometric to measure GFP silencing. Briefly, cells to be analyzed were trypsinized, washed in PBSA, resuspended in 200 μ L of 0.01% sodium azide and 2% FCS in PBSA, and analyzed using a FACScalibur (BD, Becton Dickinson, usa) flow cytometer. Data processing Using CELLQuest software (BD Co., USA)The percentage of control cells with reporter and irrelevant (negative control) shRNA was analyzed and reported as Mean Fluorescence Intensity (MFI).

anti-GFP siRNA designated si22 was obtained from Qiagen (Qiagen) (U.S.A.). The anti-GFP siRNA sequence of si22 is sense 5'-gcaagcugacccugaaguucau-3' (SEQ ID NO: 86) and antisense 5'-gaacuucagggucagcuugccg-3' (SEQ ID NO: 87). A positive control genetic construct designated pshGFP was generated by a one-step PCR reaction using the mouse U6 sequence as a template. The forward primer was 5'-TTTTAGTATATGTGCTGCCG-3' (SEQ ID NO: 88) and the reverse primer was 5'-ctcgagttccaaaaaagctgaccctgaagttcatctctcttgaagatgaacttcagggtcagccaaacaaggcttttctccaa-3' (SEQ ID NO: 89). The amplification product containing the full-length expression cassette was ligated into pGEM-T Easy. A non-related shRNA control plasmid was also constructed by the same PCR method. For this construction, the forward primer was 5'-TTTTAGTATATGTGCTGCCG-3' (SEQ ID NO: 90) and the reverse primer was 5'-ctcgagttccaaaaaaataagtcgcagcagtacaatctcttgaattgtactgctgcgacttatgaataccgcttcctcctgag-3' (SEQ ID NO: 91).

The data from one experiment is shown in fig. 34. A significant reduction in EGFP activity (RNA silencing) was observed in VERO and CHO cells for the si22 and pshGFP positive controls compared to an unrelated shRNA control. These positive controls were well-validated small dsRNA molecules (si22) or encoding shrna (pshgfp), which are known to have strong silencing activity in mammalian cells. Control RNA molecules have double-stranded regions of 20 and 21 consecutive base pairs, respectively, using only canonical base pairs and no mismatched nucleotides in the double-stranded region, and are in the range of 20-30 base pairs in length, and are typically used in mammalian cells. In contrast, hpRNA and ledRNA constructs express molecules with long dsRNA regions. Specific silencing of EGFP expression by all four constructs was observed to a significant extent in both cell types (fig. 34). The inclusion of the G: U substitution significantly improved silencing of both constructs in CHO cells. In VERO cells, only significant improvement in silencing of ledEGFP [ G: U ] constructs relative to ledEGFP [ wt ] was observed.

Similar results were obtained in a second experiment using HeLa (human) cells and measuring EGFP activity 48 hours after transfection (fig. 35).

Notably, gene silencing was observed in mammalian cells using hpRNA and ledRNA effector molecules because they have a longer double-stranded region than the conventional 20 to 30bp size range. It is also clear that modifications to replace nucleotides to create G: U base pairs significantly enhance the gene silencing effect of these longer dsRNA molecules. This effect is likely due to the fact that these structures more closely resemble endogenous prirnas (precursors of mirnas) observed in eukaryotic cells and thus improve processing of longer dsrnas for loading into RNA-induced silencing complex (RISC) effector proteins.

Example 16: RNA constructs targeting DDM1 and FANCM genes in plants

The present inventors considered methods to increase the rate at which new genetic maps and diversity (genetic gain) can be generated and explored desirable performance traits in plants. One is believed to be finding ways to increase the rate of recombination that occurs during sexual reproduction in plants. Plant breeders rely on recombination events to produce different genetic (allelic) combinations that they can search for a desired genetic profile that correlates with performance gain. However, the number of recombination events in each breeding step is extremely low relative to the number of possible genetic maps that can be explored. Furthermore, the elements that control the location in the genome where these events occur are not well understood. Thus, the inventors considered whether ledRNA delivered exogenously or endogenously by transgenic methods could be used to alter recombination rates in plants to allow for rapid increase in genetic diversity and enable faster genetic gain within breeding populations.

The plant's epigenome is affected by a series of different chemical modifications of the DNA and related proteins that organize, package, and stabilize the genome. These modifications also regulate where recombination occurs, and tight genomic packing is a strong inhibitor of recombination (Yelina et al, 2012; Melamed-Bessudo et al, 2012). Deoxydna methylation 1(DDMl) is an enzyme that regulates DNA methylation and genome packaging. Mutations in this gene can alter the position of the recombination event (Yelina et al, 2012; Melamed-Bessudo et al, 2012).

Recombination events during meiosis are tightly regulated, with only 1-2 events occurring on each chromosome to ensure proper chromosome segregation for metaphase 1. Recombination events are initiated by double strand breaks in DNA (DSB) by the enzyme spioi (Wijnker et al, 2008). This results in hundreds of DSBs along the chromosome. Although some of these DSBs result in crossovers, most are repaired by DNA repair enzymes before recombination events occur. In addition, there are many negative regulators that inhibit the progression of DSB to crossover. In the initial approach considered by the present inventors, genetic constructs encoding ledRNA molecules or conventional hairpin RNA molecules were introduced as a comparison into arabidopsis thaliana (a. thaliana) plants, which target genes encoding protein factors that could potentially affect recombination rates, such as the FANCONI anaemia supplementation GROUP M (FANCONI anaemia composition GROUP M) (FANCM).

The nucleotide sequence of the DDM1 gene of arabidopsis thaliana (a. thaliana) is provided by accession number AF143940 (jeddedelah et al, 1999). A decrease in DDMl gene expression has been shown to decrease DNA methylation in arabidopsis thaliana (a. thaliana) and increase the number and location of crossover events. (Melamed-Bessudo and Levy, 2012).

Brassica napus (Brassica napus) is an heterotetraploid species and has two DDMl genes on chromosomes a7, a9, C7 and C9 on each of the a and C subgenomes, and thus four DDMl genes overall. These genes were named BnaA07g37430D-1, BnaC07g16550D-1, BnaA09g52610D-1 and BnaC09g 07810D-1. The nucleotide sequence of the DDM1 gene BnaA07g37430D-1 of Brassica napus (Brassica napus) is provided by accession number XR-001278527 (SEQ ID NO: 93). Hairpin RNA constructs targeting 500 nucleotide regions of 4 genes corresponding to SEQ ID NO: nucleotide 650 of 93-. Based on sequence conservation between genes, the nucleotide regions used to design hpRNA and ledRNA constructs target all four DDM1 genes BnaA07g37430D-1, BnaC07g16550D-1, BnaA09g52610D-1, and BnaC09g07810D-1 present in Brassica napus (B.napus). The sequence of elements of the hpRNA construct is promoter-sense-loop sequence, comprising intron-antisense sequence of Hellsgate vector-transcription terminator/polyadenylation region. The nucleotide sequence of the chimeric DNA encoding hpRNA is provided as SEQ ID NO: 94.

A second hairpin RNA construct was prepared encoding a hairpin RNA targeting the same 500 nucleotide region and having the same structure, except that 97 cytosine nucleotides (C) of the sense sequence were replaced with thymine nucleotides (T5-T7). When the chimeric DNA is transcribed and the G: U substituted hpRNA is self-annealed, this provides nucleotide base pairing of G: U base pairs in 97/500 ═ 19.4% of the dsRNA region. The nucleotide sequence of the chimeric DNA encoding the hpRNA modified by G is provided as SEQ ID NO: 95. in addition, chimeric DNA was prepared encoding ledRNA targeting the same region of the DDM1 gene of brassica napus (b.napus). The nucleotide sequence of the chimeric DNA encoding ledRNA is provided as SEQ ID NO: 96.

to produce RNA by in vitro transcription, DNA preparations were cut with the restriction enzyme HincII, which cuts immediately after the coding region, transcribed in vitro with RNA polymerase T7, the RNA purified and then concentrated in aqueous buffer. Endogenous DDMl transcripts in brassica napus (b.napus) cotyledons were targeted using ledRNA. Cotyledons of 5-day-old seedlings aseptically grown on tissue culture medium were carefully excised and placed in petri dishes containing 2ml of Ms liquid medium (containing 2% (w/v) sucrose) and 113 μ g of ledna or 100ul of aqueous buffer as controls. The MS liquid medium used for the treatment contained Silwett-77, a surfactant (0.5. mu.l in 60 ml). The petri dish was incubated with gentle shaking on a shaker to allow the cotyledons to soak in the ledRNA-containing solution. Samples were harvested 5 and 7 hours after application of ledRNA. In parallel experiments, the upper surface of cotyledons was coated with 10. mu.g of ledRNA or buffer and incubated on wet tissue paper. Samples were collected 7 hours after application of ledRNA.

Furthermore, to target the endogenous transcript of DDM1 in the reproductive tissues of brassica napus (b.napus), brassica oleracea, brassica napus flower buds were exposed to ledRNA in the presence or absence of aliquots of the AGL1 cell suspension of the Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain, i.e. live AGL1 cells. Aqueous buffer with or without AGL1 cells was used as the respective control. AGL1 was grown in 10ml LB broth containing 25mg/ml rifampicin (rifampicin) at 28 ℃ for 2 days. Cells were harvested by centrifugation at 3000rpm for 5 minutes. The cell pellets were washed and the cells were resuspended in 2ml liquid MS medium. The flower buds were incubated in Petri dishes containing 2ml of MS broth (including 0.5. mu.l of Silwett-77 in 50ml of MS broth) and 62. mu.g of ledRNA or 62. mu.g + 50. mu.l of AGL1 culture. As a control, 50. mu.l buffer or 50. mu.l buffer + 50. mu.l AGL1 culture was used. The samples were incubated for 7 hours on a shaker with gentle shaking. Three biological replicates were used for each treatment.

Treated and control cotyledons and flower buds were washed twice in sterile distilled water, surface water was removed using tissue paper and snap frozen with liquid nitrogen. RNA was isolated from treated and control tissues, genomic DNA was removed by DNase treatment and quantified. First strand cDNA was synthesized using equal amounts of total RNA from the ledRNA treated samples and their corresponding controls. The expression of DDMl was detected using real-time fluorescent quantitative PCR (qRT-PCR method).

In the treated cotyledons soaked with ledRNA, DDM1 transcript abundance was reduced by about 83-86% at 5 hours and by 91% at 7 hours compared to the control. Similarly, a reduction of approximately 78-85% in DDMl mRNA levels was observed in cotyledons coated with ledRNA compared to controls. In the absence of Agrobacterium cells, no differences in abundance of DDMl mRNA were detected in flower buds treated with ledRNA compared to controls. However, a reduction of about 60-75% of the DDMl transcript levels was observed in flower buds treated with ledRNA in the presence of Agrobacterium, compared to their corresponding controls. When the control without Agrobacterium (Agrobacterium) was compared to the control with Agrobacterium (Agrobacterium), no significant difference in DDMl transcript levels was detected, indicating that the Agrobacterium (Agrobacterium) cells themselves did not cause a reduction in DDMl transcripts. Taken together, these results indicate that ledRNA is able to reduce endogenous DDMl transcript levels in cotyledons and flower buds, while live Agrobacterium cells appear to promote entry of ledRNA into flower buds. This accessibility of ledRNA can also be achieved by physical methods such as puncturing the outer layer of the flower buds, centrifugation or vacuum infiltration, or a combination of these methods.

Certain Arabidopsis thaliana (Arabidopsis thaliana) mutants, such as the zip4 mutant, lack meiotic crossing, resulting in erroneous separation of chromosomal homologs and thus reduced fertility and resulting in shorter siliques (fruits) that can be visually distinguished from wild-type siliques. Reducing the expression of the FANCM gene can reverse the phenotype of the zip4 mutant.

The nucleotide sequence of the FANCM gene of Arabidopsis thaliana (A. thaliana) is provided by accession number NM-001333162 (SEQ ID NO: 97). Designing and making hairpin RNA constructs targeting a 500 nucleotide region of a gene corresponding to SEQ ID NO: nucleotide number 97 and 853-1352. The sequence of elements of the construct is promoter-sense-loop sequence, comprising intron-antisense sequence-transcription terminator/polyadenylation region of Hellsgate vector. The nucleotide sequence of the chimeric DNA encoding hpRNA is provided as SEQ ID NO: 98. a second hairpin RNA construct was prepared encoding a similar hairpin RNA targeting the same 500 nucleotide region except that 102 cytosine nucleotides (C) of the sense sequence were replaced with thymine nucleotides (T5-T7). When the chimeric DNA is transcribed and thus the G: U substituted hpRNA is self-annealed, this provides nucleotide base pairing of 102/500 ═ 20.4% of the dsRNA region to G: U base pairs. The nucleotide sequence of the chimeric DNA encoding the hpRNA modified by G is provided as SEQ ID NO: 99. in addition, chimeric DNA encoding ledRNA targeting the same region of the FANCM gene of arabidopsis thaliana (a. thaliana) was prepared. The nucleotide sequence of the chimeric DNA encoding ledRNA is provided as SEQ ID NO: 100.

Napus (b. napus) has the FANCM gene on each of its a and C subgenomes, designated BnaA05g18180D-1 and BnaC05g 27760D-1. The nucleotide sequence of one of the FANCM genes of brassica napus (b.napus) is provided by accession number XM _022719486.1 as SEQ ID NO: 101). Chimeric DNA encoding hairpin RNA was designed and targeted to the 503 nucleotide region of the gene, corresponding to SEQ ID NO: 101 nucleotide 2847-3349. The sequence of elements of the construct is promoter-sense-loop sequence, comprising intron-antisense sequence-transcription terminator/polyadenylation region of Hellsgate vector. The nucleotide sequence of the chimeric DNA encoding hpRNA is provided as SEQ ID NO: 102. a second hairpin RNA construct was prepared encoding a similar hairpin RNA targeting the same 503 nucleotide region except that 107 cytosine nucleotides (C) of the sense sequence were replaced with thymine nucleotides (T5-T7). When the chimeric DNA is transcribed and the G: U substituted hpRNA is self-annealed, this provides nucleotide base pairing of G: U base pairs in 107/500 ═ 21.4% of the dsRNA region. The nucleotide sequence of the chimeric DNA encoding the hpRNA modified by G is provided as SEQ ID NO: 103. in addition, chimeric DNA encoding ledRNA targeting the same region of the FANCM gene of brassica napus (b.napus) was prepared. The nucleotide sequence of the chimeric DNA encoding ledRNA is provided as SEQ ID NO: 104.

To produce RNA by in vitro transcription, DNA preparations were cut with the restriction enzyme HincII, which cuts immediately after the coding region, transcribed in vitro with RNA polymerase T7, the RNA purified and then concentrated in aqueous buffer. ledRNA was used together with Agrobacterium tumefaciens AGL1 to target the FANCM transcript in the pre-meiotic shoots of the zip4 mutant of Arabidopsis thaliana (A. thaliana). Due to the reduced hybrid formation, the siliques of the zip4 mutant were shorter and easier to visualize than the wild type siliques, thus resulting in reduced fertility. Inhibition of FANCM in the zip4 mutant has been shown to restore fertility and restore silique length.

Arabidopsis thaliana (a. thaliana) zip4 inflorescences containing pre-meiotic shoots were contacted with ledRNA targeting FANCM and AGL1 or buffer with AGL1 as a control, in the presence of a surfactant in each case, Silwett-77 in this case. Once seed set-up is complete, siliques developing from pre-meiotic shoots are excised to determine seed number. Of the 15 siliques from the ledRNA-treated sample, two siliques showed 10 seeds, one silique had 9 seeds, while the number of seeds in the control silique was 3-6. These results indicate that the observed increase in seed number is due to ledRNA inhibition of FANCM transcript levels, resulting in an increase in meiotic crossovers and increased fertility.

Example 17: RNA constructs for fungal disease resistance

LedR against Mlo genes of barley and wheatNA

Fungal diseases, powdery mildew of cereals are caused by ascorbyl barley powdery mildew (Blumeria graminis f.sp.hordei) in barley and related wheat powdery mildew (Blumeria graminis f.sp.tritici) in wheat. Erysiphe graminis (b.graminis) is an obligate biotrophic fungal pathogen of the order Erysiphales (Glawe, 2008) that requires a plant host for propagation, involving a tight interaction between the fungus and the host cell to render the fungus vegetative from the plant. After the fungal ascospores or conidia contact the surface, the fungus initially infests the leaves, leaf sheaths, or epidermal layers of the ear. The leaves remain green and active for a period of time after infection, then become powdered, the mycelial mass grows, the leaves gradually change color and die. As the disease progresses, the fungal mycelium may develop tiny black spots, which are the sexual fruiting bodies of the fungus. Powdery mildew is distributed worldwide and is most harmful in cold, humid climates. The disease affects grain yield mainly by reducing the number of head inflorescences (heads) and reducing kernel size and weight. Currently, disease control is carried out by spraying the crops with fungicides that need to be applied frequently in cold and humid conditions, which is costly, or by growth-resistant cultivars. In addition, australian wheat powdery mildew has developed resistance.

The Mlo gene of barley and wheat encodes a Mlo polypeptide which confers susceptibility to powdery mildew (B.graminis) of the Poaceae family by an unknown mechanism. There are a number of closely related Mlo proteins encoded by the plant-unique family of Mlo genes. Each gene encodes 7 transmembrane domain proteins with unknown biochemical activity localized in the plasma membrane. Notably, only specific Mlo genes within this family are capable of acting as powdery mildew susceptibility genes and these genes encode polypeptides with conserved motifs within the cytoplasmic C-terminal domain of the Mlo protein. The mechanism of action of Mlo polypeptides as susceptibility factors for powdery mildew is not known. The emergence of native wheat mlo mutants has not been reported, probably due to the polyploid nature of wheat. However, artificially generated mlo mutants show some resistance to the disease, but often show significantly reduced grain yield or premature leaf senescence (Wang et al; Acevedo-Garcia et al, 2017).

Hexaploid wheat has 3 Mlo gene homologous sequences, designated TaMlo-A1, TaMlo-B1 and TaMlo-D1 on chromosomes 5AL, 4BL, and 4DL, respectively (Elliott et AL, 2002). The nucleotide sequences of the cdnas corresponding to these genes can be obtained under the following accession numbers: TaMlo-A1, AF361933 and AX 063298; TaMlo-B1, AF361932, AX063294 and AF 384145; and TaMlo-D1, AX 063296. A. B, D the nucleotide sequence of the gene on the genome is approximately 95-97% and 98% identical to the amino acid sequence of the encoded polypeptide, respectively. All three genes are expressed in plant leaves, and the expression quantity is increased along with the growth and maturity of plants. Thus, the present inventors designed and prepared ledRNA constructs capable of reducing the expression of all three genes, taking advantage of the degree of sequence identity between the genes and targeting gene regions with high sequence conservation.

Chimeric DNA encoding ledRNA constructs targeting all three of the genes TaMlo-A1, TaMlo-B1 and TaMlo-D1 was prepared. The above design principle for ledRNA was used to prepare genetic constructs in which the split sequence (split sequence) is the antisense sequence and the continuous sequence is the sense sequence (FIG. 1A).

Selecting a 500bp nucleotide sequence of the TaMlo target gene corresponding to the nucleotide sequence as set forth in SEQ ID NO: 1403-1569 of 136, and 916-1248. The length of the dsRNA region of each ledRNA is 500 bp; the sense sequence in the dsRNA region is an uninterrupted continuous sequence, e.g., corresponding to a sequence identical to SEQ ID NO: 1403-1569 of 136, and 916-1248. The nucleotide sequence encoding the ledRNA is provided herein as SEQ ID NO: 137.

LedRNA was prepared by in vitro transcription with T7RNA polymerase, purified and suspended in buffer. 10 μ g of gledrRNA/leaf was applied to the leaf area of wheat plants at the Zadoffs 23 growth stage using a paint brush. As a control, some leaves were treated with buffer only simulation. Treated and control leaf samples were harvested and RNA extracted. QPCR analysis of extracted RNA showed that TaMlo mRNA levels were reduced by 95.7% as a combination of three TaMlo mrnas. Plants at the growth stage of Z73 were also treated and tested. By QPCR they showed a 91% reduction in TaMlo gene expression relative to control leaf samples. The reduction in expression of the TaMlo gene observed in the treated leaf regions was specific for the treated regions-there was no reduction in TaMlo mRNA levels in the distal untreated portions of the leaves.

In barley mlo mutants, increased expression of various disease defense-related genes was observed. Thus, the level of defense-related genes encoding PR4, PR10, β -1, 3-glucanase, chitinase, germ cells and ADP-ribosylation factors in ledRNA-treated wheat leaves was determined by QPCR. None of these genes significantly changed in expression levels in the treated leaf regions relative to the control leaf regions.

To test the ability of ledRNA to increase disease resistance by reducing Mlo gene expression, spores of Erysiphe cichoracearum were applied to treated and untreated regions of the leaves. Leaves were isolated from wheat plants, as previously treated with ledRNA and kept on medium (50 mg benzimidazole and 1g agar per liter of water) to prevent leaf senescence under light. And inoculating powdery mildew spores 24h later, wherein the disease course is 5-24 d. The treated leaves showed little to no fungal mycelium growth and no leaf discoloration relative to control leaves that did not receive ledRNA, which showed extensive mycelium growth surrounded by discolored regions.

In further experiments, lower levels of ledRNA were administered to identify the minimum level of ledRNA that was effective. In the current formulations, the application of RNA at concentrations as low as 200ng/μ l (total 2 μ g per leaf) showed significant inhibition of powdery mildew lesions, indicating that the amount of inhibitory RNA can be greatly reduced while still providing inhibition of the growth and development of the fungus. In addition, leaves were inoculated 1, 2, 4, 7 and 14 days after ledRNA treatment to see how long the protection remained. Effective silencing of the endogenous gene was observed throughout the time period from the first time point at 24 hours after treatment to the last time point at 14 days after treatment, at which time the endogenous gene still showed a 91% reduction in expression. The whole plant will also be sprayed with the ledRNA formulation and tested for disease resistance after inoculation with the fungal disease agent.

Targeting the VvMLO gene of grape (vitas vinifera)LedRNA

The MLO genes of grapes (Vitis vinifera) and Vitis vinifera (Vitis pseudoreticulata) encode MLO polypeptides that confer susceptibility to fungal powdery mildew caused by ascomycete Erysiphe necator. Erysiphe necator (e.necator) is an obligate biotrophic fungal pathogen that requires a plant host for propagation, involving close interactions between the fungus and the host cell to render the fungus vegetative from the plant. There are a number of closely related MLO proteins encoded by gene families, all of which are plant-unique and encode 7 transmembrane domain proteins of unknown biochemical activity that are localized in the plasma membrane. Notably, only specific MLO genes within this family are capable of serving as powdery mildew susceptibility genes, and these genes encode polypeptides with conserved motifs within the cytoplasmic C-terminal domain of the Mlo protein. The mechanism of action of MLO polypeptides as susceptibility factors for powdery mildew is not clear.

Three different but related MLO genes targeting Vitis (Vitis) species, namely the ledRNA constructs of VvMLO3, VvMLO4 and VvMLO17 (named according to Feechan et al, Functional Plant Biology,2008,35: 1255-. For the first, e.g., 860 nucleotide sequence of the VvML03 target gene was selected, which corresponds to SEQ ID NO: nucleotide 297 of 138 and 1156. Chimeric DNAs encoding three ledRNA constructs targeting the VvMLO3, VvMLO4 and VvMLO17 genes were prepared. The above design principle for ledRNA was used to prepare genetic constructs in which the split sequence was the antisense sequence and the continuous sequence was the sense sequence (FIG. 1A). The dsRNA region of each ledRNA is 600 bp; the sense sequence in the dsRNA region is an uninterrupted continuous sequence, e.g., a sequence corresponding to SEQ ID NO: nucleotide 427 of 138 and 1156. The nucleotide sequence encoding one ledRNA is provided herein as SEQ ID NO: 139.

ledRNA is prepared by in vitro transcription and applied to the leaves of the grape (Vitis vinifera) plant, Cabernet Sauvignon variety, either alone or as a mixture of all three. Subsequently, spores of powdery mildew are applied to the treated and untreated areas of the leaves. Reduced levels of target mRNA were observed using quantitative RT-PCR. Disease progression was followed over time. Significant down-regulation of VvMlo4 was observed by applying a solution of ledRNA targeting VvMlo3, VvMlo4 or VvMlo11 at 1 μ g/ml.

LedRNA targeting fungal genes

The coding region of the Cyp51 gene for the fungal pathogen Rhizoctonia solani, for which the LedRNA construct is designed, is essential for the synthesis of ergosterol and for the survival and growth of fungi. Genetic constructs were made using the design principle of ledRNA described above, in which the split sequence was an antisense sequence and the continuous sequence was a sense sequence (FIG. 1A). A single ledRNA construct was designed to target two genes from r.solani, where the dsRNA region of ledRNA contained 350bp from each gene; the sense sequence in the dsRNA region is an uninterrupted, continuous sequence, e.g., a sequence corresponding to SEQ ID NO: 140 and nucleotide 884-1233 of SEQ ID NO: nucleotide 174 of 141 and 523. The nucleotide sequence provided herein that encodes one of the ledrnas is SEQ ID NO: 142. ledRNA was prepared by in vitro transcription and applied to the culture medium at a concentration of 5. mu.g per 100. mu.l of culture with an inoculum of R.solani mycelium. Fungal growth was measured at zero and every day of the following week by reading the optical density of the culture at 600 nm. The growth of r.solani in the culture containing ledrsccyp 51 was significantly lower than the control culture containing RNA buffer or control ledGFP (where there was no corresponding target in r.solani).

A ledRNA-encoding construct was also designed and the coding region for the CesA3 cellulose synthase gene in Phytophthora cinnammomi isolate 94.48 was prepared. Genetic constructs were made using the design principle of ledRNA described above, in which the split sequence was an antisense sequence and the continuous sequence was a sense sequence (FIG. 1A). The ledRNA construct was designed to target the CesA3 gene of Phytophthora cinnamomi, wherein the dsRNA region of ledRNA contained 500bp from the coding region of the gene; the sense sequence in the dsRNA region is an uninterrupted, continuous sequence, e.g., a sequence corresponding to SEQ ID NO: nucleotide 884-1233 of 143. The nucleotide sequence provided herein that encodes one of the ledrnas is SEQ ID NO: 144. ledRNA was transcribed in vitro and applied to the medium at a rate of 3. mu.g per 100. mu.l of culture. A large loss of directional mycelium growth was observed in cultures treated with ledRNA targeting PcCesA3 compared to mock treated (RNA buffer only) or ledrfp treated cultures. The loss of directional growth and the resulting amorphous globular growth pattern reminds cells with disrupted cell wall biosynthesis and is therefore consistent with silencing of the PcCesA3 gene.

Example 18: RNA constructs targeting other genes in plants

LedRNA targeting the Tor genes of Arabidopsis (A. thaliana) and Nicotiana benthamiana (N. benthamiana)

The rapamycin Target (TOR) gene encodes a serine-threonine protein kinase polypeptide that controls many cellular functions in eukaryotic cells, for example, in response to various hormones, stress, and nutrient availability. It is known to be the primary regulator of the regulation of the translation machinery to optimize cellular resources for growth (Abraham, 2002). At least in animals and yeast, TOR polypeptides are inactivated by the antifungal drug rapamycin, resulting in their being designated as rapamycin targets. In plants, TOR is essential for embryonic development of developing seeds, as shown by the lethality of homozygous mutants in TOR (mahfuz et al, 2006), and is involved in the coupling of growth cues to cellular metabolism. Down-regulation of TOR gene expression is thought to result in increased fatty acid synthesis, resulting in increased lipid content in plant tissues.

Using the principle of designing ledRNA whose isolated sequence is a sense sequence and whose contiguous sequence is an antisense sequence, a ledRNA construct targeting the TOR gene of Nicotiana benthamiana (Nicotiana benthamiana) was designed and prepared, and the nucleotide sequence of the cDNA protein coding region was provided as SEQ ID NO: 105 (fig. 1B). The target region is 603 nucleotides in length, corresponding to SEQ ID NO: nucleotide 2595 and 3197 of 105. The dsRNA region of ledRNA is 603bp in length; the antisense sequence in the dsRNA region is an uninterrupted continuous sequence corresponding to SEQ ID NO: nucleotide 2595 of 105 and the complement of 3197. The nucleotide sequence encoding the ledRNA is provided herein as SEQ ID NO: 106. a DNA preparation of a genetic construct encoding a ledRNA construct is cleaved with the restriction enzyme MlyI which cleaves DNA immediately after the coding region, RNA is transcribed and purified in vitro with RNA polymerase SP6 and then concentrated in aqueous buffer. A sample of ledRNA was applied to the upper surface of nicotiana benthamiana (n. After 2 and 4 days, treated leaf samples were harvested, dried and determined for total fatty acid content by quantitative Gas Chromatography (GC). Leaf samples treated with TOR ledRNA showed an increase in Total Fatty Acid (TFA) content from 2.5-3.0% (TFA weight/dry weight) observed in the control (untreated) sample to 3.5-4.0% of the ledRNA-treated sample. This represents a 17% to 60% increase in TFA content relative to the control, indicating a decrease in TOR gene expression in ledRNA treated tissues.

LedRNA targeting barley (H.vulgare) ALS gene

The acetolactate synthase (ALS) gene encodes an enzyme (EC 2.2.1.6) found in plants and microorganisms, which catalyzes the first step in the synthesis of the branched-chain amino acids leucine, valine and isoleucine. ALS enzymes catalyze the conversion of pyruvate to acetolactate, which is then further converted to branched chain amino acids by other enzymes. ALS inhibitors are used as herbicides, for example sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyloxybenzoate and sulfonylaminocarbonyltriazolinone herbicides.

To test whether ledRNA could reduce ALS gene expression by exogenous delivery of RNA to plants, genetic constructs encoding ledRNA were designed and prepared that targeted the ALS gene in barley (Hordeum vulgare). Barley (h.vulgare) ALS gene sequences are provided herein as SEQ ID NO: 107 (accession number LT 601589). The design principle for ledRNA was used to prepare genetic constructs in which the split sequence was the sense sequence and the contiguous sequence was the antisense sequence (FIG. 1B). The target region is 606 nucleotides in length, corresponding to SEQ ID NO: nucleotide 1333 of 107 and 1938. The dsRNA region of ledRNA is 606bp in length; the antisense sequence in the dsRNA region is an uninterrupted continuous sequence corresponding to SEQ ID NO: nucleotide 1333 of nucleotide 107 and the complement of 1938. The nucleotide sequence encoding the ledRNA is provided herein as SEQ ID NO: 108. the coding region is transcribed in vitro under the control of the SP6 RNA polymerase promoter.

The genetic construct encoding ledRNA was digested with the restriction enzyme MlyI, cleaved downstream of the ledRNA coding region, and transcribed in vitro with RNA polymerase SP6 according to the instructions of the transcription kit. RNA was applied to the upper surface of leaves of barley plants. RNA was extracted from the treated leaf samples (after 24 hours). The RNA samples were subjected to quantitative reverse transcription-PCR (QPCR) assays. The results show that ALS mRNA levels are reduced in ledRNA treated tissues. (Total RNA from treated and untreated plants was extracted, DNase treated, quantified and reverse transcribed for 2. mu.g using primer CTTGCCAATCTCAGCTGGATC. Forward primer TAAGGCTGACCTGTTGCTTGC and reverse primer CTTGCCAATCTCAGCTGGATC were used as templates for quantitative PCR. ALS mRNA expression was normalized to that of the barley (Hordenum chinensis) isolate H1 lycopene-cyclase gene. ALS expression was reduced by 82% in LED treated plants.

ledRNA targeting the NCED1 and NCED2 genes of wheat and barley

In plants, the plant hormone abscisic acid (ABA) is synthesized from carotenoid precursors, the first key step in its synthetic pathway being catalyzed by 9-cis epoxycarotenoid dioxygenase (NCED), the enzyme which cleaves 9-cis lutein to lutein (Schwartz et al, 1997). The hormone ABA is known to promote dormancy in seeds (Millar et al, 2006) and to be involved in other processes, such as stress response. It is thought that increasing the expression of the NCED gene increases ABA concentration, thereby promoting dormancy. Two NCED isozymes coded by different homologous genes exist in crops of wheat, barley, etc., and are named NCED1 and NCED2 respectively.

For the breakdown of ABA, the enzyme ABA-8-hydroxylase (ABA8OH-2, also known as CYP707a2) hydroxylates ABA, a step in its catabolism, resulting in dormancy and disruption of seed germination.

ledRNA constructs targeting homologous genes encoding the corresponding homologues in barley (Hordeum vulgare) HvNCED1 (accession No. AK361999, SEQ ID NO: 109) or HvNCED2 (accession No. AB 239298; SEQ ID NO: 110) and wheat were designed for transgenic expression in barley and wheat plants. These constructs used highly conserved regions of the wheat and barley NCED1 and NCED2 genes, in which the wheat and barley nucleotide sequences were about 97% identical. The above design principle for ledRNA was used to prepare genetic constructs in which the split sequence was the antisense sequence and the continuous sequence was the sense sequence (FIG. 1A). The target region is 602 nucleotides in length, corresponding to SEQ ID NO: nucleotide 435 and 1035 of 109. The length of dsRNA region of LedRNA is 602 bp; the sense sequence in the dsRNA region is an uninterrupted continuous sequence corresponding to SEQ ID NO: 110, nucleotides 435-. Nucleotide sequences encoding NCED1 and NCED2 ledRNA are provided herein as SEQ ID NOs: 111 and 112.

In a similar manner, ledRNA constructs were prepared that target the ABA-OH-2 gene (accession number DQ145933, SEQ ID NO: 113) of wheat (T.aestivum) and barley (H.vulgare). The target region is 600 nucleotides in length, corresponding to SEQ ID NO: 113, nucleotide 639-1238. The length of dsRNA region of ledRNA is 600 bp; the sense sequence in the dsRNA region is an uninterrupted continuous sequence corresponding to SEQ ID NO: 113, nucleotide 639-1238. The nucleotide sequence of the chimeric DNA encoding ledRNA is provided as SEQ ID NO: 114.

The chimeric DNA encoding ledRNA is inserted into an expression vector under the control of the Ubi gene promoter, which is constitutively expressed in most tissues including developing seeds. The expression cassette is excised and inserted into a binary vector. These are used to produce transformed wheat plants.

Transgenic wheat plants were grown to maturity, seeds were obtained therefrom, and the reduced expression of the NCED or ABA-OH-2 gene and the effect on seed dormancy corresponding to the reduced gene expression were analyzed. A range of phenotypes for which the degree of dormancy is expected to vary. To modulate the extent of the altered phenotype, modified genetic constructs are generated for expressing ledRNAs having G: U base pairs in a double-stranded RNA region, particularly for ledRNAs in which 15-25% of the nucleotides in the double-stranded region of the ledRNA are involved in the G: U base pairs, as a percentage of the total number of nucleotides in the double-stranded region.

ledRNA targeting Arabidopsis thaliana (A. thaliana) EIN2 gene

As described in example 10, the EIN2 gene of arabidopsis thaliana (a. thaliana) encodes a receptor protein involved in ethylene sensing. When germinated on ACC, EIN2 mutant seedlings showed hypocotyl elongation relative to wild type seedlings. Since the gene is expressed in seedlings shortly after seed germination, delivery of ledRNA by transgenic means, relative to exogenous delivery of pre-formed RNA, is considered the most suitable way to test the degree of down-regulation of EIN 2.

A ledRNA construct targeting the Arabidopsis thaliana (Arabidopsis thaliana) EIN2 gene (SEQ ID NO: 115) was designed, which targets a 400 nucleotide region of the mRNA of the target gene. Constructs were made by inserting the sequence encoding ledRNA (SEQ ID NO: 116) into a vector containing the 35S promoter to express ledRNA in Arabidopsis (A. thaliana) plants. Transgenic arabidopsis thaliana (a. thaliana) plants were generated and tested for reduction of EIN2 gene expression by QPCR and hypocotyl length determination in the presence of ACC. Decreased expression levels of EIN2 and increased hypocotyl length were observed in some transgenic lines of plants.

ledRNA targeting Arabidopsis (A. thaliana) CHS gene

The chalcone synthase (CHS) gene in plants encodes an enzyme that catalyzes the conversion of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone, the first guaranteed enzyme in flavonoid biosynthesis. Flavonoids are a class of organic compounds found primarily in plants, involved in defense mechanisms and stress tolerance.

A ledRNA construct targeting the Arabidopsis (Arabidopsis thaliana) CHS gene (SEQ ID NO: 117) was designed that targets 338 nucleotide regions of the target gene mRNA. Constructs were made by inserting the DNA sequence encoding ledRNA (SEQ ID NO: 118) into a vector containing the 35S promoter to express ledRNA in Arabidopsis (A. thaliana) plants. Transgenic arabidopsis (a. thaliana) plants were produced by transformation with the genetic construct in a binary vector and tested for reduction in CHS gene expression and reduction in flavonoid production by QPCR. In some transgenic lines of plants, for example in the seed coat of transgenic seeds, reduced levels of CHS expression and reduced levels of flavonoids are observed.

ledRNA targeting the LanR gene of Lupinus angustifolia (Lupinus angustifolius)

The LanR gene of lupin angustifolia (Lupinus angustifolius L.) encodes a polypeptide related to the sequence of the tobacco N gene conferring resistance to Tobacco Mosaic Virus (TMV).

A chimeric DNA (accession No.: XM-019604347, SEQ ID NO: 119) for generating a ledRNA molecule targeting the LanR gene of Lupinus angustifolia (L.angustifolius) was designed and prepared. The above design principle for ledRNA was used to prepare genetic constructs in which the split sequence was the antisense sequence and the continuous sequence was the sense sequence (FIG. 1A). The nucleotide sequence encoding the ledRNA is provided herein as SEQ ID NO: 120. LedRNA was produced by in vitro transcription, purified and concentrated, and aliquots of RNA were applied to leaves of plants containing the LanR gene lupin angustifolius (l. Virus samples were applied to treated and untreated plants and disease symptoms were compared after a few days.

LedRNAi targeting VRN2 gene endows wheat with vernalization reactivity

Wheat VRN2A, VRN2B and VRN2D candidate genes in TGACv1_ scaffold _374416_5AL, TGACv1_ scaffold _320642_4BL and TGACv1_ scaffold _342601_4DL identified as homologs of the wheat zct 1 gene (Genbank accession number AAS58481.1) were identified as targets for designing ledtrnai constructs. The 309bp region of the VRN2B gene was used for the dsRNA region of the ledRNAi construct designated LedTaVRN 2. Led RNAi was generated by in vitro transcription using T7 RNA polymerase and diluted in water. The solution is used for impregnating wheat grains for germination at 4 ℃ for 3 days. Seeds of vernalization sensitive wheat variety CSIRO W7 were used. The treated seeds were planted in soil and the resulting plants were observed over time for a shift from vegetative growth to flower development. The time of flowering is recorded, which is indicated by the emergence of the ear from the sheath of the inflorescence, and the number of leaves on the stem at the time of flowering is recorded. Seed-derived plants incubated with LedTaVRN2 flowering an average of at least 17 days earlier than seed-derived plants incubated with buffer or non-specific dsRNA control alone. Furthermore, at flowering time, seed derived plants incubated with LedTaVRN2 had on average a 2.3 reduction in leaf number on the main stem, indicating that nodes dedicated to leaf production were reduced and more nodes dedicated to flowers/grains.

Example 19: RNA constructs targeting insect genes

Introduction to the design reside in

Aphids are sap-sucking insects which cause substantial and sometimes severe damage to plants directly by feeding the plant sap and, in some cases, indirectly by transmitting various viruses which cause diseases in plants. While in some cases Bt toxins are effective in protecting crop plants from chewing insects, it is not generally effective against sap-sucking insects. The use of plant cultivars containing resistance genes may be an effective method for controlling aphids, however, most resistance genes are highly specific for certain aphid species or biotypes and resistance is often over-developed as new biotypes evolve rapidly through genetic or epigenetic changes. Furthermore, resistance genes are not accessible in many crops or may not be present for certain common aphid species, such as the green peach aphid, which infests a broad host species. At present, aphids are mainly prevented and treated by frequently applying pesticides causing aphid resistance. For example, only one pesticide mode of action group in australia is still effective against myzus persicae, as myzus persicae has managed to acquire resistance to all other registered insecticides.

RNAi-mediated gene silencing has been shown in several studies to be useful as a research tool in many aphid species, for a review see Scott et al, 2013; yu et al, 2016, but have not been shown to be effective in protecting plants from aphid infestations. In these studies, dsRNA targeting key genes involved in aphid growth and development, infestation, or feeding processes were delivered by direct injection into aphids or by feeding the aphids on artificial diets containing dsRNA.

To test the potential of modified RNAi molecules, such as the ledRNA molecules described herein, to combat sap-sucking insects, the inventors selected the green peach aphid (Myzus persicae) as a model for sap-sucking insects for several reasons. First, myzus persicae is a polyphagic insect that infects host plant species widely throughout the world, including major food and horticultural crops. Second, myzus persicae is a major cause of transmission of some destructive viruses, such as beet west yellow virus, which has posed a serious hazard in some rape-growing areas. First 2 aphid genes were selected as down-regulated target genes, 1 encoding a key effector protein (CO02) and the second encoding an activated protein kinase C receptor (Rack-1). The C002 protein is an aphid salivary gland protein, which is essential for aphids to feed their host plants (Mutti et al, 2006; Mutti et al, 2008). Rack1 is an intracellular receptor that binds to activated protein kinase C, an enzyme primarily involved in the signal transduction cascade (McCahill et al, 2002; Seddas et al, 2004). MpC002 is expressed predominantly in aphid salivary glands, and MpRack1 is expressed predominantly in the intestine. In previous studies, the use of RNAi by direct injection or artificial diet resulted in the death of several aphid species tested (Pitino et al, 2011; Pitino and Hogenhout, 2012; Yu et al, 2016).

Materials and methods: aphid cultures and plant material

Peach aphids (Myzus persicae) were collected in the west australia. Aphids were raised on radish plants (Raphanus sativus L.) in a feeding room under ambient light prior to each experiment. Aphids were transferred to experimental artificial diet cages with fine paintbrushes.

The composition of the artificial diet used for aphid feeding was the same as described in Dadd and Mittler (1966). The device for artificial diet of aphids uses plastic tubes with a diameter of 1cm and a height of 1 cm. 100 μ l of artificial aphid diet with or without ledRNA was enclosed between two paraffin films to create a diet sachet. At the top of the pouch, there was a chamber for aphids to move around and feed from the diet by piercing their stylets through the top layer of the stretched parafilm. 8 nymphs, one or two, were gently transferred to the aphid chamber using a fine paint brush. The experiment was carried out in a growth chamber at 20 ℃.

The tobacco leaves and radish leaves used in one experiment were collected from plants grown in soil at 22 ℃ under a 16 hour light/8 hour dark cycle. In experiments involving the cutting of radish leaves, small radish leaves (2-4 cm) attached to a fragment (about 2cm in length) of the stem were cut ²). To keep the leaves fresh, the stems were inserted into a medium containing 1.5 g of Ba per 100 ml of water in a 5cm diameter petri dishcto agar and 1.16 g Aquasol. Aphids were transferred to the leaves with a fine paint brush. Dishes with leaves and aphids were kept in growth cabinets at 20 ℃ under a 16 hour light/8 hour dark cycle.

Double-stranded RNA (dsrna) is prepared by in vitro RNA transcription of a DNA template comprising one or more T7 promoters and T7 RNA polymerase using standard methods.

MpC002 and MpRack-1 genes and LedRNA constructs

The peach aphid MpC002 and MpRack-1 genes tested as target genes were the same as described by Pitino et al (2011; 2012). The DNA sequences of two genes, MpC002(> MYZPE13164_0_ v1.0_000024990.1|894nt) and MpRack-1(> MYZPE13164_0_ v1.0_000198310.1|960nt), were obtained from the NCBI website. The cDNA sequences of these two genes are provided herein as SEQ ID NOs: 123 and 124. The ledRNA constructs were designed in the same manner as described in the previous examples. The DNA sequence encoding the ledRNA molecule is provided herein as SEQ ID NO: 125 and 126, which serve as transcription templates for ledRNA synthesis. Vector DNA encoding ledRNA molecules targeting MpC002 and MpRack-1 genes was introduced into e.coli strain DH5 α to prepare plasmid DNA for RNA transcription in vitro, and into e.coli strain HT115 for transcription in vivo (in bacteria).

Efficacy of ledRNA molecules on reducing aphid Performance

To examine whether ledRNA targeting MpC002 or the MpRack-1 gene affected aphid performance, each ledRNA was delivered to aphids via the artificial diet route as described in example 1. In each experiment, 10 biological replicates were set up; each biological copy had 8 1-2 years old myzus persicae nymphs. Controls in each experiment used an equal concentration of unrelated ledRNA, ledGFP.

At a lower concentration of 50 ng/. mu.l of each ledRNA molecule, aphid survival after feeding from an artificial diet containing MpC002 or MpRack-1ledRNA did not differ significantly from control ledGFP. However, ledRNA targeting MpC002 gene significantly (P <0.05) reduced the reproduction rate of Myzus persicae (FIG. 37). The average number of nymphs produced per adult aphid was reduced by about 75% compared to the number of nymphs produced by adult aphids maintained on a control diet with control ledRNA. At higher concentrations of 200 ng/. mu.l, ledRNA targeting MpC002 or MpRack-1 increased aphid mortality (FIG. 37B). After 24 hours, a reduction in aphid survival was also observed in diets including MpC002 or MpRack-1ledRNAs and continued for 5 days of the experiment. The results show that the use of ledRNA targeting the essential gene of aphid can cause death of aphid and reduce reproduction of aphid. The potency of each ledRNA was compared to a double stranded RNA molecule targeting the same region of the target gene (dsRNAi), which comprises separate but annealed sense and antisense RNA strands.

Uptake of ledRNA molecules by aphids

To follow the uptake and distribution of ledRNA within aphids, the ledRNA targeting either MpC002 or MpRack-1 gene was labeled with Cya3 (cyanine dye labeled nucleotide triphosphate) during the synthesis described in example 1. Cyr3 marker has been reported to have no effect on the biological function of conventional dsRNA molecules and thus can be used as a marker for fluorescence detection. Aphids that had been fed labeled ledRNA were examined using confocal microscopy using a Leica EL6000 microsystem instrument. Cyr3 labeled ledRNA targeting MpC002 or MpRack-1 could be detected within hours of artificial diet feeding, subsequently in the reproductive system, and even in the neonatal nymph, which is the offspring of the adult already reared. The results show that the gene which plays a key role in the function or reproduction of the digestive system by aphid can become an effective target of ledRNA molecule by feeding.

led RNA stability

To test the stability of ledRNA in diet and recovered from aphid feeding, RNA was recovered from artificial diet and aphid honeydew after feeding a diet containing the tagged ledRNA molecule. The RNA samples were electrophoresed on a gel and examined by fluorescence detection. The ledMpC002 RNA clearly showed a single product of approximately 700bp on an agarose gel before feeding. RNA recovered from the artificial diet showed RNA smears of size 100-700bp, indicating some degradation but still mostly intact after 25 days of diet exposure at room temperature. RNA recovered from the honeydew of the aphid showed fluorescence in the region of 350-700bp RNA and was therefore largely intact. Although some ledRNA was degraded, most of the ledRNA molecules remained intact for a considerable period of time in the artificial diet as well as in the aphid honeydew. This degree of stability of the ledRNA molecule should allow the ledRNA to be active and remain active when administered exogenously.

Uptake of marker ledRNA by plant leaves

Cy 3-labeled ledMpC002 RNA was applied to the upper surface of tobacco leaves to see if it could penetrate the leaf tissue. 10 microliter of Cy 3-labeled ledMpC002 (concentration 1. mu.g/. mu.l) was smeared in a circle having a diameter of 2cm and the area of application was marked with a black marker. Images of leaf fluorescence under 525nm excitation were captured over a 5 hour period using a Leica EL 6000 microsystem instrument, and stained tissue was compared to non-stained tissue. Within 1 hour after application, the Cy3 marker was clearly detectable in mesophyllic tissue, thus the Cy3 marker clearly penetrated the waxy cuticle layer on the leaf surface. The fluorescence level increased at 2 hours and remained until the 5 hour time point. It is unclear whether the ledRNA molecule enters the cell or the nucleus. However, since sap-sucking insects feed on particularly phloem sieve molecules from plant leaves and stems, the delivery of RNA into plant cells is not essential for aphid gene silencing. Experiments have shown that ledRNA molecules are found in plant tissues by topical application.

Local LedRNA uptake by aphids

Cy 3-labeled ledGFP RNA was spread over radish leaves to see if aphids could take up the topically applied ledRNA from the plants. Ten microliters of each Cy 3-labeled ledGFP (concentration 10. mu.g/. mu.l) was spread on small excised radish leaves (. about.2 cm) ²). Control leaves were coated with equal amounts of unlabeled ledGFP. The marked and control radish leaves were infested with eight aphids of different developmental stages. Images of leaf and aphid fluorescence were captured using the method described above for tobacco leaves. There was no detectable fluorescence in control leaves and aphids, whereas leaves coated with Cy 3-labeled ledGFP were highly fluorescent. Within 24 hours after feeding on leaves with Cy 3-labeled ledRNA, aphids showed strong fluorescence throughout the body, but were more pronounced in the intestine and legs than in other body parts. Experiments show that the aphidsThe ledRNA molecules can be taken up from the plant for topical application.

Screening of other aphid RNAi target genes

To identify more aphid target genes, a total of 16 aphid genes were evaluated for their suitability as targets of RNAi. The candidate gene selected is involved in development, reproduction, feeding or detoxification of aphids. Conventional dsrna (dsrnai) targeting each gene by containing sense and antisense sequences corresponding to regions of the target gene mRNA were supplemented into the aphid artificial diet at a concentration of 2 micrograms of RNA per microliter of diet. The effect on aphid survival and reproductive rate was used to determine the suitability of aphid RNAi target genes. Of the 16 genes studied, 9 showed a decrease in aphid survival and/or reproduction. In addition to MpC002 and MpRack-1, other suitable target genes are the genes encoding the following polypeptides and the types of functions they have in aphids: tubulin (accession number XM _022321900.1, cell structure), insulin-related peptide (XM _022313196.1, embryonic development), ATPase subunit V (XM _022312248.1, energy metabolism), notch kyphosis (XM _022313819.1, growth and development), ecdysone-triggering hormone (XM _022323100.1, development-ecdysis), short neuropeptide F (XM _022314068.1, nervous system) and leukocyte kinin (XM _022308286.1, water balance and food intake). For most genes, the effect of RNAi on aphid reproduction appears to be more robust and stronger than the effect on survival, i.e. greater on reproduction.

Trans-generation action of exogenous RNAi on aphids

To examine how long the effect of RNAi persists, artificial diet supplemented with dsRNAi targeting MpC002, MpRack-1, MpGhb, or supplemented with control dsGFP was fed on two-or three-year-old developmental stage aphids for 10 days. The surviving aphids were then transferred to cut radish leaves without the use of RNA. For all three genes, up to 6 days, each surviving aphid produced significantly lower numbers of nymphs than aphids fed with control dsGFP RNA molecules or water. For MpC002 and MpRack-1dsRNA, lower reproduction rates on radish leaves were maintained for at least 9 days. To investigate whether dsRNAi affects offspring, aphids that were born within three days on radish leaves and were not fed directly on an RNA-containing diet were transferred to freshly cut radish leaves and their survival and production rates were monitored for 15 days. Although there was no significant difference in survival rates, aphids that were all born on diets with MpC002, MpRack-1 or MpGh dsRNA produced significantly fewer numbers of aphids than those that were born on maternal aphids on diets with control dsGFP or water. In conclusion, the effect caused by feeding the dsRNA molecule to the parent aphid persists in the latter aphids.

Conclusion

The objective of this study was to use the ledRNA design to test the use of exogenous RNAi for control of aphids, a major group of sap-sucking pests, a worldwide problem, and to identify suitable target genes. Aphids are known to have an RNAi mechanism to process foreign RNA (Scott et al, 2013; Yu et al, 2016). Here, oral delivery via artificial diet containing ledRNA molecules targeting MpC002 or MpRack-1 genes could cause aphid death and reduce aphid reproduction. These molecules were tested against two different target genes, one encoding the effector protein C002 and the other encoding the receptor for activated protein kinase (Rack-1), which is essential for the feeding and development of Myzus persicae (Myzus persicae). ledRNA molecules targeting these genes significantly reduced aphid reproduction when added to artificial diets at concentrations as low as 50 ng/. mu.l. At higher concentrations of 200 ng/. mu.l ledRNA also increased aphid mortality. When ledRNA uptake was studied using the Cy3 marker, ledRNA molecules were observed within hours of feeding the artificial diet, in the aphid cuticle, subsequently in the reproductive system, and even in the newborn nymph as progeny of the fed adults. As shown by the results of conventional dsRNA, the effect of ledRNA on aphid reproduction may last at least two generations.

It was also shown that ledRNA molecules remained largely intact in artificial diets for at least three and half weeks. Most of the intact ledRNA molecules were also found in the aphid honeydew, the excretion product of aphids. The marked ledRNA is applied to the leaves of the plant, enters the phloem where the aphids feed, and is detected in the aphids. Taken together, these results indicate the strong potential of ledRNA for controlling aphids and other sap-sucking insects, including by exogenous delivery via diet, providing a practical method for managing aphids and other sap-sucking insects. These RNA molecules can also be expressed in transgenic plants, using promoters that favor RNA synthesis in phloem tissue to control aphids and other sap-sucking insects. Furthermore, the use of ledRNA [ G: U ] or hairpin [ G: U ] RNA comprising 10-30% G: U base pairs in the dsRNA region of the molecule is expected to provide better control, based on increasing the level of accumulation of these dsRNA molecules by reducing the self-silencing of the transgenes encoding these dsRNA molecules.

Example 20: RNA constructs targeting other insect genes

LedRNA targeting insect genes

Helicoverpa armigera (Helicoverpa armigera) is an insect pest of the order Lepidoptera, also known as Helicoverpa armigera or corn borer. Cotton bollworm (h. armigera) larvae feed on a variety of plants, including many important cultivated crops, and cause considerable crop damage worth billions of dollars per year. Larvae are omnivorous and world-wide pests that feed on a wide range of plant species including cotton, corn, tomatoes, chickpeas, pigeon peas, alfalfa, rice, sorghum, and cowpea.

Cotton bollworm (h. armigera) ABC transporter gene (ABCwhite) was selected as the target gene with an easily detectable phenotype to test the ledRNA and ledRNA (G: U) constructs in insect larvae. ABC transporters belong to the ATP-binding cassette transporter superfamily-for example, 54 different ABC transporter genes have been identified in the Helicoverpa (Helicoverpa) genome. ABC transporters encode membrane-bound proteins that carry any one or more of a variety of molecules across the membrane. Proteins use the energy released by ATP hydrolysis to transport molecules across membranes. Some ABC transporters are associated with plant secondary metabolite degradation in cotton bollworm h. The ABCwhite protein translocates the eye pigment and pteridine pathway precursors to pigment granules in the eye, and the knockout mutant presents white eyes.

The nucleotide sequence of the ABCwhite gene is provided as SEQ ID NO: 127 (accession number: KU 754476). To test whether ledRNA could reduce ABCwhite gene expression by exogenously delivering RNA into larval diet, genetic constructs encoding ledRNA were designed and prepared. The design principle for ledRNA was used to prepare genetic constructs in which the split sequence was the sense sequence and the contiguous sequence was the antisense sequence (FIG. 1B). The target region is 603 nucleotides in length, corresponding to SEQ ID NO: nucleotide 496-1097 of 127. The dsRNA region of ledRNA is 603bp in length; the antisense sequence in the dsRNA region is an uninterrupted continuous sequence corresponding to SEQ ID NO: nucleotide 496 of 127 and the complement of 1097. The nucleotide sequence encoding the ledRNA is provided herein as SEQ ID NO: 128. the coding region is transcribed in vitro under the control of the T7 RNA polymerase promoter.

The genetic construct encoding ledRNA was digested with the restriction enzyme SnaBI, cleaved downstream of the ledRNA coding region, and transcribed in vitro with RNA polymerase T7 according to the instructions of the transcription kit. RNA was added to the artificial diet and provided to cotton bollworm (h.

Corresponding ledRNA constructs with G: U base pairs in the double-stranded stem were prepared and compared with canonical base-paired ledRNA.

LedRNA targeting genes in ants

Argentina argentea (Linepihema humile), commonly known as Argentina argentea, is an insect pest that is widespread in several continents. An Argentina argentea (L.humile) gene encoding a Pheromone Biosynthesis Activating Neuropeptide (PBAN) neuropeptide-like (LOC105673224) was selected as a target gene and involved in communication between pheromones and insects.

The nucleotide sequence of the PBAN gene is provided as SEQ ID NO: 129 (accession number: XM _ 012368710). To test whether ledRNA could reduce PBAN gene expression by delivering the RNA exogenously to the diet in the form of a decoy, a genetic construct encoding ledRNA targeting the gene was designed and prepared. The design principle for ledRNA was used to prepare genetic constructs in which the split sequence was the sense sequence and the contiguous sequence was the antisense sequence (FIG. 1B). The target region is 540 nucleotides in length, corresponding to SEQ ID NO: nucleotide 136 of 129 and 675. The dsRNA region of ledRNA is 540bp in length; the antisense sequence in the dsRNA region is an uninterrupted continuous sequence corresponding to SEQ ID NO: nucleotide 136 of 129 and the complement of 675. The nucleotide sequence encoding the ledRNA is provided herein as SEQ ID NO: 130. the coding region is transcribed in vitro under the control of the T7 RNA polymerase promoter.

The genetic construct encoding ledRNA was digested with the restriction enzyme SnaBI, cleaved downstream of the ledRNA coding region, and transcribed in vitro with RNA polymerase T7 according to the instructions of the transcription kit. RNA was coated on corn flour for oral delivery to argentina ant (l.humile).

LedRNA targeting lucilia cuprina (L.cuprina) gene

Lucilia cuprina (Lucilia cuprina) is an insect pest, more commonly known as macadamia ovis copperna. Belongs to the family of the aphididae (Calliphoridae) and is a member of the order Diptera (Diptera) of the insects. Five target genes were selected for ledRNA construct testing, namely genes encoding the V-type proton ATPase catalytic subunit A of Lucilia cuprina (L.cuprina) (accession XM-023443547), RNAse 1/2 (accession XM-023448015), chitin synthase (accession XM-023449557), ecdysone receptor (EcR; accession U75355), and gamma-tubulin 1/1-like (accession XM-023449717). Each genetic construct was made using the design principle of ledRNA, where the split sequence was the sense sequence and the contiguous sequence was the antisense sequence (FIG. 1B). In each case, the target region is about 600 nucleotides in length, and the antisense sequence in the dsRNA region is an uninterrupted continuous sequence. The nucleotide sequence encoding ledRNA targeting the ATPase-A gene is provided herein as SEQ ID NO: 131. the nucleotide sequence encoding ledRNA targeting rnase 1/2 gene is provided herein as SEQ ID NO: 132. the nucleotide sequence encoding ledRNA targeting the chitin synthase gene is provided herein as SEQ ID NO: 133. the nucleotide sequence encoding the ledRNA targeting the EcR gene is provided herein as SEQ ID NO: 134. the nucleotide sequence encoding ledRNA targeting the γ -tubulin 1/1-like gene is provided herein as SEQ ID NO: 135. in each construct, the coding region was transcribed in vitro under the control of the T7 RNA polymerase promoter.

Example 21 transgene-derived ledRNA accumulates at high levels in stably transformed plants

A DNA fragment encoding the ledRNA sequence targeting the mRNA of either the GUS reporter gene or the Arabidopsis EIN2 gene was synthesized and cloned into pART7 to form the p35S: ledRNA: Ocs3' polyadenylation region/terminator expression cassette for expression in plant cells. The fragment was then excised with NotI and inserted into the NotI site of pART27 to form ledGUS and ledEIN2 vectors for plant transformation. ledGUS constructs and existing hpGUS constructs designed to produce long hpRNAs with 563bp dsRNA stem and 1113nt loop were transformed into GUS expressing tobacco line PPGH24, respectively, by Agrobacterium-mediated transformation. RNA samples from independent transformants that showed strong GUS silencing or little or no significant reduction in GUS activity were used in Northern blot hybridization assays to detect transgene-encoded hpGUS or ledGUS RNA. As shown in FIG. 38, the hybridization signals detected from ledGUS transformed plants were much stronger than those detected from hpGUS transformed plants showing strong GUS silencing (indicated by "-" in FIG. 38). In fact, most of the hybridization signals of hpGUS RNA samples were non-specific background signals, which were also observed from RNA of control, untransformed plants (WT). Several strong hybridizing bands were observed for ledGUS lines, presumably due to some partial processing of full-length ledRNA.

The nucleotide sequence of the genetic construct encoding ledGUS is shown in SEQ ID NO 5. Nucleotides 1-17 correspond to the T7 RNA polymerase promoter for in vitro RNA synthesis, nucleotides 18-270 correspond to the 5 'portion of the GUS antisense sequence, nucleotides 271-430 correspond to the loop 1 sequence, nucleotides 431-933 correspond to the GUS sense sequence, nucleotides 934-1093 correspond to the loop 2 sequence, and nucleotides 1094-1343 correspond to the 3' portion of the GUS antisense sequence.

In a similar manner, the ledEIN2 and hpEIN2 constructs were introduced into the Col-0 ecotype of Arabidopsis plants, respectively, by Agrobacterium-mediated transformation. The hpEIN2 construct encoding hpEIN2[ wt ] RNA was as previously described and contained 200bp sense and antisense EIN2 sequences in an inverted repeat configuration, separated by the PDK intron. The nucleotide sequence of the genetic construct encoding ledEIN2 is shown in SEQ ID NO 116. Nucleotides 37-225 correspond to the 5 'portion of the antisense sequence of EIN2, nucleotides 226-373 correspond to the loop 1 sequence, nucleotides 374-773 correspond to the sense sequence of EIN2, nucleotides 774-893 correspond to the loop 2 sequence, and nucleotides 894-1085 correspond to the 3' portion of the antisense sequence of EIN 2. Nucleotides 37-225 (antisense) are complementary to nucleotides 374-.

RNA samples from primary independent transformants were used for Northern blot hybridization analysis. As shown in fig. 39, ledEIN2 plants showed stronger hybridization signals than hpEIN2 plants for larger RNA molecules (fig. 39, top panel), indicating that ledEIN 2-derived RNA accumulated at higher levels than hpEIN 2-derived RNA. For processed RNA (siRNA) in the size range of 20-25 nucleotides, siRNA abundance was detected in ledEIN2 plants more than in hpEIN2 plants (fig. 39, lower panel), and the amount of siRNA correlated well with the abundance of larger RNA molecules. These results indicate that transgene-derived ledRNA is processed to some extent by Dicer into siRNA, but not completely. The ledRNA transgene was also shown to produce more siRNA than the corresponding hpRNA transgene.

These results indicate that expression of the ledRNA construct in plant cells results in higher levels of accumulation of unprocessed and processed transcripts compared to the corresponding hpRNA construct. This is believed to indicate an increased stability of the ledRNA molecule.

Example 22 hairpin RNAs are potent precursors of circular RNAs in plants

Circular RNA (circRNA) is a covalently linked closed loop with no free 5' and 3' ends or polyadenylation sequences as the 3' region. They are typically non-coding in that they do not encode a polypeptide and, therefore, are not translated. circRNA is relatively resistant to digestion by rnases, particularly exonucleases (e.g., RNase R). circRNA of viral or viroid origin or satellite RNA associated with viruses has long been observed in plants and animals. For example, the subviral RNA pathogen Potato Spindle Tuber Viroid (Potato Spindle tube Viroid) in plants has a circular RNA genome of approximately 360nt in size. In plants, such satellite RNAs are typically capable of being replicated in the presence of helper viruses. In contrast, viroids are completely dependent on host functions, which include endogenous plant RNA polymerase for their replication.

The use of deep sequencing techniques of RNA in combination with specially designed bioinformatics tools has now identified large amounts of cirrrna from plant and animal genomes. Thousands of putative circrnas have been identified in plants including arabidopsis, rice and soybean, which tend to exhibit tissue-specific or biotic and abiotic stress-responsive expression patterns, but the biological function of circrnas in plants has not been demonstrated. The tissue-specific or stress-responsive expression patterns of many putative plant circrnas suggest that they may have a potential role in plant development and defense responses, but this has not been demonstrated.

The common consensus on the biogenesis of circrnas is that they are formed by reverse splicing of introns, i.e. the splicing machinery "reverse splices" pre-mRNA and covalently links spliced exons together. Thus, the endogenous intron splicing mechanism is crucial for the current model of circRNA biogenesis. This biogenesis model is based primarily on studies of mammalian systems in which most exon circrnas are shown to contain canonical intron splicing signals that include common GT/AG intron border dinucleotides. In animals, intron regions flanking the exon circRNA often contain short inverted repeats of the sequence of the transposable element, and this leads to the suggestion that complementary intron sequences promote circRNA formation. Indeed, vector systems for expressing circRNA in animals have been developed based on naturally occurring exon-intron sequences and a concatameric intron containing complementary TE repeats. However, the role of the complementary flanking sequences in circRNA formation in plants is not clear, since the proportion of identified exon circRNA with such flanking intron sequences is very low, varying from 0.3% in Arabidopsis to 6.2% in rice.

Long hairpin RNA (hpRNA) transgenes have been widely used to induce gene silencing or RNA interference in plants (Wesley et al, 2001). hpRNA transgene constructs typically comprise inverted repeats having complementary sense and antisense sequences relative to the promoter sequence and a spacer sequence therebetween to separate and link the sense and antisense sequences. The spacer also stabilizes the inverted repeat structure of the DNA plasmid in bacterial cells during vector construction. Thus, it is expected that RNA transcripts from a typical hpRNA transgene will form a stem-loop structure with a double-stranded (ds) stem of base-paired sense and antisense sequences and a "loop" corresponding to the spacer sequence. Such RNA transcripts are also referred to as self-complementary RNAs due to the ability of the sense and antisense regions to anneal by base pairing to form the dsRNA or stem region of the molecule.

Loop fragments from long hpRNA accumulate in plant cells and are resistant to RNase R

A transgene was prepared encoding a long hpRNA targeting GUS mRNA with 563bp sense and antisense sequences and a 1113bp spacer (fig. 40, GUShp 1100). A second transgene was also prepared encoding a shorter hpRNA targeting the same GUS mRNA, with 93bp sense and antisense sequences and a 93bp spacer (GUSHP 93-1). Both constructs were introduced separately into nicotiana benthamiana leaf cells for transient expression of hairpin RNA, and also used to transform arabidopsis plants for stable integration and heritable transgene expression. As previously reported, both constructs produced different RNA fragments of the loop sequence of the expected size upon introduction into and expression in plant cells (FIG. 41; Wang et al, 2008; Shen et al, 2015). In this study, the inventors wished to determine whether the loop sequence was converted into a circular RNA.

A third construct with the Arabidopsis U6 promoter, instead of the 35S promoter, was prepared for expression of the shorter hpRNA (GUSHp 93-2). A fourth GUS hpRNA construct was also prepared, which included the PDK intron as a spacer sequence (GUShpPDK in figure 40). This construct encodes a hairpin RNA in which the intron is expected to splice after transcription, leaving a much shorter loop sequence. These constructs were also introduced into nicotiana benthamiana leaves to examine whether loop sequences could be detected and whether they formed circular RNA. The dsRNA stem and loop sequences in these constructs were both derived from the GUS coding sequence and no known intron sequences were introduced. Constructs, as well as genetic constructs encoding and expressing the cucumovirus 2b protein as a Viral Suppressor Protein (VSP) to enhance transgene expression, were introduced into nicotiana benthamiana leaves using agrobacterium-mediated infiltration in the presence or absence of a construct expressing the target GUS, respectively. The accumulation and size of loop fragments were analyzed using Northern blot hybridization assay. A representative photograph of an autoradiogram of a Northern blot is shown in FIG. 42.

As shown in FIG. 42, a long loop fragment of GUSHP1100 was readily detected in Agrobacterium-infiltrated samples as previously reported (Shen et al, 2015). To test whether the loop fragment is circular, RNA samples were treated with RNase R and electrophoresed on polyacrylamide gels. RNase R treatment 10. mu.g total RNA (or 50ng in vitro transcript) was mixed with RNase R buffer and water in a total volume of 20. mu.l. The mixture was heated in boiling water for 3 minutes, rapidly cooled on ice, then 0.5. mu.l RNase R was added and the tube was incubated at 37 ℃ for 10 minutes. The enzyme was inactivated and the remaining RNA was recovered by precipitation with ethanol. RNase R treatment degraded most of the RNA as indicated by a sharp decrease in ethidium bromide stained material in the gel (FIG. 42, bottom panel). Some ribosomal RNA fragments were still visible in the gel as determined by all RNase R treatments, indicating partial resistance of certain RNA species to RNase R. Despite the total RNA depletion in RNase R treated samples, the loop fragments of approximately 1100nt were still abundant, only reduced by an amount of about 24% compared to untreated samples. This indicates that the loop fragment is relatively resistant to RNase R digestion and is therefore circular in structure. The amount of loop RNA was reduced by 24% relative to the untreated sample due to the residual amount of endonuclease activity in the commercially available RNase R enzyme or due to the decrease in RNA recovery after RNase R digestion in the ethanol precipitation step.

The RNase R treatment assay was repeated, containing 50ng of in vitro transcribed RNA corresponding to the loop sequence as a linear RNA control. In addition, hpGUS1100 infiltrated Nicotiana benthamiana RNA samples were treated with two rounds of RNase R treatment to more rigorously test for RNase R resistance. It was observed that 76% of the loop fragments from GUSHP1100 infiltrated Nicotiana benthamiana leaves remained after one round of RNase R treatment, while only about 8.5% of the linear in vitro transcripts remained. Two-fold RNase R treatment further reduced the loop-derived material, but did not eliminate it. It should also be noted that the RNA bands corresponding to the loop sequences from the nicotiana benthamiana samples appeared larger on the gel blot than in vitro transcripts, which is consistent with circular RNA, which reportedly migrates slower than linear RNA molecules in gel electrophoresis with the same number of nucleotides. From these experiments, it was concluded that a loop sequence of about 1100 nucleotides is circular.

Northern blot hybridization analysis of GUshp93-1 and GUshpPDK-infiltrated Nicotiana benthamiana RNA samples also detected RNA molecules corresponding in size to the loop sequence length. For the GUSHp93-1 and GUSHp93-2 constructs, more loop fragments were produced by GUSHp93-2 directed by the U6 promoter than GUSHp93-1 driven by the 35S promoter, indicating that the U6 promoter has stronger transcriptional activity than the 35S promoter in Nicotiana benthamiana leaf cells, or that the molecule is somehow more stable.

The spacer sequence of the GUShpPDK construct included a 0.76kb sized spliceable PDK intron, and thus the initial transcript of this construct contained a loop of approximately 0.8 kb. Northern blots were treated to remove the GUS probe and re-probed with the full-length antisense probe against the PDK intron sequence. The PDK probe hybridizes strongly to unknown RNA species, which is observed as a strong band on all lanes. RNase A treatment reduced but did not completely eliminate the non-specific band. Nevertheless, although the abundance of the fragments appeared to be relatively weak, intron-specific bands of PDK of the expected size could be detected in GUShpPDK-infiltrated RNA samples, probably because the intron sequences were spliced out of most of the GUShpPDK initial transcripts. To examine whether the PDK loop fragment was circular, RNA from GUSHpPDK-infiltrated Nicotiana benthamiana leaves was treated with RNase R. Non-specific hybridizing bands were almost completely removed by RNase R treatment. In contrast, although abundance could not be easily compared to untreated samples due to strong signal from non-specific bands, PDK intron bands were easily detected after RNase R treatment. Taken together, these results indicate that hpRNA transcripts are efficient precursors for circular RNA formation and suggest that circular RNAs correspond to the entire circular sequence.

RNase R resistance loop fragments can also accumulate in stably transformed Arabidopsis thaliana plants

hpGUS347 and two hpGFP constructs (FIG. 40) were used to transform the ecotype Col-0 of Arabidopsis plants and two plants expressing the transgenes selected for each construct. The hpGUS347 construct was used in this experiment as a control for hpGFP constructs designed to contain a miR165/166 binding site for testing miRNA sponge function (discussed in example 24). Transgenic plants of the T2 generation were analyzed for accumulation of RNA molecules produced from the hpGUS347 construct, in particular to examine the loop sequences and whether they are circular. Bands corresponding to the loop of hpGUS347 transcripts were detected in both RNase R-treated and untreated RNA samples from both hpGUS347 lines. As for RNA samples of Agrobacterium-infiltrated Nicotiana benthamiana tissue, the band intensity of RNase R-treated samples appeared to be slightly reduced compared to untreated samples, but most of the RNA signal was retained. RT-qPCR analysis using primers designed to detect circRNA confirmed the presence of circRNA in RNase R treated hpGUS347 samples, which was slightly less abundant than the untreated samples. These results indicate that expression of a stably integrated hpRNA transgene that produces hairpin RNA also produces circRNA from the loop sequence.

Excision of the loop of the hpRNA transcript at the dsRNA stem-loop junction and formation of a circular RNA

To further confirm the circular nature of RNA molecules derived from loop sequences and characterize their linker sequences, loop sequences were amplified from GUShp1100, GUShp93 and GUShp pdk-infiltrated samples by RT-PCR using oligonucleotide primers that can amplify putative linker sequences. The RT-PCR product was then cloned into pGEM-T Easy vector and sequenced to confirm the nucleotide sequence of the junction. The position of the nucleotides for loop cleavage and ligation of the circular RNA varies somewhat, with the 5 'site located within the 3' end of the dsRNA stem and the 3 'site located near the 3' end of the loop, but the 5 'site shows a clear preference for G nucleotides 10 nucleotides from the 3' end of the dsRNA stem. It is noted that the excision and ligation sites of the PDK intron circular RNA follow the same pattern as those from GUShp1100 and GUShp93 RNA, and are located outside the canonical intron splice sites. It was concluded that the formation of circular RNA was determined by the stem-loop structure, independent of intron splicing. It was also concluded that, at least in this example, the hairpin RNA was processed to release and circularize the loop sequence by 5 'cleavage within the 3' end of the dsRNA stem and 3 'cleavage near the 3' end of the loop sequence, with a covalent bond formed between the 5 'and 3' ends of the cleaved sequence.

Example 23 hpRNA expressed in Saccharomyces cerevisiae was not processed to circular RNA

Yeast species saccharomyces cerevisiae is a eukaryotic organism and possesses the same intron splicing machinery as all eukaryotes. Since the current consensus model for circular RNA formation is based on intron splicing, the inventors investigated whether hpRNA can form circular RNA in saccharomyces cerevisiae as it does in plant cells. To generate a construct for expression of hpRNA, the inverted repeat region of GUSHp1100 was excised from the plant expression vector and inserted into a yeast expression vector under the control of the yeast ADH1 promoter (FIG. 43), and the resulting genetic construct was introduced into s.cerevisiae cells. As shown in FIG. 43, Northern blot hybridization analysis of RNA extracted from each of three independent transgenic yeast strains detected a high molecular weight band corresponding to the GUSHP1100 transcript. This indicates that the GUSHP1100 transcript was not processed in Saccharomyces cerevisiae, but still maintained full length. To confirm this, the response of Saccharomyces cerevisiae-expressed and Nicotiana benthamiana-expressed GUSHp1100 transcripts to RNase R treatment were compared. As shown in FIG. 44, Saccharomyces cerevisiae-expressed RNA showed high molecular weight bands highly sensitive to RNase R treatment and thus was not circular. That is, the yeast RNA sample did not exhibit circular molecules derived from loop sequences as produced in Nicotiana benthamiana cells. The results showed that the GUSHP1100 transcript expressed in Saccharomyces cerevisiae was not processed and remained full-length. By gel electrophoresis, the size of the Saccharomyces cerevisiae RNA band appeared to be larger than the in vitro GUSHP1100 transcript, probably due to the 5 'and 3' UTR and poly (A) sequences present in the Saccharomyces cerevisiae expressed RNA but not in the in vitro transcript. Thus, the presence of an intron splicing machinery in s.cerevisiae is insufficient to allow processing of the hpRNA loop and formation of circular RNA, as occurs in plant cells.

In a similar manner, the genetic construct GUShp347 was introduced into and expressed in saccharomyces cerevisiae. Northern blot hybridization analysis again showed that hpRNA appeared to be full-length and did not appear to be processed, at least not having cleavage of loop sequences or dsRNA regions.

The inventors concluded that s.cerevisiae and its related budding yeast without Dicer enzyme (Drinnenberg et al, 2003) are advantageous as organisms for the production of full-length hairpins and ledrnas, including the modified RNA molecules described herein. Such full-length RNAs are useful when unprocessed dsRNA is desired, for example, for silencing gene activity by topical application to insects.

Example 24 hpRNA Loop can be used as an effective "sponge" to inhibit miRNA function "

It has been found that some circular RNAs in animals contain multiple sequences that are complementary to specific mirnas, and thus serve as binding sites for those mirnas, known as miRNA "sponges". The inventors tested whether the circular RNA produced by the long hpRNA construct could act as a miRNA sponge in plant cells. Two GFP hpRNA constructs were designed (fig. 40) with the same GUS sequence-derived spacer, except that one sequence was modified to have two arabidopsis miR165/166 binding sites. The construct GFPhp [ G: U ] has an inverted repeat sequence with the same antisense sequence as the second (control) construct GFPhp [ WT ], but with a modified sense sequence in which all cytosine nucleotides are replaced by thymine. Thus, transcripts of GFPhp [ G: U ] will form a dsRNA region corresponding to the GFP sequence, except that approximately 25% of the base pairs are G: U base pairs. Another construct, GFPhp [ WT ], encodes a hairpin RNA with a fully canonical base-paired dsRNA stem of the same length as the hairpin of GFPhp [ G: U ], and was used as a control (FIG. 40). GUS hpRNA construct GUSH 347, comprising a spacer region without a miR165/166 binding site, was included as a second control.

Arabidopsis thaliana was transformed with each of the constructs, and transgenic plants of the three constructs were obtained. Transformed plants were visually examined for phenotypes associated with the reduction of miR165/166, including the unique folding of leaves into "horns". As expected, GUShp347 transformed plants did not show a phenotype associated with miR165/166 inhibition. Also, no clear phenotype was observed in GFPhp [ WT ] transformed plants. In contrast, most GFPhp [ G: U ] plants exhibit varying levels of a phenotype associated with miR165/166 inhibition, including the trumpet phenotype.

Northern blot hybridization was performed on RNA extracted from GFPhp [ G: U ] transformed plants with a range of mild, moderate and strong to severe phenotypes to examine the accumulation of hpRNA expression. The probe used was a full length antisense RNA corresponding to GUS mRNA. The probe has 822bp continuous sequence complementarity with the sense and adjacent loops of GUSHp347 transcript. The probe has less sequence complementarity with GFPhp transcripts which share a 228bp loop region in GUS derived sequences, in three non-contiguous regions of 49, 109 and 70bp length flanking two miRNA binding sequences. As shown in FIG. 45B, very large amounts of GFP hpRNA molecules were detected in GFPhp [ G: U ] plants, and the amount of RNA molecules detected in Northern blots was positively correlated with the severity of the phenotype. GFPhp [ WT ] plants exhibit low accumulation levels of hpRNA molecules, which are detectable only in Northern blot analysis, consistent with relatively low transcription levels of conventional hpRNA transgenes compared to G: U modified hpRNA transgenes. That is, as shown in the above examples, hpRNA [ G: U ] transgenes are less self-silenced compared to the corresponding hpRNA [ WT ] transgene.

RT-qPCR was used to quantify the accumulation of circular RNA molecules derived from loop sequences. The results indicated that large amounts of circRNA were present in GFPhp [ G: U ] transgenic plants, which correlated with the level of full-length hpRNA accumulation (FIG. 45C). Northern blot hybridization analysis detects small RNAs ranging in size from 20 to 25nt, confirming the down-regulation of miR165/166 in GFPhp [ G: U ] plants. The extent of reduction correlated with the amount of hpRNA and circRNA and the severity of the phenotype. Expression analysis of the miR165/166 target gene using RT-qPCR showed that inhibition of the target gene by miR165/166 was released in plants exhibiting strong miR165/166 down-regulation and severe phenotype. Taken together, these results indicate that hpRNA loops can be used as specific miRNA sponges to inhibit miRNA function in plants.

The inventors also contemplate the use of circular RNA produced at high levels in plant cells as a stabilizing molecule to translate into a means of producing high levels of polypeptide. To initiate cap-independent translation, an Internal Ribosome Entry Site (IRES) is desirably used. Many IRES sequences have been identified.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The present application claims priority from AU2018902840, filed 8/3/2018, AU2018902896, filed 8/2018, PCT/AU2018/051015, filed 9/17/2018, and AU 2019900941, filed 3/20/2019, the disclosures of which are incorporated herein by reference.

All publications discussed and/or cited herein are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Sequence listing

<110> Federal research and technology organization

<120> RNA molecule comprising non-canonical base pairs

<130> 526425PCT

<150> AU 2018902840

<151> 2018-08-03

<150> AU 2018902896

<151> 2018-08-08

<150> PCT/AU2018/051015

<151> 2018-09-17

<150> AU 2019900941

<151> 2019-03-04

<160> 144

<170> PatentIn version 3.5

<210> 1

<211> 1229

<212> RNA

<213> Artificial Sequence

<220>

<223> GFP ledRNA

<400> 1

gggugucgcc cucgaacuuc accucggcgc gggucuugua guugccgucg uccuugaaga 60

agauggugcg cuccuggacg uagccuucgg gcauggcgga cuugaagaag ucgugcugcu 120

ucaugugguc gggguagcgg cugaagcacu gcacgccgua ggugaaggug gucacgaggg 180

ugggccaggg cacgggcagc uugccggugg ugcagaugaa cuucaggguc agcuugccgu 240

agguggcauc gcccucgccc ucgccggaca cgcugaacuu guggccguuu acgucgccgu 300

ccagcucgac caggaugggc accaccccgg ugaacagcuc cucgcccuug cucacuaugg 360

aucaacuagg gaucccccug aaguucaucu gcaccaccgg caagcugccc gugcccuggc 420

ccacccucgu gaccaccuuc accuacggcg ugcagugcuu cagccgcuac cccgaccaca 480

ugaagcagca cgacuucuuc aaguccgcca ugcccgaagg cuacguccag gagcgcacca 540

ucuucuucaa ggacgacggc aacuacaaga cccgcgccga ggugaaguuc gagggcgaca 600

cccuggugaa ccgcaucgag cugaagggca ucgacuucaa ggaggacggc aacauccugg 660

ggcacaagcu ggaguacaac uacaacagcc acaacgucua uaucauggcc gacaagcaga 720

agaacggcau caaggugaac uucaagaucc gccacaacau cgaggacggc agcgugcagc 780

ucgccgacca cuaccagcag aacaccccca ucggcgacgg ccccgugcug cugccaagcu 840

uuaggugauc caagcuugau ccgggcuuua cuuguacagc ucguccaugc cgagagugau 900

cccggcggcg gucacgaacu ccagcaggac caugugaucg cgcuucucgu uggggucuuu 960

gcucagggcg gacugggugc ucagguagug guugucgggc agcagcacgg ggccgucgcc 1020

gaugggggug uucugcuggu aguggucggc gagcugcacg cugccguccu cgauguugug 1080

gcggaucuug aaguucaccu ugaugccguu cuucugcuug ucggccauga uauagacguu 1140

guggcuguug uaguuguacu ccagcuugug ccccaggaug uugccguccu ccuugaaguc 1200

gaugcccuuc agcucgaugc gguucacca 1229

<210> 2

<211> 1326

<212> RNA

<213> Artificial Sequence

<220>

<223> GUS ledRNA

<400> 2

gggaacagac gcgugguuac agucuugcgc gacaugcguc accacgguga uaucguccac 60

ccagguguuc ggcguggugu agagcauuac gcugcgaugg auuccggcau aguuaaagaa 120

aucauggaag uaagacugcu uuuucuugcc guuuucgucg guaaucacca uucccggcgg 180

gauagucugc caguucaguu cguuguucac acaaacggug auacguacac uuuucccggc 240

aauaacauac ggcgugacau cggcuucaaa uggcguauag ccgcccugau gcuccaucac 300

uuccugauua uugacccaca cuuugccgua augagugacc gcaucgaaac gcagcacgau 360

acgcuggccu gcccaaccuu ucgguauaaa gacuucgcgc ugauaccaga cgugccguau 420

guuauugccg ggaaaagugu acguaucacc guuuguguga acaacgaacu gaacuggcag 480

acuaucccgc cgggaauggu gauuaccgac gaaaacggca agaaaaagca gucuuacuuc 540

caugauuucu uuaacuaugc cggaauccau cgcagcguaa ugcucuacac cacgccgaac 600

accugggugg acgauaucac cguggugacg caugucgcgc aagacuguaa ccacgcgucu 660

guucccgacu ggcagguggu ggccaauggu gaugucagcg uugaacugcg ugaugcggau 720

caacaggugg uugcaacugg acaaggcacu agcgggacuu ugcaaguggu gaauccgcac 780

cucuggcaac cgggugaagg uuaucucuau gaacugugcg ucacagccaa aagccagaca 840

gagugugaua ucuacccgcu ucgcgucggc auccggucag uggcagugaa gggccaacag 900

uuccugauua accacaaacc guucuacuuu acuggcuuug gucgucauga agaugcggac 960

uuacguggca aaggauucga uaacgugcug auggugcacg accacgcauu aauggacugg 1020

auuggggcca acuccuaccg uaccucgcau uacccuuacg cugaagagau gcucgaugug 1080

guuaaucagg aacuguuggc ccuucacugc cacugaccgg augccgacgc gaagcgggua 1140

gauaucacac ucugucuggc uuuuggcugu gacgcacagu ucauagagau aaccuucacc 1200

cgguugccag aggugcggau ucaccacuug caaagucccg cuagugccuu guccaguugc 1260

aaccaccugu ugauccgcau cacgcaguuc aacgcugaca ucaccauugg ccaccaccug 1320

ccaguc 1326

<210> 3

<211> 1485

<212> RNA

<213> Artificial Sequence

<220>

<223> FAD2.1 ledRNA

<400> 3

gaagaucugu agccucucgc ggucauugua gauugggccg uaagggucau agugacaugc 60

aaagcgauca uaaugucggc cagaaacauu gaaagccaag uacaaaggcc agccaagagu 120

aagggugauc guaagugaaa uaacccggcc ugguggauug uucaaguacu uggaauacca 180

uccgaguugu gauuucggcu uaggcacaaa aaccucaucg cgcucgagug agccaguguu 240

ggaguggugg cgacgaugac uauauuucca agagaaguag ggcaccauca gagcagagug 300

gaggauaagc ccgacagugu caucaaccca cugguaguca cuaaaggcau gguggccaca 360

uucgugcgca auaacccaaa uaccagugca aacacaaccc ugacaaaucc aguaaauagg 420

ccaugcaagg uagcaauccu aggcacucug cucugauggu gcccuacuuc ucuuggaaau 480

auagucaucg ucgccaccac uccaacacug gcucacucga gcgcgaugag guuuuugugc 540

cuaagccgaa aucacaacuc ggaugguauu ccaaguacuu gaacaaucca ccaggccggg 600

uuauuucacu uacgaucacc cuuacucuug gcuggccuuu guacuuggcu uucaauguuu 660

cuggccgaca uuaugaucgc uuugcauguc acuaugaccc uuacggccca aucuacaaug 720

accgcgagag gcuacagauc uuccuuucug augcuggagu uauuggagcu gguuaucuac 780

uauaucguau ugccuuggua aaagggcuag cuuggcucgu guguauguau ggcguaccac 840

uccuaaucgu gaacggcuuc cuugucuuga ucacuuauuu gcagcacacu cacccgucau 900

ugccucacua cgauucaucc gaaugggauu ggcuaagggg agcuuuggca accgucgaca 960

gagacuaugg cauucuaaac aaggucuucc acaacaucac cgauacucac guaguccacc 1020

aucuguucuc gaccaugcca cacucuagag ugaugcuuca ucuuucucca cauagauaca 1080

cucuuuugcu ucccuccaca uugccuugaa aaccgggguu ccgucaaauu gguaguaguc 1140

uccgaguaau ggcuugacug cuuuuguugc cuccauugca uuguagugug gcauggucga 1200

gaacagaugg uggacuacgu gaguaucggu gauguugugg aagaccuugu uuagaaugcc 1260

auagucucug ucgacgguug ccaaagcucc ccuuagccaa ucccauucgg augaaucgua 1320

gugaggcaau gacgggugag ugugcugcaa auaagugauc aagacaagga agccguucac 1380

gauuaggagu gguacgccau acauacacac gagccaagcu agcccuuuua ccaaggcaau 1440

acgauauagu agauaaccag cuccaauaac uccagcauca gaaag 1485

<210> 4

<211> 1258

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA construct encoding GFP ledRNA

<400> 4

taatacgact cactataggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt 60

gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt 120

gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt 180

gaaggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt 240

cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg 300

gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc 360

gcccttgctc actatggatc aactagggat ccccctgaag ttcatctgca ccaccggcaa 420

gctgcccgtg ccctggccca ccctcgtgac caccttcacc tacggcgtgc agtgcttcag 480

ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta 540

cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt 600

gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga 660

ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat 720

catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga 780

ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc 840

cgtgctgctg ccaagcttta ggtgatccaa gcttgatccg ggctttactt gtacagctcg 900

tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc 960

ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc 1020

agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg 1080

ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg 1140

gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg 1200

ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccattgt cgggatac 1258

<210> 5

<211> 1346

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA construct encoding Gus ledRNA

<400> 5

taatacgact cactataggg aacagacgcg tggttacagt cttgcgcgac atgcgtcacc 60

acggtgatat cgtccaccca ggtgttcggc gtggtgtaga gcattacgct gcgatggatt 120

ccggcatagt taaagaaatc atggaagtaa gactgctttt tcttgccgtt ttcgtcggta 180

atcaccattc ccggcgggat agtctgccag ttcagttcgt tgttcacaca aacggtgata 240

cgtacacttt tcccggcaat aacatacggc gtgacatcgg cttcaaatgg cgtatagccg 300

ccctgatgct ccatcacttc ctgattattg acccacactt tgccgtaatg agtgaccgca 360

tcgaaacgca gcacgatacg ctggcctgcc caacctttcg gtataaagac ttcgcgctga 420

taccagacgt gccgtatgtt attgccggga aaagtgtacg tatcaccgtt tgtgtgaaca 480

acgaactgaa ctggcagact atcccgccgg gaatggtgat taccgacgaa aacggcaaga 540

aaaagcagtc ttacttccat gatttcttta actatgccgg aatccatcgc agcgtaatgc 600

tctacaccac gccgaacacc tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag 660

actgtaacca cgcgtctgtt cccgactggc aggtggtggc caatggtgat gtcagcgttg 720

aactgcgtga tgcggatcaa caggtggttg caactggaca aggcactagc gggactttgc 780

aagtggtgaa tccgcacctc tggcaaccgg gtgaaggtta tctctatgaa ctgtgcgtca 840

cagccaaaag ccagacagag tgtgatatct acccgcttcg cgtcggcatc cggtcagtgg 900

cagtgaaggg ccaacagttc ctgattaacc acaaaccgtt ctactttact ggctttggtc 960

gtcatgaaga tgcggactta cgtggcaaag gattcgataa cgtgctgatg gtgcacgacc 1020

acgcattaat ggactggatt ggggccaact cctaccgtac ctcgcattac ccttacgctg 1080

aagagatgct cgatgtggtt aatcaggaac tgttggccct tcactgccac tgaccggatg 1140

ccgacgcgaa gcgggtagat atcacactct gtctggcttt tggctgtgac gcacagttca 1200

tagagataac cttcacccgg ttgccagagg tgcggattca ccacttgcaa agtcccgcta 1260

gtgccttgtc cagttgcaac cacctgttga tccgcatcac gcagttcaac gctgacatca 1320

ccattggcca ccacctgcca gtcaac 1346

<210> 6

<211> 1512

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA construct encoding FAD2.1 ledRNA

<400> 6

atttaggtga cactatagaa gatctgtagc ctctcgcggt cattgtagat tgggccgtaa 60

gggtcatagt gacatgcaaa gcgatcataa tgtcggccag aaacattgaa agccaagtac 120

aaaggccagc caagagtaag ggtgatcgta agtgaaataa cccggcctgg tggattgttc 180

aagtacttgg aataccatcc gagttgtgat ttcggcttag gcacaaaaac ctcatcgcgc 240

tcgagtgagc cagtgttgga gtggtggcga cgatgactat atttccaaga gaagtagggc 300

accatcagag cagagtggag gataagcccg acagtgtcat caacccactg gtagtcacta 360

aaggcatggt ggccacattc gtgcgcaata acccaaatac cagtgcaaac acaaccctga 420

caaatccagt aaataggcca tgcaaggtag caatcctagg cactctgctc tgatggtgcc 480

ctacttctct tggaaatata gtcatcgtcg ccaccactcc aacactggct cactcgagcg 540

cgatgaggtt tttgtgccta agccgaaatc acaactcgga tggtattcca agtacttgaa 600

caatccacca ggccgggtta tttcacttac gatcaccctt actcttggct ggcctttgta 660

cttggctttc aatgtttctg gccgacatta tgatcgcttt gcatgtcact atgaccctta 720

cggcccaatc tacaatgacc gcgagaggct acagatcttc ctttctgatg ctggagttat 780

tggagctggt tatctactat atcgtattgc cttggtaaaa gggctagctt ggctcgtgtg 840

tatgtatggc gtaccactcc taatcgtgaa cggcttcctt gtcttgatca cttatttgca 900

gcacactcac ccgtcattgc ctcactacga ttcatccgaa tgggattggc taaggggagc 960

tttggcaacc gtcgacagag actatggcat tctaaacaag gtcttccaca acatcaccga 1020

tactcacgta gtccaccatc tgttctcgac catgccacac tctagagtga tgcttcatct 1080

ttctccacat agatacactc ttttgcttcc ctccacattg ccttgaaaac cggggttccg 1140

tcaaattggt agtagtctcc gagtaatggc ttgactgctt ttgttgcctc cattgcattg 1200

tagtgtggca tggtcgagaa cagatggtgg actacgtgag tatcggtgat gttgtggaag 1260

accttgttta gaatgccata gtctctgtcg acggttgcca aagctcccct tagccaatcc 1320

cattcggatg aatcgtagtg aggcaatgac gggtgagtgt gctgcaaata agtgatcaag 1380

acaaggaagc cgttcacgat taggagtggt acgccataca tacacacgag ccaagctagc 1440

ccttttacca aggcaatacg atatagtaga taaccagctc caataactcc agcatcagaa 1500

agcccgggac tc 1512

<210> 7

<211> 732

<212> DNA

<213> Aequorea victoria

<400> 7

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccttcaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

agcccggatc tc 732

<210> 8

<211> 1855

<212> DNA

<213> Escherichia coli

<400> 8

atggtccgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60

ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120

gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180

cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240

ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300

aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360

tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg 420

cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttac 480

ttccatgatt tctttaacta tgccggaatc catcgcagcg taatgctcta caccacgccg 540

aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg taaccacgcg 600

tctgttcccg actggcaggt ggtggccaat ggtgatgtca gcgttgaact gcgtgatgcg 660

gatcaacagg tggttgcaac tggacaaggc actagcggga ctttgcaagt ggtgaatccg 720

cacctctggc aaccgggtga aggttatctc tatgaactgt gcgtcacagc caaaagccag 780

acagagtgtg atatctaccc gcttcgcgtc ggcatccggt cagtggcagt gaagggccaa 840

cagttcctga ttaaccacaa accgttctac tttactggct ttggtcgtca tgaagatgcg 900

gacttacgtg gcaaaggatt cgataacgtg ctgatggtgc acgaccacgc attaatggac 960

tggattgggg ccaactccta ccgtacctcg cattaccctt acgctgaaga gatgctcgac 1020

tgggcagatg aacatggcat cgtggtgatt gatgaaactg ctgctgtcgg ctttaacctc 1080

tctttaggca ttggtttcga agcgggcaac aagccgaaag aactgtacag cgaagaggca 1140

gtcaacgggg aaactcagca agcgcactta caggcgatta aagagctgat agcgcgtgac 1200

aaaaaccacc caagcgtggt gatgtggagt attgccaacg aaccggatac ccgtccgcaa 1260

gtgcacggga atatttcgcc actggcggaa gcaacgcgta aactcgaccc gacgcgtccg 1320

atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag cgatctcttt 1380

gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga tttggaaacg 1440

gcagagaagg tactggaaaa agaacttctg gcctggcagg agaaactgca tcagccgatt 1500

atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta caccgacatg 1560

tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc 1620

agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata 1680

ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg accgcaaacc gaagtcggcg 1740

gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc gcagcaggga 1800

ggcaaacaat gaatcaacaa ctctcctggc gcaccatcgt cggctacagc ctcgg 1855

<210> 9

<211> 1152

<212> DNA

<213> Nicotiana benthamiana

<400> 9

atgggagctg gtggtaatat gtctcttgta accagcaaga ctggcgaaaa gaagaatcct 60

cttgaaaagg taccaacctc aaagcctcct ttcacagttg gtgatatcaa gaaggccatc 120

ccacctcact gctttcagcg gtctctcgtt cgttcgttct cctatgttgt gtatgacctt 180

ttactggtgt ccgtcttcta ctacattgcc accacttact tccacctcct cccgtcccca 240

tattgctacc ttgcatggcc tatttactgg atttgtcagg gttgtgtttg cactggtatt 300

tgggttattg cgcacgaatg tggccaccat gcctttagtg actaccagtg ggttgatgac 360

actgtcgggc ttatcctcca ctctgctctg atggtgccct acttctcttg gaaatatagt 420

catcgtcgcc accactccaa cactggctca ctcgagcgcg atgaggtttt tgtgcctaag 480

ccgaaatcac aactcggatg gtattccaag tacttgaaca atccaccagg ccgggttatt 540

tcacttacga tcacccttac tcttggctgg cctttgtact tggctttcaa tgtttctggc 600

cgacattatg atcgctttgc atgtcactat gacccttacg gcccaatcta caatgaccgc 660

gagaggctac agatcttcct ttctgatgct ggagttattg gagctggtta tctactatat 720

cgtattgcct tggtaaaagg gctagcttgg ctcgtgtgta tgtatggcgt accactccta 780

atcgtgaacg gcttccttgt cttgatcact tatttgcagc acactcaccc gtcattgcct 840

cactacgatt catccgaatg ggattggcta aggggagctt tggcaaccgt cgacagagac 900

tatggcattc taaacaaggt cttccacaac atcaccgata ctcacgtagt ccaccatctg 960

ttctcgacca tgccacacta caatgcaatg gaggcaacaa aagcagtcaa gccattactc 1020

ggagactact accaatttga cggaaccccg gttttcaagg caatgtggag ggaagcaaaa 1080

gagtgtatct atgtggagaa agatgaagca tcacaaggca aaggtgtttt ctggtacaaa 1140

aacaaattct ga 1152

<210> 10

<211> 225

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS sense region for constructs encoding hairpin RNA molecules

targeting the GUS mRNA

<400> 10

cctcgaggat cctcgcgtcg gcatccggtc agtggcagtg aagggcgaac agttcctgat 60

taaccacaaa ccgttctact ttactggctt tggtcgtcat gaagatgcgg acttgcgtgg 120

caaaggattc gataacgtgc tgatggtgca cgaccacgca ttaatggact ggattggggc 180

caactcctac cgtacctcgc attaccctta cgaagcttgg taccc 225

<210> 11

<211> 216

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS sense region for the construct encoding the hairpin RNA

molecule hpGUS[G:U]

<400> 11

ccctcgagtt gtgttggtat ttggttagtg gtagtgaagg gtgaatagtt tttgattaat 60

tataaattgt tttattttat tggttttggt tgttatgaag atgtggattt gtgtggtaaa 120

ggatttgata atgtgttgat ggtgtatgat tatgtattaa tggattggat tggggttaat 180

ttttattgta ttttgtatta tttttatggg tacccc 216

<210> 12

<211> 216

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS sense region for constructs encoding the hairpin RNA molecule

hpGUS[1:4]

<400> 12

ccctcgagtc gggtccgcaa ccgctcactg ggagtcaagc gcgtacactt cgtgaataag 60

cactaacggt tgtacattag tgggtttcgt cctcaagaac atggggagtt gggtgccaat 120

ggaatcgtta aggtggtgaa ggtccaccac ctcgctttat tggtctgcat tcggggcaag 180

tccaacccta cgtcggattt cccataccgg tacccc 216

<210> 13

<211> 216

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS sense region for constructs encoding the hairpin RNA molecule

hpGUS[2:10]

<400> 13

ccctcgagtc gcgtcgcgat ccggtctctg gcagtgttgg gcgaactctt cctgatatac 60

cacaaagggt tctactaaac tggcttacgt cgtcatctag atgcggtgtt gcgtgggtaa 120

ggattcctta acgtgcacat ggtgcagcac cacgcaaaaa tggactccat tggggcgtac 180

tcctacgcta cctcgctata cccttagcgg tacccc 216

<210> 14

<211> 240

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of nucleotides 781-1020 of the protein coding region

of the GUS gene

<400> 14

gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa gggcgaacag 60

ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga agatgcggac 120

ttgcgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt aatggactgg 180

attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg 240

<210> 15

<211> 463

<212> RNA

<213> Artificial Sequence

<220>

<223> hairpin structure (including its loop) of the hpGUS[wt] RNA

<400> 15

ggauccucgc gucggcaucc ggucaguggc agugaagggc gaacaguucc ugauuaacca 60

caaaccguuc uacuuuacug gcuuuggucg ucaugaagau gcggacuugc guggcaaagg 120

auucgauaac gugcugaugg ugcacgacca cgcauuaaug gacuggauug gggccaacuc 180

cuaccguacc ucgcauuacc cuuacgaagc uugguacccc agcuuguugg gaagcugggu 240

ucgaaaucga uaagcuucgu aaggguaaug cgagguacgg uaggaguugg ccccaaucca 300

guccauuaau gcguggucgu gcaccaucag cacguuaucg aauccuuugc cacgcaaguc 360

cgcaucuuca ugacgaccaa agccaguaaa guagaacggu uugugguuaa ucaggaacug 420

uucgcccuuc acugccacug accggaugcc gacgcgagga ucc 463

<210> 16

<211> 457

<212> RNA

<213> Artificial Sequence

<220>

<223> hairpin structure (including its loop) of the hpGUS[G:U] RNA

<400> 16

cucgaguugu guugguauuu gguuaguggu agugaagggu gaauaguuuu ugauuaauua 60

uaaauuguuu uauuuuauug guuuugguug uuaugaagau guggauuugu gugguaaagg 120

auuugauaau guguugaugg uguaugauua uguauuaaug gauuggauug ggguuaauuu 180

uuauuguauu uuguauuauu uuuaugggua ccccagcuug uugggaagcu ggguucgaaa 240

ucgauaagcu ucguaagggu aaugcgaggu acgguaggag uuggccccaa uccaguccau 300

uaaugcgugg ucgugcacca ucagcacguu aucgaauccu uugccacgca aguccgcauc 360

uucaugacga ccaaagccag uaaaguagaa cgguuugugg uuaaucagga acuguucgcc 420

cuucacugcc acugaccgga ugccgacgcg aggaucc 457

<210> 17

<211> 457

<212> RNA

<213> Artificial Sequence

<220>

<223> hairpin structure (including its loop) of the hpGUS[1:4] RNA

<400> 17

cucgagucgg guccgcaacc gcucacuggg agucaagcgc guacacuucg ugaauaagca 60

cuaacgguug uacauuagug gguuucgucc ucaagaacau ggggaguugg gugccaaugg 120

aaucguuaag guggugaagg uccaccaccu cgcuuuauug gucugcauuc ggggcaaguc 180

caacccuacg ucggauuucc cauaccggua ccccagcuug uugggaagcu ggguucgaaa 240

ucgauaagcu ucguaagggu aaugcgaggu acgguaggag uuggccccaa uccaguccau 300

uaaugcgugg ucgugcacca ucagcacguu aucgaauccu uugccacgca aguccgcauc 360

uucaugacga ccaaagccag uaaaguagaa cgguuugugg uuaaucagga acuguucgcc 420

cuucacugcc acugaccgga ugccgacgcg aggaucc 457

<210> 18

<211> 457

<212> RNA

<213> Artificial Sequence

<220>

<223> hairpin structure (including its loop) of the hpGUS[2:10] RNA

<400> 18

cucgagucgc gucgcgaucc ggucucuggc aguguugggc gaacucuucc ugauauacca 60

caaaggguuc uacuaaacug gcuuacgucg ucaucuagau gcgguguugc guggguaagg 120

auuccuuaac gugcacaugg ugcagcacca cgcaaaaaug gacuccauug gggcguacuc 180

cuacgcuacc ucgcuauacc cuuagcggua ccccagcuug uugggaagcu ggguucgaaa 240

ucgauaagcu ucguaagggu aaugcgaggu acgguaggag uuggccccaa uccaguccau 300

uaaugcgugg ucgugcacca ucagcacguu aucgaauccu uugccacgca aguccgcauc 360

uucaugacga ccaaagccag uaaaguagaa cgguuugugg uuaaucagga acuguucgcc 420

cuucacugcc acugaccgga ugccgacgcg aggaucc 457

<210> 19

<211> 4851

<212> DNA

<213> Arabidopsis thaliana

<400> 19

atctctctct ttcgatggaa ctgagctctt tctctctttc ctcttctttt ctctctctat 60

ctctatctct cgtagcttga taagagtttc tctcttttga agatccgttt ctctctctct 120

cactgagact attgttgtta ggtcaacttg cgatcatggc gatttcgaag gtctgaagct 180

gatttcgaat ggtttggaga tatccgtagt ggttaagcat atggaagtct atgttctgct 240

cttggttgct ctgttagggc ttcctccatt tggaccaact tagctgaatg ttgtatgatc 300

tctctccttg aagcagcaaa taagaagaag gtctggtcct taacttaaca tctggttact 360

agaggaaact tcagctatta ttaggtaaag aaagactgta cagagttgta taacaagtaa 420

gcgttagagt ggctttgttt gcctcggtga tagaagaacc gactgattcg ttgttgtgtg 480

ttagctttgg agggaatcag atttcgcgag ggaaggtgtt ttagatcaaa tctgtgaatt 540

ttactcaact gaggctttta gtgaaccacg actgtagagt tgaccttgaa tcctactctg 600

agtaattata ttatcagata gatttaggat ggaagctgaa attgtgaatg tgagacctca 660

gctagggttt atccagagaa tggttcctgc tctacttcct gtccttttgg tttctgtcgg 720

atatattgat cccgggaaat gggttgcaaa tatcgaagga ggtgctcgtt tcgggtatga 780

cttggtggca attactctgc ttttcaattt tgccgccatc ttatgccaat atgttgcagc 840

tcgcataagc gttgtgactg gtaaacactt ggctcagatc tgcaatgaag aatatgacaa 900

gtggacgtgc atgttcttgg gcattcaggc ggagttctca gcaattctgc tcgaccttac 960

catggttgtg ggagttgcgc atgcacttaa ccttttgttt ggggtggagt tatccactgg 1020

agtgtttttg gccgccatgg atgcgttttt atttcctgtt ttcgcctctt tccttgaaaa 1080

tggtatggca aatacagtat ccatttactc tgcaggcctg gtattacttc tctatgtatc 1140

tggcgtcttg ctgagtcagt ctgagatccc actctctatg aatggagtgt taactcggtt 1200

aaatggagag agcgcattcg cactgatggg tcttcttggc gcaagcatcg tccctcacaa 1260

tttttatatc cattcttatt ttgctgggga aagtacatct tcgtctgatg tcgacaagag 1320

cagcttgtgt caagaccatt tgttcgccat ctttggtgtc ttcagcggac tgtcacttgt 1380

aaattatgta ttgatgaatg cagcagctaa tgtgtttcac agtactggcc ttgtggtact 1440

gacttttcac gatgccttgt cactaatgga gcaggtattt atgagtccgc tcattccagt 1500

ggtctttttg atgctcttgt tcttctctag tcaaattacc gcactagctt gggctttcgg 1560

tggagaggtc gtcctgcatg acttcctgaa gatagaaata cccgcttggc ttcatcgtgc 1620

tacaatcaga attcttgcag ttgctcctgc gctttattgt gtatggacat ctggtgcaga 1680

cggaatatac cagttactta tattcaccca ggtcttggtg gcaatgatgc ttccttgctc 1740

ggtaataccg cttttccgca ttgcttcgtc gagacaaatc atgggtgtcc ataaaatccc 1800

tcaggttggc gagttcctcg cacttacaac gtttttggga tttctggggt tgaatgttgt 1860

ttttgttgtt gagatggtat ttgggagcag tgactgggct ggtggtttga gatggaatac 1920

cgtgatgggc acctcgattc agtacaccac tctgcttgta tcgtcatgtg catccttatg 1980

cctgatactc tggctggcag ccacgccgct gaaatctgcg agtaacagag cggaagctca 2040

aatatggaac atggatgctc aaaatgcttt atcttatcca tctgttcaag aagaggaaat 2100

tgaaagaaca gaaacaagga ggaacgaaga cgaatcaata gtgcggttgg aaagcagggt 2160

aaaggatcag ttggatacta cgtctgttac tagctcggtc tatgatttgc cagagaacat 2220

tctaatgacg gatcaagaaa tccgttcgag ccctccagag gaaagagagt tggatgtaaa 2280

gtactctacc tctcaagtta gtagtcttaa ggaagactct gatgtaaagg aacagtctgt 2340

attgcagtca acagtggtta atgaggtcag tgataaggat ctgattgttg aaacaaagat 2400

ggcgaaaatt gaaccaatga gtcctgtgga gaagattgtt agcatggaga ataacagcaa 2460

gtttattgaa aaggatgttg aaggggtttc atgggaaaca gaagaagcta ccaaagctgc 2520

tcctacaagc aactttactg tcggatctga tggtcctcct tcattccgca gcttaagtgg 2580

ggaaggggga agtgggactg gaagcctttc acggttgcaa ggtttgggac gtgctgcccg 2640

gagacactta tctgcgatcc ttgatgaatt ttggggacat ttatatgatt ttcatgggca 2700

attggttgct gaagccaggg caaagaaact agatcagctg tttggcactg atcaaaagtc 2760

agcctcttct atgaaagcag attcgtttgg aaaagacatt agcagtggat attgcatgtc 2820

accaactgcg aagggaatgg attcacagat gacttcaagt ttatatgatt cactgaagca 2880

gcagaggaca ccgggaagta tcgattcgtt gtatggatta caaagaggtt cgtcaccgtc 2940

accgttggtc aaccgtatgc agatgttggg tgcatatggt aacaccacta ataataataa 3000

tgcttacgaa ttgagtgaga gaagatactc tagcctgcgt gctccatcat cttcagaggg 3060

ttgggaacac caacaaccag ctacagttca cggataccag atgaagtcat atgtagacaa 3120

tttggcaaaa gaaaggcttg aagccttaca atcccgtgga gagatcccga catcgagatc 3180

tatggcgctt ggtacattga gctatacaca gcaacttgct ttagccttga aacagaagtc 3240

ccagaatggt ctaacccctg gaccagctcc tgggtttgag aattttgctg ggtctagaag 3300

catatcgcga caatctgaaa gatcttatta cggtgttcca tcttctggca atactgatac 3360

tgttggcgca gcagtagcca atgagaaaaa atatagtagc atgccagata tctcaggatt 3420

gtctatgtcc gcaaggaaca tgcatttacc aaacaacaag agtggatact gggatccgtc 3480

aagtggagga ggagggtatg gtgcgtctta tggtcggtta agcaatgaat catcgttata 3540

ttctaatttg gggtcacggg tgggagtacc ctcgacttat gatgacattt ctcaatcaag 3600

aggaggctac agagatgcct acagtttgcc acagagtgca acaacaggga ccggatcgct 3660

ttggtccaga cagccctttg agcagtttgg tgtagcggag aggaatggtg ctgttggtga 3720

ggagctcagg aatagatcga atccgatcaa tatagacaac aacgcttctt ctaatgttga 3780

tgcagaggct aagcttcttc agtcgttcag gcactgtatt ctaaagctta ttaaacttga 3840

aggatccgag tggttgtttg gacaaagcga tggagttgat gaagaactga ttgaccgggt 3900

agctgcacga gagaagttta tctatgaagc tgaagctcga gaaataaacc aggtgggtca 3960

catgggggag ccactaattt catcggttcc taactgtgga gatggttgcg tttggagagc 4020

tgatttgatt gtgagctttg gagtttggtg cattcaccgt gtccttgact tgtctctcat 4080

ggagagtcgg cctgagcttt ggggaaagta cacttacgtt ctcaaccgcc tacagggagt 4140

gattgatccg gcgttctcaa agctgcggac accaatgaca ccgtgctttt gccttcagat 4200

tccagcgagc caccagagag cgagtccgac ttcagctaac ggaatgttac ctccggctgc 4260

aaaaccggct aaaggcaaat gcacaaccgc agtcacactt cttgatctaa tcaaagacgt 4320

tgaaatggca atctcttgta gaaaaggccg aaccggtaca gctgcaggtg atgtggcttt 4380

cccaaagggg aaagagaatt tggcttcggt tttgaagcgg tataaacgtc ggttatcgaa 4440

taaaccagta ggtatgaatc aggatggacc cggttcaaga aaaaacgtga ctgcgtacgg 4500

atcattgggt tgaagaagaa gaacattgtg agaaatctca tgatcaaagt gacgtcgaga 4560

gggaagccga agaatcaaaa ctctcgcttt tgattgctcc tctgcttcgt taattgtgta 4620

ttaagaaaag aagaaaaaaa atggattttt gttgcttcag aatttttcgc tctttttttc 4680

ttaatttggt tgtaatgtta tgtttatata catatatcat catcatagga ccatagctac 4740

aaaccgaatc cggtttgtgt aattctatgc ggaatcataa agaaatcgtc ggtttgaaat 4800

gttaaatctc ctaaaccgga tctctgcacg tagctgacac atcgacgcta g 4851

<210> 20

<211> 1703

<212> DNA

<213> Arabidopsis thaliana

<400> 20

gttgcaaata tataaatcaa tcaaaagatt taaaacccac cattcaatct tggtaagtaa 60

cgaaaaaaaa gggaagcaag aagaaccaca gaaaaggggg ctaacaacta gacacgtaga 120

tcttcatctg cccgtccatc taacctacca cactctcatc ttctttttcc cgtgtcagtt 180

tgttatataa gctctcactc tccggtatat ttccaaatac acctaacttg tttagtacac 240

aacagcaaca tcaaactcta ataaacccaa gttggtgtat actataatgg tgatggctgg 300

tgcttcttct ttggatgaga tcagacaggc tcagagagct gatggacctg caggcatctt 360

ggctattggc actgctaacc ctgagaacca tgtgcttcag gcggagtatc ctgactacta 420

cttccgcatc accaacagtg aacacatgac cgacctcaag gagaagttca agcgcatgtg 480

cgacaagtcg acaattcgga aacgtcacat gcatctgacg gaggaattcc tcaaggaaaa 540

cccacacatg tgtgcttaca tggctccttc tctggacacc agacaggaca tcgtggtggt 600

cgaagtccct aagctaggca aagaagcggc agtgaaggcc atcaaggagt ggggccagcc 660

caagtcaaag atcactcatg tcgtcttctg cactacctcc ggcgtcgaca tgcctggtgc 720

tgactaccag ctcaccaagc ttcttggtct ccgtccttcc gtcaagcgtc tcatgatgta 780

ccagcaaggt tgcttcgccg gcggtactgt cctccgtatc gctaaggatc tcgccgagaa 840

caatcgtgga gcacgtgtcc tcgttgtctg ctctgagatc acagccgtta ccttccgtgg 900

tccctctgac acccaccttg actccctcgt cggtcaggct cttttcagtg atggcgccgc 960

cgcactcatt gtggggtcgg accctgacac atctgtcgga gagaaaccca tctttgagat 1020

ggtgtctgcc gctcagacca tccttccaga ctctgatggt gccatagacg gacatttgag 1080

ggaagttggt ctcaccttcc atctcctcaa ggatgttccc ggcctcatct ccaagaacat 1140

tgtgaagagt ctagacgaag cgtttaaacc tttggggata agtgactgga actccctctt 1200

ctggatagcc caccctggag gtccagcgat cctagaccag gtggagataa agctaggact 1260

aaaggaagag aagatgaggg cgacacgtca cgtgttgagc gagtatggaa acatgtcgag 1320

cgcgtgcgtt ctcttcatac tagacgagat gaggaggaag tcagctaagg atggtgtggc 1380

cacgacagga gaagggttgg agtggggtgt cttgtttggt ttcggaccag gtctcactgt 1440

tgagacagtc gtcttgcaca gcgttcctct ctaaacagaa cgcttgcctt ctatctgcct 1500

acctacctac gcaaaacttt aatcctgtct tatgttttat ataatataat cattatatgt 1560

ttacgcaata attaaggaag aattcatttg atgtgatatg tgatatgtgc tggacaggtc 1620

tattcgactg tttttgtact ctcttttttg tgtcttttta caatattaaa tctatgggtc 1680

ttgaatgaca tcaaatcttt gtt 1703

<210> 21

<211> 229

<212> DNA

<213> Arabidopsis thaliana

<400> 21

cctcgaggat cctctagacc tcagctaggg tttatccaga gaatggttcc tgctctactt 60

cctgtccttt tggtttctgt cggatatatt gatcccggga aatgggttgc aaatatcgaa 120

ggaggtgctc gtttcgggta tgacttggtg gcaattactc tgcttttcaa ttttgccgcc 180

atcttatgcc aatatgttgc agctcgcata agcgttaagc ttggtaccc 229

<210> 22

<211> 218

<212> DNA

<213> Artificial Sequence

<220>

<223> fragment comprising the 200nt sense sequence of EIN2

<400> 22

cctcgagtct agattttagt tagggtttat ttagagaatg gtttttgttt tattttttgt 60

ttttttggtt tttgttggat atattgattt tgggaaatgg gttgtaaata ttgaaggagg 120

tgtttgtttt gggtatgatt tggtggtaat tattttgttt tttaattttg ttgttatttt 180

atgttaatat gttgtagttt gtataagtgt tggtaccc 218

<210> 23

<211> 230

<212> DNA

<213> Arabidopsis thaliana

<400> 23

cctcgaggat ccgttgtctg ctctgagatc acagccgtta ccttccgtgg tccctctgac 60

acccaccttg actccctcgt cggtcaggct cttttcagtg atggcgccgc cgcactcatt 120

gtggggtcgg accctgacac atctgtcgga gagaaaccca tctttgagat ggtgtctgcc 180

gctcagacca tccttccaga ctctgatggt gctctagaag cttggtaccc 230

<210> 24

<211> 219

<212> DNA

<213> Artificial Sequence

<220>

<223> fragment comprising the 200nt sense sequence of CHS

<400> 24

cctcgaggtt gtttgttttg agattatagt tgttattttt tgtggttttt ttgatattta 60

ttttgatttt tttgttggtt aggttttttt tagtgatggt gttgttgtat ttattgtggg 120

gttggatttt gatatatttg ttggagagaa atttattttt gagatggtgt ttgttgttta 180

gattattttt ttagattttg atggtgtcta gaggtaccc 219

<210> 25

<211> 219

<212> DNA

<213> Artificial Sequence

<220>

<223> fragment comprising the 200nt antisense sequence of EIN2

<400> 25

caagcttaat gtttatgtga gttgtaatat attggtataa gatggtggta aaattgaaaa 60

gtagagtaat tgttattaag ttatatttga aatgagtatt ttttttgata tttgtaattt 120

attttttggg attaatatat ttgatagaaa ttaaaaggat aggaagtaga gtaggaatta 180

ttttttggat aaattttagt tgaggttcta gaggatccc 219

<210> 26

<211> 219

<212> DNA

<213> Artificial Sequence

<220>

<223> fragment comprising the 200nt antisense sequence of CHS

<400> 26

caagcttcta gagtattatt agagtttgga aggatggttt gagtggtaga tattatttta 60

aagatgggtt tttttttgat agatgtgtta gggtttgatt ttataatgag tgtggtggtg 120

ttattattga aaagagtttg attgatgagg gagttaaggt gggtgttaga gggattatgg 180

aaggtaatgg ttgtgatttt agagtagata atggatccc 219

<210> 27

<211> 300

<212> DNA

<213> Arabidopsis thaliana

<400> 27

agtaattata ttatcagata gatttaggat ggaagctgaa attgtgaatg tgagacctca 60

gctagggttt atccagagaa tggttcctgc tctacttcct gtccttttgg tttctgtcgg 120

atatattgat cccgggaaat gggttgcaaa tatcgaagga ggtgctcgtt tcgggtatga 180

cttggtggca attactctgc ttttcaattt tgccgccatc ttatgccaat atgttgcagc 240

tcgcataagc gttgtgactg gtaaacactt ggctcagatc tgcaatgaag aatatgacaa 300

<210> 28

<211> 300

<212> DNA

<213> Arabidopsis thaliana

<400> 28

tccgtatcgc taaggatctc gccgagaaca atcgtggagc acgtgtcctc gttgtctgct 60

ctgagatcac agccgttacc ttccgtggtc cctctgacac ccaccttgac tccctcgtcg 120

gtcaggctct tttcagtgat ggcgccgccg cactcattgt ggggtcggac cctgacacat 180

ctgtcggaga gaaacccatc tttgagatgg tgtctgccgc tcagaccatc cttccagact 240

ctgatggtgc catagacgga catttgaggg aagttggtct caccttccat ctcctcaagg 300

<210> 29

<211> 240

<212> DNA

<213> Arabidopsis thaliana

<400> 29

tcttcattgc agatctgagc caagtgttta ccagtcacaa cgcttatgcg agctgcaaca 60

tattggcata agatggcggc aaaattgaaa agcagagtaa ttgccaccaa gtcatacccg 120

aaacgagcac ctccttcgat atttgcaacc catttcccgg gatcaatata tccgacagaa 180

accaaaagga caggaagtag agcaggaacc attctctgga taaaccctag ctgaggtctc 240

<210> 30

<211> 300

<212> DNA

<213> Arabidopsis thaliana

<400> 30

gatggaaggt gagaccaact tccctcaaat gtccgtctat ggcaccatca gagtctggaa 60

ggatggtctg agcggcagac accatctcaa agatgggttt ctctccgaca gatgtgtcag 120

ggtccgaccc cacaatgagt gcggcggcgc catcactgaa aagagcctga ccgacgaggg 180

agtcaaggtg ggtgtcagag ggaccacgga aggtaacggc tgtgatctca gagcagacaa 240

cgaggacacg tgctccacga ttgttctcgg cgagatcctt agcgatacgg aggacagtac 300

<210> 31

<211> 4035

<212> DNA

<213> Arabidopsis thaliana

<400> 31

atgggatcta gggttccaat agaaaccatc gaagaagacg gcgaattcga ttgggaagca 60

gcagtcaaag aaatcgactt ggcttgtctt aaaaccacaa acgcttcttc ttcttcgtca 120

tcccatttca ctcctttggc taatccacca attacggcaa atctcactaa gccacctgcg 180

aagagacaat ctactctcga taaattcatc ggcagaaccg aacataaacc ggagaatcat 240

caagttgttt ccgagtgtgg tgttaacgat aacgataata gtcctttagt tgggattgat 300

cctgaggcag ctaaaacttg gatttatcca gtgaatggga gtgttccttt aagagattat 360

cagtttgcta taacgaagac tgctttgttt tcgaatacat tggtggcttt gcctacggga 420

cttggtaaaa cgcttatagc tgcggttgtt atgtataatt acttcagatg gtttccacaa 480

ggtaaaatag tatttgcggc gccttctagg cctcttgtga tgcagcagat tgaggcgtgt 540

cataatattg ttggaatacc acaagaatgg acgattgact tgacgggtca gacatgtcct 600

tcgaaaagag cttttttgtg gaaaagcaaa cgggttttct ttgtcactcc acaagtgtta 660

gagaaggata tacagtcagg aacatgtctt actaactact tggtttgctt ggtgatcgac 720

gaggcacatc gagctttagg gaattattct tattgtgttg tagttcgtga gttgatggcg 780

gtaccgatac agctgagaat actggctctt actgcaactc ctggatcaaa gacacaggcc 840

atccagggta tcattgataa tttgcagata tccacacttg aatatcgaaa tgagagtgac 900

catgatgttt gcccttatgt ccacgacaga aaattagaag tcatcgaggt tcccttgggt 960

caagatgcag atgatgtatc gaaacgcctg tttcatgtta tacgtccata tgcagtcagg 1020

cttaaaaact ttggggttaa tctaaataga gatatacaaa ctttaagtcc acacgaagta 1080

cttatggcaa gggataagtt tcgtcaagca cctctaccag gccttcccca tgtaaatcac 1140

ggagatgtag aatcttgctt tgcagctctt atcactcttt atcatattcg taagctcctt 1200

tctagtcatg gaataagacc agcgtatgag atgctagaag agaaattgaa agaagggcca 1260

tttgctaggt tgatgagtaa gaatgaagat attaggatga cgaagctttt gatgcagcaa 1320

aggttgtcac atggagcacc aagcccaaaa ttgtcgaaga tgttagaaat actggttgat 1380

catttcaaag tgaaagatcc gaagacatca cgggtcatta ttttctcaaa tttcagagga 1440

agcgtaagag acataatgaa cgcattaagt aatattggag atatggtcaa agcaactgag 1500

tttattggtc aaagttcagg taagacattg aaaggccagt cgcaaaaaat tcagcaggct 1560

gttttggaga aatttagagc tggggggttc aatgttattg tcgcaacatc tattggtgaa 1620

gaaggcttgg atatcatgga agttgaccta gttatatgtt ttgatgctaa tgtatctcct 1680

ctgaggatga ttcaacggat gggaagaact ggaaggaaaa ataatggtcg agttgtagtt 1740

cttgcttgtg aaggatcaga aaagaacagc tatatgcgaa agcaagcaag tggacgggct 1800

attaaaaaac acatgcggaa tggaggaaca aatagtttta attttcatcc tagtccaagg 1860

atgattcccc atgtttataa gccagaagtt cagcatgttg agttttcaat caagcaattc 1920

gttccacgtg gaaagaaact acaagaggag tatgccactg agactccagc tttccagaaa 1980

aagcttacac ctgcagagac gcatatgctc gctaagtatt acaacaaccc cgatgaggaa 2040

aagttgagag tgtccttaat tgcgttccct cacttccaga cattgccatc caaggtgcac 2100

aaagtaatgc attcacgtca aacaggcatg ttaattgacg ctatgcagca cttgcaagag 2160

ccaacttttt cagaacagag taaaagcttc ttcactgagt ttcgagctcc tttgggtgaa 2220

agagaagagc ttgatacagg tctgagggtt actaatgatc caaaagatct acactctgtc 2280

cgtgatttgg aagtcaacac atcacagaga aaggcaaaac aagttgaatc tcccacaagc 2340

accttagaga caacagagaa ggattacgaa gaatcttcac ccacacaccg ttatcttttc 2400

agttcagaat gtgcatccgt tgatactctg gggaacgtct tcgtaatgcc agttcctctt 2460

ttattctttc ctaatgttct ggagtcagac aatacgcctc tgcctaaaac agaaaaacaa 2520

cattcttgcc ggaatacatc tcacattgac ttagttccag tagatacttc ggaaaaacat 2580

cggcaagata atatctcatg caagttaaag gaaagattct cgccagacgg tgccagcgag 2640

acactagaga ctcatagcct tgtgaaaagg aactccacca gagtaggtga agatgatgta 2700

gcgaattctg ttggagaaat tgtgttatca tcggatgaag atgactgtga gggattggag 2760

cttagtccac ggctcactaa cttcatcaag agcggcattg ttccagagtc acctgtctat 2820

gaccaagggg aagcgaacag agaagaagat cttgaatttc ctcagctttc ttcacccatg 2880

aggttcagta acgaattggc aggagagtct tctttccctg agagaaaggt tcagcataag 2940

tgcaacgatt ataacattgt gtctacaacc actgaattga gaactcctca gaaggaggta 3000

ggtttggcca acggaacaga atgcttggct gtttctccta ttcctgagga ttggagaact 3060

cccttggcga atctgacaaa cacaaacagc agcgctcgca aagattggcg ggtgagttct 3120

ggagaaaagt tagaaactct tcgacagcct cgcaagttga agagactacg tagacttgga 3180

gattgctcga gtgctgtaaa ggagaattat cctggtatta cagaggcaga ccatatcaga 3240

tctcgttctc gcggtaaaaa gcacattaga ggtaagaaga agatgatcat ggatgatgat 3300

gtccaagtct tcattgacga ggaagctgag gtctcttcgg gagcagagat gtcggctgat 3360

gagaacgaag atgtgactgg cgattcattt gaagatagtt tcatagatga cggaacaatg 3420

cctacagcaa atactcaagc cgagtctggt aaagttgaca tgatggctgt ttacaggcgt 3480

tctcttctca gccagtcacc attaccggcg agatttcgtg atttagccgc atcaagtctg 3540

agtccttatt ctgctggacc cttgacgaga ataaatgaga gcagaagcga ctcagataaa 3600

tcattgtctt ctcttcgaac accaaaaaca acaaactctg agtcaaacca agatgcaatg 3660

atgataggaa acctttcggt agtacaaatc tcgtcagata gccggaaaag gaaatttagc 3720

ttatgcaact cggcgaatgc ccccgtgatt aacttagaaa gcaagtttgc agctcatgca 3780

caagccacgg agaaggaaag ccatgaaggc gtgagaagca atgcaggtgc gttagagtac 3840

aatgatgatg atgatgatgc attctttgcg acactagact ttgatgcaat ggaagcacaa 3900

gccacattgt tattgtcgaa acagagatcc gaagcaaaag agaaagaaga cgcaacggtt 3960

atacctaatc caggcatgca gagaagtgat ggtatggaga aagatgcacc atcttttgat 4020

cttggtctgt ggtga 4035

<210> 32

<211> 2310

<212> DNA

<213> Brassica napus

<400> 32

atgtcaaatg aaaataaaaa tataaaaact aaatttcatc ctagttcaag gatgattccc 60

catgtttata agccagaagt tcagcatgtt aagttttcga tcgagcaatt cattccacgt 120

ggaaagaagc tacaagatga gcctgccact gagactccag ctttcaagaa aaagcttaca 180

ccggaagaga tggatatgct cgccaagtat ttcaaaccca acgaggaaaa gtggagagtt 240

tccttgattg ctttccctca cttccaaaca ttgccatcca aagtgcacaa agtaatgcat 300

tcacgccaaa caagcatatt aattgatgct atgcagcatc tgcaagagac aactttgaca 360

gagcaaagta aaagtttctt cattaagtat ggagctcctt tggctgaaag agatgagctt 420

gacgcaggtc tgagggttgg tgatgatccg aaagatttac cctcttccga tgatttggat 480

gtcaacacat cacagagaaa ggcaaaacaa attttagaat ctcccacaag cacattagag 540

actacagaga aggatttcga agcatcttca cccacacact gttatctttt cagttcagaa 600

tgtgcgtccg ttgatactct ggggaaggtc tttgtattgc cggttcctct ctcattctct 660

tctaatgtac cagggtcaga ctgcgtggga agagaaaaag aactttcttc cccgaataag 720

tcccacactg acgttgttcc gatagatagt tcctcaaaac atcggcaaga taatatttca 780

tgcaagttaa agcaaggatt cttgccagat tgtgccaacg agactttgga gtcccaaagc 840

cttttgaaaa ggcactccac cgatgtaggt aaaggagata tagagaattg tgctggagaa 900

attatgatat catcggatga agaagacgac tgtgaggatt tggagcttag tccaaggctc 960

actaacttca tcaagagtgg cgttgttcca gattcacctg tctatgacca agttgcatac 1020

gaagcaaaca gagaagaaga ccttgatctt ccacacacga gtttaactaa tgaattggca 1080

gaagagccat cgacacctga gaaaaaggtt cacattgctt ctacggccaa tgaattcaga 1140

actcctcaga aggaagaaga tttagccaac gaaacagaaa gcttcgctgt ttctccaatg 1200

cctgaggagt ggagaactcc cttggcgaat atcaccaacg caagcagcag cgctagcaaa 1260

gattggcgcg tgagttcggg agaaaagtca gaaactcttc gacagcctcg caagttgaag 1320

agacttcgta gacttggaga ttgctcgagt gctgtgaagg agaataatcc tggtattgca 1380

aagacagacc atatcagatc tcgttctcgc agtgtaaaga acataagagg taaaatgatt 1440

ctgtatttcc ttttgctctg tgttcaaggc aagaagaaga tacgcgcgga taataatgct 1500

agaatcttca ttgaagcgga agctgaggtg tcttcggaat cagaaatgtc ggttgatgag 1560

aacgtagatt tgaccagcga ttcatttgaa gatagcttca tagatgacgg tacaatgcct 1620

acagcaaata ctcaagccga gtgtgctaaa gttgacatga tggccgttta caggtatata 1680

tcgaatcaaa acaagtcttt cttctactat gatttactaa gaatcataag ctatggtttc 1740

cacagacgtt ctctactcag ccaatcacca ttaccggcaa gatttcgtga tgtagctgca 1800

tcaagtccga gtccttattc ttctggtctc ttgaagacaa taaatgagag cagaagcgac 1860

tcagataaat cattgtcttc tcttagaacc ccacaaacaa cgaacaacga gtcaaacaag 1920

gatgcaatgg ccacaggaga cctttcggta gcacaaatct caacagacag ccggaaaagg 1980

aaattcagct tatgcaactc agcgaatgtc ccagtgatta acttggaaaa caagtttgaa 2040

gctcatgcac aagccacgga gaaggaaagc catgaaggtc cgagaagcaa tgcaggtgca 2100

tcacagtaca aggatgagga tgaagatgat gatgcattct acgcgacact ggactttgat 2160

gccatggaag cgcatgcgac attgctattg tcgaaacaaa ggtcagaaac gaaaacaaaa 2220

gaagatgcat cggtgaaacc tcatttgggc aatcagagga atgatggttt gccgaaggat 2280

gggccatctt ttgatcttgg tttgtggtga 2310

<210> 33

<211> 1822

<212> DNA

<213> Artificial Sequence

<220>

<223> hpFANCM-At[wt]

<400> 33

ggctcgagaa ccgaattcta atacgactca ctatagggtc aggaacatgt cttactaact 60

acttggtttg cttggtgatc gacgaggcac atcgagcttt agggaattat tcttattgtg 120

ttgtagttcg tgagttgatg gcggtaccga tacagctgag aatactggct cttactgcaa 180

ctcctggatc aaagacacag gccatccagg gtatcattga taatttgcag atatccacac 240

ttgaatatcg aaatgagagt gaccatgatg tttgccctta tgtccccgac agaaaattag 300

aagtcatcga ggttcccttg ggtcaagatg cagatgatgt atcgaaacgc ctgtttcatg 360

ttatacgtcc atatgcagtc aggcttaaaa actttggggt taatctaaat agagatatac 420

aaactttaag tccacacgaa gtacttatgg caagggataa gtttcgtcaa gcacctctac 480

caggccttcc ccatgtaaat cacggagatg tagaatcttg ctttgcagct cttatcaggt 540

aaggaaataa ttattttctt ttttcctttt agtataaaat agttaagtga tgttaattag 600

tatgattata ataatatagt tgttataatt gtgaaaaaat aatttataaa tatattgttt 660

acataaacaa catagtaatg taaaaaaata tgacaagtga tgtgtaagac gaagaagata 720

aaagttgaga gtaagtatat tatttttaat gaatttgatc gaacatgtaa gatgatatac 780

tagcattaat atttgtttta atcataatag taattctagc tggtttgatg aattaaatat 840

caatgataaa atactatagt aaaaataaga ataaataaat taaaataata tttttttatg 900

attaatagtt tattatataa ttaaatatct ataccattac taaatatttt agtttaaaag 960

ttaataaata ttttgttaga aattccaatc tgcttgtaat ttatcaataa acaaaatatt 1020

aaataacaag ctaaagtaac aaataatatc aaactaatag aaacagtaat ctaatgtaac 1080

aaaacataat ctaatgctaa tataacaaag cgcaagatct atcattttat atagtattat 1140

tttcaatcaa cattcttatt aatttctaaa taatacttgt agttttatta acttctaaat 1200

ggattgacta ttaattaaat gaattagtcg aacatgaata aacaaggtaa catgatagat 1260

catgtcattg tgttatcatt gatcttacat ttggattgat tacagttgat aagagctgca 1320

aagcaagatt ctacatctcc gtgatttaca tggggaaggc ctggtagagg tgcttgacga 1380

aacttatccc ttgccataag tacttcgtgt ggacttaaag tttgtatatc tctatttaga 1440

ttaaccccaa agtttttaag cctgactgca tatggacgta taacatgaaa caggcgtttc 1500

gatacatcat ctgcatcttg acccaaggga acctcgatga cttctaattt tctgtcgggg 1560

acataagggc aaacatcatg gtcactctca tttcgatatt caagtgtgga tatctgcaaa 1620

ttatcaatga taccctggat ggcctgtgtc tttgatccag gagttgcagt aagagccagt 1680

attctcagct gtatcggtac cgccatcaac tcacgaacta caacacaata agaataattc 1740

cctaaagctc gatgtgcctc gtcgatcacc aagcaaacca agtagttagt aagacatgtt 1800

cctgaccccg ggatccaagc tt 1822

<210> 34

<211> 1822

<212> DNA

<213> Artificial Sequence

<220>

<223> hpFANCM-At[G:U]

<400> 34

ggctcgagaa ccgaattcta atacgactca ctatagggtt aggaatatgt tttattaatt 60

atttggtttg tttggtgatt gatgaggtat attgagtttt agggaattat ttttattgtg 120

ttgtagtttg tgagttgatg gtggtattga tatagttgag aatattggtt tttattgtaa 180

tttttggatt aaagatatag gttatttagg gtattattga taatttgtag atatttatat 240

ttgaatattg aaatgagagt gattatgatg tttgttttta tgtttttgat agaaaattag 300

aagttattga ggtttttttg ggttaagatg tagatgatgt attgaaatgt ttgttttatg 360

ttatatgttt atatgtagtt aggtttaaaa attttggggt taatttaaat agagatatat 420

aaattttaag tttatatgaa gtatttatgg taagggataa gttttgttaa gtatttttat 480

taggtttttt ttatgtaaat tatggagatg tagaattttg ttttgtagtt tttattaggt 540

aaggaaataa ttattttctt ttttcctttt agtataaaat agttaagtga tgttaattag 600

tatgattata ataatatagt tgttataatt gtgaaaaaat aatttataaa tatattgttt 660

acataaacaa catagtaatg taaaaaaata tgacaagtga tgtgtaagac gaagaagata 720

aaagttgaga gtaagtatat tatttttaat gaatttgatc gaacatgtaa gatgatatac 780

tagcattaat atttgtttta atcataatag taattctagc tggtttgatg aattaaatat 840

caatgataaa atactatagt aaaaataaga ataaataaat taaaataata tttttttatg 900

attaatagtt tattatataa ttaaatatct ataccattac taaatatttt agtttaaaag 960

ttaataaata ttttgttaga aattccaatc tgcttgtaat ttatcaataa acaaaatatt 1020

aaataacaag ctaaagtaac aaataatatc aaactaatag aaacagtaat ctaatgtaac 1080

aaaacataat ctaatgctaa tataacaaag cgcaagatct atcattttat atagtattat 1140

tttcaatcaa cattcttatt aatttctaaa taatacttgt agttttatta acttctaaat 1200

ggattgacta ttaattaaat gaattagtcg aacatgaata aacaaggtaa catgatagat 1260

catgtcattg tgttatcatt gatcttacat ttggattgat tacagttgat aagagctgca 1320

aagcaagatt ctacatctcc gtgatttaca tggggaaggc ctggtagagg tgcttgacga 1380

aacttatccc ttgccataag tacttcgtgt ggacttaaag tttgtatatc tctatttaga 1440

ttaaccccaa agtttttaag cctgactgca tatggacgta taacatgaaa caggcgtttc 1500

gatacatcat ctgcatcttg acccaaggga acctcgatga cttctaattt tctgtcgggg 1560

acataagggc aaacatcatg gtcactctca tttcgatatt caagtgtgga tatctgcaaa 1620

ttatcaatga taccctggat ggcctgtgtc tttgatccag gagttgcagt aagagccagt 1680

attctcagct gtatcggtac cgccatcaac tcacgaacta caacacaata agaataattc 1740

cctaaagctc gatgtgcctc gtcgatcacc aagcaaacca agtagttagt aagacatgtt 1800

cctgaccccg ggatccaagc tt 1822

<210> 35

<211> 1818

<212> DNA

<213> Artificial Sequence

<220>

<223> hpFANCM-Bn[wt]

<400> 35

ggatccttgg tacctaatac gactcactat agggagaaat tatgatatca tcggatgaag 60

aagacgactg tgaggatttg gagcttagtc caaggctcac taacttcatc aagagtggcg 120

ttgttccaga ttcacctgtc tatgaccaag ttgcatacga agcaaacaga gaagaagacc 180

ttgatcttcc acacacgagt ttaactaatg aattggcaga agagccatcg acacctgaga 240

aaaaggttca cattgcttct acggccaatg aattcagaac cccaacgaag gaagaagatt 300

tagccaacga aacagaaagc ttcgctgttt ctccaatgcc tgaggagtgg agaactccct 360

tggcgaatat caccaacgca agcagcagcg ctagcaaaga ttggcgcgtg agttcgggag 420

aaaagtcaga aactcttcga cagcctcgca agttgaagag acttcgtaga cttggagatt 480

gctcgagtgc tgtgaaggag aataatcctg gtattgcaaa gacagaccat atcgtaagga 540

aataattatt ttcttttttc cttttagtat aaaatagtta agtgatgtta attagtatga 600

ttataataat atagttgtta taattgtgaa aaaataattt ataaatatat tgtttacata 660

aacaacatag taatgtaaaa aaatatgaca agtgatgtgt aagacgaaga agataaaagt 720

tgagagtaag tatattattt ttaatgaatt tgatcgaaca tgtaagatga tatactagca 780

ttaatatttg ttttaatcat aatagtaatt ctagctggtt tgatgaatta aatatcaatg 840

ataaaatact atagtaaaaa taagaataaa taaattaaaa taatattttt ttatgattaa 900

tagtttatta tataattaaa tatctatacc attactaaat attttagttt aaaagttaat 960

aaatattttg ttagaaattc caatctgctt gtaatttatc aataaacaaa atattaaata 1020

acaagctaaa gtaacaaata atatcaaact aatagaaaca gtaatctaat gtaacaaaac 1080

ataatctaat gctaatataa caaagcgcaa gatctatcat tttatatagt attattttca 1140

atcaacattc ttattaattt ctaaataata cttgtagttt tattaacttc taaatggatt 1200

gactattaat taaatgaatt agtcgaacat gaataaacaa ggtaacatga tagatcatgt 1260

cattgtgtta tcattgatct tacatttgga ttgattacag gatatggtct gtctttgcaa 1320

taccaggatt attctccttc acagcactcg agcaatctcc aagtctacga agtctcttca 1380

acttgcgagg ctgtcgaaga gtttctgact tttctcccga actcacgcgc caatctttgc 1440

tagcgctgct gcttgcgttg gtgatattcg ccaagggagt tctccactcc tcaggcattg 1500

gagaaacagc gaagctttct gtttcgttgg ctaaatcttc ttccttcgtt ggggttctga 1560

attcattggc cgtagaagca atgtgaacct ttttctcagg tgtcgatggc tcttctgcca 1620

attcattagt taaactcgtg tgtggaagat caaggtcttc ttctctgttt gcttcgtatg 1680

caacttggtc atagacaggt gaatctggaa caacgccact cttgatgaag ttagtgagcc 1740

ttggactaag ctccaaatcc tcacagtcgt cttcttcatc cgatgatatc ataatttctc 1800

tctagaaagg atcccggg 1818

<210> 36

<211> 1818

<212> DNA

<213> Artificial Sequence

<220>

<223> hpFANCM-Bn[G:U]

<400> 36

ggatccttgg tacctaatac gactcactat agggagaaat tatgatatta ttggatgaag 60

aagatgattg tgtggatttg gagtttagtt taaggtttat taattttatt aagagtggtg 120

ttgttttaga tttatttgtt tatgattaag ttgtatatga agtaaatagt gaagaagatt 180

ttgatttttt atatatgagt ttaattaatg aattggtaga agagttattg atatttgaga 240

aaaaggttta tattgttttt atggttaatg aatttagaat tttaatgaag gaagaagatt 300

tagttaatga aatagaaagt tttgttgttt ttttaatgtt tgaggagtgg agaatttttt 360

tggtgaatat tattaatgta agtagtagtg ttagtaaaga ttggtgtgtg agtttgggag 420

aaaagttaga aattttttga tagttttgta agttgaagag attttgtaga tttggagatt 480

gtttgagtgt tgtgaaggag aataattttg gtattgtaaa gatagattat attgtaagga 540

aataattatt ttcttttttc cttttagtat aaaatagtta agtgatgtta attagtatga 600

ttataataat atagttgtta taattgtgaa aaaataattt ataaatatat tgtttacata 660

aacaacatag taatgtaaaa aaatatgaca agtgatgtgt aagacgaaga agataaaagt 720

tgagagtaag tatattattt ttaatgaatt tgatcgaaca tgtaagatga tatactagca 780

ttaatatttg ttttaatcat aatagtaatt ctagctggtt tgatgaatta aatatcaatg 840

ataaaatact atagtaaaaa taagaataaa taaattaaaa taatattttt ttatgattaa 900

tagtttatta tataattaaa tatctatacc attactaaat attttagttt aaaagttaat 960

aaatattttg ttagaaattc caatctgctt gtaatttatc aataaacaaa atattaaata 1020

acaagctaaa gtaacaaata atatcaaact aatagaaaca gtaatctaat gtaacaaaac 1080

ataatctaat gctaatataa caaagcgcaa gatctatcat tttatatagt attattttca 1140

atcaacattc ttattaattt ctaaataata cttgtagttt tattaacttc taaatggatt 1200

gactattaat taaatgaatt agtcgaacat gaataaacaa ggtaacatga tagatcatgt 1260

cattgtgtta tcattgatct tacatttgga ttgattacag gatatggtct gtctttgcaa 1320

taccaggatt attctccttc acagcactcg agcaatctcc aagtctacga agtctcttca 1380

acttgcgagg ctgtcgaaga gtttctgact tttctcccga actcacgcgc caatctttgc 1440

tagcgctgct gcttgcgttg gtgatattcg ccaagggagt tctccactcc tcaggcattg 1500

gagaaacagc gaagctttct gtttcgttgg ctaaatcttc ttccttcgtt ggggttctga 1560

attcattggc cgtagaagca atgtgaacct ttttctcagg tgtcgatggc tcttctgcca 1620

attcattagt taaactcgtg tgtggaagat caaggtcttc ttctctgttt gcttcgtatg 1680

caacttggtc atagacaggt gaatctggaa caacgccact cttgatgaag ttagtgagcc 1740

ttggactaag ctccaaatcc tcacagtcgt cttcttcatc cgatgatatc ataatttctc 1800

tctagaaagg atcccggg 1818

<210> 37

<211> 2274

<212> DNA

<213> Brassica napus

<400> 37

atggttagtc tgcgctccac agaaaacact ccggcttcgg aaatggccag cgacggcaaa 60

acggagaaag atggctccgg cgactcaccc acttctgttc tcagcgatga ggaaaactgt 120

gaagagaaaa ctgctactgt tgctgtagag gaagagatac ttctagccaa gaatggagat 180

tcgtctctta tctctgaggc catggctcag gaagaagagc agcttctcaa aatccgggaa 240

gatgaagaga ttgctaaacg tgctgctggc tctggtgaag ctcctgatct gaatgatact 300

cagtttacta aacttgatga gctcttgacc caaacccagc tctactctga gtttctcctt 360

gagaaaatgg aggatatcac caaaaatggg atagaaggtg agacccaaaa ggccgagcct 420

gagcctgagc ctgagcccga gaagaaaggc cgtggacgta aaagaaaggc tgctcctcag 480

ggcgacagta tgaaggctaa gaaagctgtt gctgctatga tttcaagatc caaagaaggc 540

cgtgaatctg ccgactcaga tctgacagag gaagaaagag tcatgaaaga gcagggtgaa 600

cttgttcctc ttctgactgg cggaaagtta aagtcttatc agctcaaagg tgtcaaatgg 660

ctgatatcat tgtggcaaaa tggtttgaat ggaattttag ctgatcaaat gggtcttgga 720

aagacaattc aaaccattgg tttcctatca cacctcaaag gaaatgggtt ggatggtcca 780

tatctagtca ttgccccact ctctactctt tcaaactgga tgaatgagat cgctaggttc 840

acgccttcca ttaatgcaat catttaccat ggagataaga aagaaaggga tgagctcagg 900

aagaggcaca tgcccagaac tgttggtccg aagttcccta tagtcataac ttcttatgag 960

gttgctatga atgatgctaa aaagaatctg cggcactatc catggaaata tgttgtgatt 1020

gatgagggtc acaggttgaa aaaccacaag tgtaaactgc tgagggagct aagatacttg 1080

aatatggaga acaaacttct gctgacagga acacctctgc aaaataattt gtctgagctt 1140

tggtcactgt tgaattttat tctgcctgac atctttgcat cacatgacga atttgaatca 1200

tggtttgatt tttctggaaa gaataataat gaagcaacta aggaagaagg agaagagaaa 1260

agaagagctc aagtggttgc gaaacttcat aatatactac gacctttcat cctccggaga 1320

atgaaatgtg atgttgagct ctcacttccc cggaaaaaag agattatcat ctatgctaca 1380

atgacggacc atcagaagaa gttccaggaa catcttgtga accacacctt ggaagcacac 1440

attagagatg atactgtccg aggtcatggc ttgaagggaa agcttaacaa tcttgctatt 1500

caacttcgaa agaactgcaa ccatcctgac cttcttgtgg ggcaactaga tggctcatat 1560

ctctacccac ctttggaaga cattgtggga cagtgcggta aattccgctt attggagaga 1620

ttgcttgttc ggttatttgc caaaaatcac agagtcctta tcttctccca gtggacaaaa 1680

atactggaca ttatggatta ctacttcagt gagaaggggt ttgaggtttg ccgaatcgac 1740

ggtagtgtga aactagaaga aaggagaaga cagatccaag aattcaatga tgagaagagc 1800

aactgcagga tatttcttct cagtaccaga gccggaggac tcggaattaa tcttactgct 1860

gcagatacat gcatcctcta cgatagcgat tggaaccctc aaatggactt gcaagccatg 1920

gacagatgcc acagaattgg tcagacaaaa cctgttcatg tttacaggct tgcgacggct 1980

cagtcaatag agggccgagt tctgaaacga gcatacagta agcttaagct ggaacatgtg 2040

gttattggca aggggcagtt tcatcaagaa cgtgccaagt cttcaacacc gttagaggaa 2100

gatgacatac tggcgttgct taaggacgac gaaaatgctg aagataaact gatacaaacc 2160

gacataagcg aggaggatct tgacagggtg cttgaccgta gtgatctgat gattacctta 2220

ccgggcgaga ctcaagcaca tgaagctttt ccagtgaagg gtccgggttg ggaa 2274

<210> 38

<211> 1824

<212> DNA

<213> Artificial Sequence

<220>

<223> hpDDM1-Bn[wt]

<400> 38

ggatccttgg tacctaatac gactcactat agggagctgt tgctgctatg atttcaagat 60

ccaaagaagg ccgtgaatct gccgactcag atctgacaga ggaagaaaga gtcatgaatg 120

agcagggtga acttgttcct cttctgactg gcggaaagtt aaagtcttat cagctcaaag 180

gtgtcaaatg gctgatatca ttgtggcaaa atggtttgaa tggaatttta gctgatcaaa 240

tgggtcttgg aaagacaatt caaaccattg gtttcctatc accccaacaa ggaaatgggt 300

tggatggtcc atatctagtc attgccccac tctctactct ttcaaaagcg attggaaccc 360

tcaaatggac ttgcaagcca tggacagatg ccacagaatt ggtcagacaa aacctgttca 420

tgtttacagg cttgcgacgg ctcagtcaat agagggccga gttctgaaac gagcatacag 480

taagcttaag ctggaacatg tggttattgg caaggggcag tttcatcaag aacgtggtaa 540

ggaaataatt attttctttt ttccttttag tataaaatag ttaagtgatg ttaattagta 600

tgattataat aatatagttg ttataattgt gaaaaaataa tttataaata tattgtttac 660

ataaacaaca tagtaatgta aaaaaatatg acaagtgatg tgtaagacga agaagataaa 720

agttgagagt aagtatatta tttttaatga atttgatcga acatgtaaga tgatatacta 780

gcattaatat ttgttttaat cataatagta attctagctg gtttgatgaa ttaaatatca 840

atgataaaat actatagtaa aaataagaat aaataaatta aaataatatt tttttatgat 900

taatagttta ttatataatt aaatatctat accattacta aatattttag tttaaaagtt 960

aataaatatt ttgttagaaa ttccaatctg cttgtaattt atcaataaac aaaatattaa 1020

ataacaagct aaagtaacaa ataatatcaa actaatagaa acagtaatct aatgtaacaa 1080

aacataatct aatgctaata taacaaagcg caagatctat cattttatat agtattattt 1140

tcaatcaaca ttcttattaa tttctaaata atacttgtag ttttattaac ttctaaatgg 1200

attgactatt aattaaatga attagtcgaa catgaataaa caaggtaaca tgatagatca 1260

tgtcattgtg ttatcattga tcttacattt ggattgatta cagtcacgtt cttgatgaaa 1320

ctgccccttg ccaataacca catgttccag cttaagctta ctgtatgctc gtttcagaac 1380

tcggccctct attgactgag ccgtcgcaag cctgtaaaca tgaacaggtt ttgtctgacc 1440

aattctgtgg catctgtcca tggcttgcaa gtccatttga gggttccaat cgcttttgaa 1500

agagtagaga gtggggcaat gactagatat ggaccatcca acccatttcc ttgttggggt 1560

gataggaaac caatggtttg aattgtcttt ccaagaccca tttgatcagc taaaattcca 1620

ttcaaaccat tttgccacaa tgatatcagc catttgacac ctttgagctg ataagacttt 1680

aactttccgc cagtcagaag aggaacaagt tcaccctgct ctttcatgac tctttcttcc 1740

tctgtcagat ctgagtcggc agattcacgg ccttctttgg atcttgaaat catagcagca 1800

acagcttcta gaaaggatcc cggg 1824

<210> 39

<211> 1823

<212> DNA

<213> Artificial Sequence

<220>

<223> hpDDM1-Bn[G:U]

<400> 39

ggatccttgg tacctaatac gactcactat agggttgttg ttgttatgat tttaagattt 60

aaagaaggtt gtgaatttgt tgatttagat ttgatagagg aagaaagagt tatgaatgag 120

tagggtgaat ttgttttttt tttgattggt ggaaagttaa agttttatta gtttaaaggt 180

gttaaatggt tgatattatt gtggtaaaat ggtttgaatg gaattttagt tgattaaatg 240

ggttttggaa agataattta aattattggt tttttattat attttaaagg aaatgggttg 300

gatggtttat atttagttat tgttttattt tttatttttt taaaagtgat tggaattttt 360

aaatggattt gtaagttatg gatagatgtt atagaattgg ttagataaaa tttgtttatg 420

tttataggtt tgtgatggtt tagttaatag agggttgagt tttgaaatga gtatatagta 480

agtttaagtt ggaatatgtg gttattggta aggggtagtt ttattaagaa tgtgtggtaa 540

ggaaataatt attttctttt ttccttttag tataaaatag ttaagtgatg ttaattagta 600

tgattataat aatatagttg ttataattgt gaaaaaataa tttataaata tattgtttac 660

ataaacaaca tagtaatgta aaaaaatatg acaagtgatg tgtaagacga agaagataaa 720

agttgagagt aagtatatta tttttaatga atttgatcga acatgtaaga tgatatacta 780

gcattaatat ttgttttaat cataatagta attctagctg gtttgatgaa ttaaatatca 840

atgataaaat actatagtaa aaataagaat aaataaatta aaataatatt tttttatgat 900

taatagttta ttatataatt aaatatctat accattacta aatattttag tttaaaagtt 960

aataaatatt ttgttagaaa ttccaatctg cttgtaattt atcaataaac aaaatattaa 1020

ataacaagct aaagtaacaa ataatatcaa actaatagaa acagtaatct aatgtaacaa 1080

aacataatct aatgctaata taacaaagcg caagatctat cattttatat agtattattt 1140

tcaatcaaca ttcttattaa tttctaaata atacttgtag ttttattaac ttctaaatgg 1200

attgactatt aattaaatga attagtcgaa catgaataaa caaggtaaca tgatagatca 1260

tgtcattgtg ttatcattga tcttacattt ggattgatta cagtcacgtt cttgatgaaa 1320

ctgccccttg ccaataacca catgttccag cttaagctta ctgtatgctc gtttcagaac 1380

tcggccctct attgactgag ccgtcgcaag cctgtaaaca tgaacaggtt ttgtctgacc 1440

aattctgtgg catctgtcca tggcttgcaa gtccatttga gggttccaat cgcttttgaa 1500

agagtagaga gtggggcaat gactagatat ggaccatcca acccatttcc ttgttggggt 1560

gataggaaac caatggtttg aattgtcttt ccaagaccca tttgatcagc taaaattcca 1620

ttcaaaccat tttgccacaa tgatatcagc catttgacac ctttgagctg ataagacttt 1680

aactttccgc cagtcagaag aggaacaagt tcaccctgct ctttcatgac tctttcttcc 1740

tctgtcagat ctgagtcggc agattcacgg ccttctttgg atcttgaaat catagcagca 1800

acagttctag aaaggatccc ggg 1823

<210> 40

<211> 720

<212> DNA

<213> Artificial Sequence

<220>

<223> EGFP

<400> 40

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccttcaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210> 41

<211> 1262

<212> DNA

<213> Artificial Sequence

<220>

<223> hpEGFP[wt]

<400> 41

gctagctaat acgactcact atagggcagc agcacggggc cgtcgccgat gggggtgttc 60

tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag 120

ttcaccttga tgccgttctt ctgcttgtcg gccatgatgt atacgttgtg gctgttgaag 180

ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc 240

tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg 300

ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg 360

aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaagcg 420

aaggtggtca ctagtgtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc 480

agggtctaga ccgcgtcggc atccggtcag tggcagtgaa gggcgaacag ttcctgatta 540

gggggatgaa gctacctggt ccgaaccaca aaccgttcta ctttactggc tttggtcgtc 600

atgaagatgc ggacttgcgt ggcaaaggat tcgataacgt gctgatggtg cacgaccacg 660

cattaatgga ctttaccttt taatggggaa tgaagctacc tggtccgaac tcctaccgta 720

cctcgcatta cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtat 780

ttaggtgaca ctatagccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 840

cccacactag tgaccacctt cgcttacggc gtgcagtgct tcagccgcta ccccgaccac 900

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 960

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1020

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1080

gggcacaagc tggagtacaa cttcaacagc cacaacgtat acatcatggc cgacaagcag 1140

aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 1200

ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgccgtcg 1260

ac 1262

<210> 42

<211> 1262

<212> DNA

<213> Artificial Sequence

<220>

<223> hpEGFP[G:U]

<400> 42

gctagctaat acgactcact atagggcagc agcacggggc cgtcgccgat gggggtgttc 60

tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag 120

ttcaccttga tgccgttctt ctgcttgtcg gccatgatgt atacgttgtg gctgttgaag 180

ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc 240

tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg 300

ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg 360

aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaagcg 420

aaggtggtca ctagtgtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc 480

agggtctaga ccgcgtcggc atccggtcag tggcagtgaa gggcgaacag ttcctgatta 540

gggggatgaa gctacctggt ccgaaccaca aaccgttcta ctttactggc tttggtcgtc 600

atgaagatgc ggacttgcgt ggcaaaggat tcgataacgt gctgatggtg cacgaccacg 660

cattaatgga ctttaccttt taatggggaa tgaagctacc tggtccgaac tcctaccgta 720

cctcgcatta cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtat 780

ttaggtgaca ctatagcctt gaagtttatt tgtattattg gtaagttgtt tgtgttttgg 840

tttatattag tgattatttt tgtttatggt gtgtagtgtt ttagttgtta ttttgattat 900

atgaagtagt atgatttttt taagtttgtt atgtttgaag gttatgttta ggagtgtatt 960

atttttttta aggatgatgg taattataag atttgtgttg aggtgaagtt tgagggtgat 1020

attttggtga attgtattga gttgaagggt attgatttta aggaggatgg taatattttg 1080

gggtataagt tggagtataa ttttaatagt tataatgtat atattatggt tgataagtag 1140

aagaatggta ttaaggtgaa ttttaagatt tgttataata ttgaggatgg tagtgtgtag 1200

tttgttgatt attattagta gaatattttt attggtgatg gttttgtgtt gttgttgtcg 1260

ac 1262

<210> 43

<211> 1259

<212> DNA

<213> Artificial Sequence

<220>

<223> ledEGFP[wt]

<400> 43

gctagctaat acgactcact atagggtgtc gccctcgaac ttcacctcgg cgcgggtctt 60

gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc 120

ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc 180

gtaggtgaag gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat 240

gaacttcagg gtctagaccg cgtcggcatc cggtcagtgg cagtgaaggg cgaacagttc 300

ctgattaggg ggatgaagct acctggtccg aaccacaaac cgttctactt tactggcttt 360

ggtcgtcatg aagatgcgga cttgcgtggc aaaggattcg accctgaagt tcatctgcac 420

caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca 480

gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 540

cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 600

cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 660

cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 720

cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 780

caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 840

cgacggcccc gtgctgctgc ctaacgtgct gatggtgcac gaccacgcat taatggactt 900

taccttttaa tggggaatga agctacctgg tccgaactcc taccgtacct cgcattaccc 960

ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggcag cagcacgggg 1020

ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg 1080

atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata 1140

tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc 1200

ttgaagtcga tgcccttcag ctcgatgcgg ttcaccattg tcgggataca ctcgtcgac 1259

<210> 44

<211> 1259

<212> DNA

<213> Artificial Sequence

<220>

<223> ledEGFP[G:U]

<400> 44

gctagctaat acgactcact atagggtgtc gccctcgaac ttcacctcgg cgcgggtctt 60

gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc 120

ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc 180

gtaggtgaag gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat 240

gaacttcagg gtctagaccg cgtcggcatc cggtcagtgg cagtgaaggg cgaacagttc 300

ctgattaggg ggatgaagct acctggtccg aaccacaaac cgttctactt tactggcttt 360

ggtcgtcatg aagatgcgga cttgcgtggc aaaggattcg attttgaagt ttatttgtat 420

tattggtaag ttgtttgtgt tttggtttat ttttgtgatt atttttattt atggtgtgta 480

gtgttttagt tgttattttg attatatgaa gtagtatgat ttttttaagt ttgttatgtt 540

tgaaggttat gtttaggagt gtattatttt ttttaaggat gatggtaatt ataagatttg 600

tgttgaggtg aagtttgagg gtgatatttt ggtgaattgt attgagttga agggtattga 660

ttttaaggag gatggtaata ttttggggta taagttggag tataattata atagttataa 720

tgtttatatt atggttgata agtagaagaa tggtattaag gtgaatttta agatttgtta 780

taatattgag gatggtagtg tgtagtttgt tgattattat tagtagaata tttttattgg 840

tgatggtttt gtgttgttgt ttaacgtgct gatggtgcac gaccacgcat taatggactt 900

taccttttaa tggggaatga agctacctgg tccgaactcc taccgtacct cgcattaccc 960

ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggcag cagcacgggg 1020

ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg 1080

atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata 1140

tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc 1200

ttgaagtcga tgcccttcag ctcgatgcgg ttcaccattg tcgggataca ctcgtcgac 1259

<210> 45

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<223> hpGUS[G:U]

<400> 45

ttgtgttggt atttggttag tggtagtgaa gggtgaatag tttttgatta attataaatt 60

gttttatttt attggttttg gttgttatga agatgtggat ttgtgtggta aaggatttga 120

taatgtgttg atggtgtatg attatgtatt aatggattgg attggggtta atttttattg 180

tattttgtat tatttttatg 200

<210> 46

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<223> hpGUS[1:4]

<400> 46

tcgggtccgc aaccgctcac tgggagtcaa gcgcgtacac ttcgtgaata agcactaacg 60

gttgtacatt agtgggtttc gtcctcaaga acatggggag ttgggtgcca atggaatcgt 120

taaggtggtg aaggtccacc acctcgcttt attggtctgc attcggggca agtccaaccc 180

tacgtcggat ttcccatacc 200

<210> 47

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<223> hpGUS[2:10]

<400> 47

tcgcgtcgcg atccggtctc tggcagtgtt gggcgaactc ttcctgatat accacaaagg 60

gttctactaa actggcttac gtcgtcatct agatgcggtg ttgcgtgggt aaggattcct 120

taacgtgcac atggtgcagc accacgcaaa aatggactcc attggggcgt actcctacgc 180

tacctcgcta tacccttagc 200

<210> 48

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<223> hpEIN2[G:U]

<400> 48

gattttagtt agggtttatt tagagaatgg tttttgtttt attttttgtt tttttggttt 60

ttgttggata tattgatttt gggaaatggg ttgtaaatat tgaaggaggt gtttgttttg 120

ggtatgattt ggtggtaatt attttgtttt ttaattttgt tgttatttta tgttaatatg 180

ttgtagtttg tataagtgtt 200

<210> 49

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<223> hpCHS[G:U]

<400> 49

gttgtttgtt ttgagattat agttgttatt ttttgtggtt tttttgatat ttattttgat 60

ttttttgttg gttaggtttt ttttagtgat ggtgttgttg tatttattgt ggggttggat 120

tttgatatat ttgttggaga gaaatttatt tttgagatgg tgtttgttgt ttagattatt 180

tttttagatt ttgatggtgt 200

<210> 50

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<223> hpEIN2[G:U/U:G]

<400> 50

aatgtttatg tgagttgtaa tatattggta taagatggtg gtaaaattga aaagtagagt 60

aattgttatt aagttatatt tgaaatgagt attttttttg atatttgtaa tttatttttt 120

gggattaata tatttgatag aaattaaaag gataggaagt agagtaggaa ttattttttg 180

gataaatttt agttgaggtt 200

<210> 51

<211> 199

<212> DNA

<213> Artificial Sequence

<220>

<223> hpCHS[G:U/U:G]

<400> 51

gtattattag agtttggaag gatggtttga gtggtagata ttattttaaa gatgggtttt 60

tttttgatag atgtgttagg gtttgatttt ataatgagtg tggtggtgtt attattgaaa 120

agagtttgat tgatgaggga gttaaggtgg gtgttagagg gattatggaa ggtaatggtt 180

gtgattttag agtagataa 199

<210> 52

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS-WT-F

<400> 52

cctcgaggat cctcgcgtcg gcatccggtc 30

<210> 53

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS-WT-R

<400> 53

gggtaccaag cttcgtaagg gtaatgcgag gta 33

<210> 54

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS-GU-F

<400> 54

ccctcgagtt gtgttggtat ttggttagtg gtagtgaagg gtgaatagtt tttgattaat 60

tataaattgt tttattttat tggttttggt tgttatgaag atgtggattt gtgtggta 118

<210> 55

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS-GU-R

<400> 55

ggggtaccca taaaaataat acaaaataca ataaaaatta accccaatcc aatccattaa 60

tacataatca tacaccatca acacattatc aaatccttta ccacacaaat ccacatct 118

<210> 56

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS-4M-F

<400> 56

ccctcgagtc gggtccgcaa ccgctcactg ggagtcaagc gcgtacactt cgtgaataag 60

cactaacggt tgtacattag tgggtttcgt cctcaagaac atggggagtt gggtgcca 118

<210> 57

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS-4M-R

<400> 57

ggggtaccgg tatgggaaat ccgacgtagg gttggacttg ccccgaatgc agaccaataa 60

agcgaggtgg tggaccttca ccaccttaac gattccattg gcacccaact ccccatgt 118

<210> 58

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS-10M-F

<400> 58

ccctcgagtc gcgtcgcgat ccggtctctg gcagtgttgg gcgaactctt cctgatatac 60

cacaaagggt tctactaaac tggcttacgt cgtcatctag atgcggtgtt gcgtgggt 118

<210> 59

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<223> GUS-10M-R

<400> 59

ggggtaccgc taagggtata gcgaggtagc gtaggagtac gccccaatgg agtccatttt 60

tgcgtggtgc tgcaccatgt gcacgttaag gaatccttac ccacgcaaca ccgcatct 118

<210> 60

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward primer (35S-F3)

<400> 60

tggctcctac aaatgccatc 20

<210> 61

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse primer (GUSwt-R2)

<220>

<221> R

<222> (3)..(4)

<223> A or G

<220>

<221> R

<222> (9)..(9)

<223> A or G

<220>

<221> R

<222> (13)..(13)

<223> A or G

<400> 61

carraactrt tcrcccttca c 21

<210> 62

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward primer (GUSgu-R2)

<400> 62

caaaaactat tcacccttca c 21

<210> 63

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer (GUS4m-R2)

<220>

<221> R

<222> (4)..(4)

<223> A or G

<220>

<221> R

<222> (7)..(7)

<223> A or G

<220>

<221> R

<222> (9)..(9)

<223> A or G

<220>

<221> R

<222> (13)..(13)

<223> A or G

<220>

<221> R

<222> (15)..(15)

<223> A or G

<220>

<221> R

<222> (19)..(19)

<223> A or G

<400> 63

cacraartrt acrcrcttra c 21

<210> 64

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward primer (35S-F2)

<400> 64

gaggatctaa cagaactcgc 20

<210> 65

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer (35S-R1)

<400> 65

ctctccaaat gaaatgaact tcc 23

<210> 66

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> EIN2wt-F

<400> 66

cctcgaggat cctctagacc tcagctaggg tttatc 36

<210> 67

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> EIN2wt-R

<400> 67

gggtaccaag cttaacgctt atgcgagctg caa 33

<210> 68

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> CHSwt-F

<400> 68

cctcgaggat ccgttgtctg ctctgagatc ac 32

<210> 69

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> CHSwt-R

<400> 69

gggtaccaag cttctagagc accatcagag tctggaag 38

<210> 70

<211> 119

<212> DNA

<213> Artificial Sequence

<220>

<223> EIN2gu-F

<400> 70

cctcgagtct agattttagt tagggtttat ttagagaatg gtttttgttt tattttttgt 60

ttttttggtt tttgttggat atattgattt tgggaaatgg gttgtaaata ttgaaggag 119

<210> 71

<211> 119

<212> DNA

<213> Artificial Sequence

<220>

<223> EIN2gu-R

<400> 71

gggtaccaac acttatacaa actacaacat attaacataa aataacaaca aaattaaaaa 60

acaaaataat taccaccaaa tcatacccaa aacaaacacc tccttcaata tttacaacc 119

<210> 72

<211> 119

<212> DNA

<213> Artificial Sequence

<220>

<223> CHSgu-F

<400> 72

cctcgaggtt gtttgttttg agattatagt tgttattttt tgtggttttt ttgatattta 60

ttttgatttt tttgttggtt aggttttttt tagtgatggt gttgttgtat ttattgtgg 119

<210> 73

<211> 119

<212> DNA

<213> Artificial Sequence

<220>

<223> CHSgu-R

<400> 73

gggtacctct agacaccatc aaaatctaaa aaaataatct aaacaacaaa caccatctca 60

aaaataaatt tctctccaac aaatatatca aaatccaacc ccacaataaa tacaacaac 119

<210> 74

<211> 120

<212> DNA

<213> Artificial Sequence

<220>

<223> asEIN2gu-F

<400> 74

caagcttaat gtttatgtga gttgtaatat attggtataa gatggtggta aaattgaaaa 60

gtagagtaat tgttattaag ttatatttga aatgagtatt ttttttgata tttgtaattt 120

<210> 75

<211> 120

<212> DNA

<213> Artificial Sequence

<220>

<223> asEIN2gu-R

<400> 75

gggatcctct agaacctcaa ctaaaattta tccaaaaaat aattcctact ctacttccta 60

tccttttaat ttctatcaaa tatattaatc ccaaaaaata aattacaaat atcaaaaaaa 120

<210> 76

<211> 120

<212> DNA

<213> Artificial Sequence

<220>

<223> asCHSgu-F

<400> 76

caagcttcta gagtattatt agagtttgga aggatggttt gagtggtaga tattatttta 60

aagatgggtt tttttttgat agatgtgtta gggtttgatt ttataatgag tgtggtggtg 120

<210> 77

<211> 120

<212> DNA

<213> Artificial Sequence

<220>

<223> asCHSgu-R

<400> 77

gggatccatt atctactcta aaatcacaac cattaccttc cataatccct ctaacaccca 60

ccttaactcc ctcatcaatc aaactctttt caataataac accaccacac tcattataaa 120

<210> 78

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> CHS-200-F2

<400> 78

gacatgcctg gtgctgacta 20

<210> 79

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> CHS-200-R2

<400> 79

ccttagcgat acggaggaca 20

<210> 80

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Actin2-For

<400> 80

tccctcagca cattccagca 20

<210> 81

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Actin2-Rev

<400> 81

gatcccattc ataaaacccc ag 22

<210> 82

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Top-35S-F2

<220>

<221> Y

<222> (8)..(8)

<223> C or T

<220>

<221> Y

<222> (11)..(11)

<223> C or T

<220>

<221> Y

<222> (14)..(14)

<223> C or T

<220>

<221> Y

<222> (17)..(17)

<223> C or T

<400> 82

agaaaatytt ygtyaayatg gtgg 24

<210> 83

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Top-35S-R2

<220>

<221> R

<222> (4)..(4)

<223> A or G

<220>

<221> R

<222> (6)..(7)

<223> A or G

<220>

<221> R

<222> (9)..(9)

<223> A or G

<220>

<221> R

<222> (12)..(12)

<223> A or G

<400> 83

tcartrrara trtcacatca atcc 24

<210> 84

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Link-35S-F2

<220>

<221> Y

<222> (1)..(2)

<223> C or T

<220>

<221> Y

<222> (5)..(5)

<223> C or T

<220>

<221> Y

<222> (10)..(10)

<223> C or T

<400> 84

yyatyattgy gataaaggaa agg 23

<210> 85

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Link-EIN2-R2

<220>

<221> R

<222> (6)..(6)

<223> A or G

<220>

<221> R

<222> (14)..(14)

<223> A or G

<400> 85

taattrccac caartcatac cc 22

<210> 86

<211> 22

<212> RNA

<213> Artificial Sequence

<220>

<223> sense si22

<400> 86

gcaagcugac ccugaaguuc au 22

<210> 87

<211> 22

<212> RNA

<213> Artificial Sequence

<220>

<223> antisense si22

<400> 87

gaacuucagg gucagcuugc cg 22

<210> 88

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward primer

<400> 88

ttttagtata tgtgctgccg 20

<210> 89

<211> 83

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer

<400> 89

ctcgagttcc aaaaaagctg accctgaagt tcatctctct tgaagatgaa cttcagggtc 60

agccaaacaa ggcttttctc caa 83

<210> 90

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer

<400> 90

ttttagtata tgtgctgccg 20

<210> 91

<211> 83

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer

<400> 91

ctcgagttcc aaaaaaataa gtcgcagcag tacaatctct tgaattgtac tgctgcgact 60

tatgaatacc gcttcctcct gag 83

<210> 92

<211> 88

<212> RNA

<213> Artificial Sequence

<220>

<223> dsRNA molecules

<400> 92

aagugauuug ugaauggauu cgguuaaguu agugauaggu aucacgcugg ccauuacuga 60

caugaccgga uucguucgcg agucacuu 88

<210> 93

<211> 2653

<212> DNA

<213> Brassica napus

<400> 93

agaaaaatcg aaaaatgtga cagtgcgtct tttcacttaa taccctcgtt ttgaatttgc 60

tctcggaaag cgtctgagag agtgttcggt gatttctccc gccgcttggg gttttttccg 120

ttaccggaat atccttctcc tccgatggtt agtctgcgct ccacagaaaa cactccggct 180

tcggaaatgg ccagcgacgg caaaacggag aaagatggct ccggcgactc acccacttct 240

gttctcagcg atgaggaaaa ctgtgaagag aaaactgcta ctgttgctgt agaggaagag 300

atacttctag ccaagaatgg agattcgtct cttatctctg aggccatggc tcaggaagaa 360

gagcagcttc tcaaaatccg ggaagatgaa gagattgcta aacgtgctgc tggctctggt 420

gaagctcctg atctgaatga tactcagttt actaaacttg atgagctctt gacccaaacc 480

cagctctact ctgagtttct ccttgagaaa atggaggata tcaccaaaaa tgggatagaa 540

ggtgagaccc aaaaggccga gcctgagcct gagcctgagc ccgagaagaa aggccgtgga 600

cgtaaaagaa aggctgctcc tcagggcgac agtatgaagg ctaagaaagc tgttgctgct 660

atgatttcaa gatccaaaga aggccgtgaa tctgccgact cagatctgac agaggaagaa 720

agagtcatga aagagcaggg tgaacttgtt cctcttctga ctggcggaaa gttaaagtct 780

tatcagctca aaggtgtcaa atggctgata tcattgtggc aaaatggttt gaatggaatt 840

ttagctgatc aaatgggtct tggaaagaca attcaaacca ttggtttcct atcacacctc 900

aaaggaaatg ggttggatgg tccatatcta gtcattgccc cactctctac tctttcaaac 960

tggatgaatg agatcgctag gttcacgcct tccattaatg caatcattta ccatggagat 1020

aagaaagaaa gggatgagct caggaagagg cacatgccca gaactgttgg tccgaagttc 1080

cctatagtca taacttctta tgaggttgct atgaatgatg ctaaaaagaa tctgcggcac 1140

tatccatgga aatatgttgt gattgatgag ggtcacaggt tgaaaaacca caagtgtaaa 1200

ctgctgaggg agctaagata cttgaatatg gagaacaaac ttctgctgcc aggaacacct 1260

ctgcaaaata atttgtctga gcttcggtca ctgttgaatt ttattctgcc tgacatcttt 1320

gcatcacatg acgaatttga atcatggttt gatttttctt gaaagaataa taatgaagca 1380

actaaggaag aaggagaaga gaaaagaaga gctcaagtgg ttgcgaaact tcataatata 1440

ctacgacctt tcatcctccg gagaatgaaa tgtgatgttg agctctcact tccccggaaa 1500

aaagagatta tcatctatgc tacaatgacg gaccatcaga agaagttcca ggaacatctt 1560

gtgaaccaca ccttggaagc acacattaga gatgatactg tccgaggtca tggcttgaag 1620

ggaaagctta acaatcttgc tattcaactt cgaaagaact gcaaccatcc tgaccttctt 1680

gtggggcaac tagatggctc atatctctac ccacctttgg aagacattgt gggacagtgc 1740

ggtaaattcc gcttattgga gagattgctt gttcggttat ttgccaaaaa tcacagagtc 1800

cttatcttct cccagtggac aaaaatactg gacattatgg attactactt cagtgagaag 1860

gggtttgagg tttgccgaat cgacggtagt gtgaaactag aagaaaggag aagacagatc 1920

caagaattca atgatgagaa gagcaactgc aggatatttc ttctcagtac cagagccgga 1980

ggactcggaa ttaatcttac tgctgcagat acatgcatcc tctacgatag cgattggaac 2040

cctcaaatgg acttgcaagc catggacaga tgccacagaa ttggtcagac aaaacctgtt 2100

catgtttaca ggcttgcgac ggctcagtca atagagggcc gagttctgaa acgagcatac 2160

agtaagctta agctggaaca tgtggttatt ggcaaggggc agtttcatca agaacgtgcc 2220

aagtcttcaa caccgttaga ggaagatgac atactggcgt tgcttaagga cgacgaaaat 2280

gctgaagata aactgataca aaccgacata agcgaggagg atcttgacag ggtgcttgac 2340

cgtagtgatc tgatgattac cttaccgggc gagactcaag cacatgaagc ttttccagtg 2400

aagggtccgg gttgggaagt ggtctcgtct agctcagctg gagggatgct gtcttccctc 2460

aacagttaga accactcttt gcaaaaccac ttcggtgtgt ttttttttcc ggaacataac 2520

cggttacttt tgcctgctac tcggaagttt taacttgaaa ccttggaaac atctgatgaa 2580

aacaattgcg gatattatgt tattagacta tttatttatg ccttttgaaa tttggcagta 2640

attttttagt taa 2653

<210> 94

<211> 1773

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting

a DDM1 gene of B. napus

<400> 94

gggagctgtt gctgctatga tttcaagatc caaagaaggc cgtgaatctg ccgactcaga 60

tctgacagag gaagaaagag tcatgaatga gcagggtgaa cttgttcctc ttctgactgg 120

cggaaagtta aagtcttatc agctcaaagg tgtcaaatgg ctgatatcat tgtggcaaaa 180

tggtttgaat ggaattttag ctgatcaaat gggtcttgga aagacaattc aaaccattgg 240

tttcctatca ccccaacaag gaaatgggtt ggatggtcca tatctagtca ttgccccact 300

ctctactctt tcaaaagcga ttggaaccct caaatggact tgcaagccat ggacagatgc 360

cacagaattg gtcagacaaa acctgttcat gtttacaggc ttgcgacggc tcagtcaata 420

gagggccgag ttctgaaacg agcatacagt aagcttaagc tggaacatgt ggttattggc 480

aaggggcagt ttcatcaaga acgtggtaag gaaataatta ttttcttttt tccttttagt 540

ataaaatagt taagtgatgt taattagtat gattataata atatagttgt tataattgtg 600

aaaaaataat ttataaatat attgtttaca taaacaacat agtaatgtaa aaaaatatga 660

caagtgatgt gtaagacgaa gaagataaaa gttgagagta agtatattat ttttaatgaa 720

tttgatcgaa catgtaagat gatatactag cattaatatt tgttttaatc ataatagtaa 780

ttctagctgg tttgatgaat taaatatcaa tgataaaata ctatagtaaa aataagaata 840

aataaattaa aataatattt ttttatgatt aatagtttat tatataatta aatatctata 900

ccattactaa atattttagt ttaaaagtta ataaatattt tgttagaaat tccaatctgc 960

ttgtaattta tcaataaaca aaatattaaa taacaagcta aagtaacaaa taatatcaaa 1020

ctaatagaaa cagtaatcta atgtaacaaa acataatcta atgctaatat aacaaagcgc 1080

aagatctatc attttatata gtattatttt caatcaacat tcttattaat ttctaaataa 1140

tacttgtagt tttattaact tctaaatgga ttgactatta attaaatgaa ttagtcgaac 1200

atgaataaac aaggtaacat gatagatcat gtcattgtgt tatcattgat cttacatttg 1260

gattgattac agtcacgttc ttgatgaaac tgccccttgc caataaccac atgttccagc 1320

ttaagcttac tgtatgctcg tttcagaact cggccctcta ttgactgagc cgtcgcaagc 1380

ctgtaaacat gaacaggttt tgtctgacca attctgtggc atctgtccat ggcttgcaag 1440

tccatttgag ggttccaatc gcttttgaaa gagtagagag tggggcaatg actagatatg 1500

gaccatccaa cccatttcct tgttggggtg ataggaaacc aatggtttga attgtctttc 1560

caagacccat ttgatcagct aaaattccat tcaaaccatt ttgccacaat gatatcagcc 1620

atttgacacc tttgagctga taagacttta actttccgcc agtcagaaga ggaacaagtt 1680

caccctgctc tttcatgact ctttcttcct ctgtcagatc tgagtcggca gattcacggc 1740

cttctttgga tcttgaaatc atagcagcaa cag 1773

<210> 95

<211> 1773

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a hairpin RNAi (hpRNA) construct with G:U

basepairs, targeting a DDM1 gene of B. napus

<400> 95

gggttgttgt tgttatgatt ttaagattta aagaaggttg tgaatttgtt gatttagatt 60

tgatagagga agaaagagtt atgaatgagt agggtgaatt tgtttttttt ttgattggtg 120

gaaagttaaa gttttattag tttaaaggtg ttaaatggtt gatattattg tggtaaaatg 180

gtttgaatgg aattttagtt gattaaatgg gttttggaaa gataatttaa attattggtt 240

ttttattata ttttaaagga aatgggttgg atggtttata tttagttatt gttttatttt 300

ttattttttt aaaagtgatt ggaattttta aatggatttg taagttatgg atagatgtta 360

tagaattggt tagataaaat ttgtttatgt ttataggttt gtgatggttt agttaataga 420

gggttgagtt ttgaaatgag tatatagtaa gtttaagttg gaatatgtgg ttattggtaa 480

ggggtagttt tattaagaat gtgtggtaag gaaataatta ttttcttttt tccttttagt 540

ataaaatagt taagtgatgt taattagtat gattataata atatagttgt tataattgtg 600

aaaaaataat ttataaatat attgtttaca taaacaacat agtaatgtaa aaaaatatga 660

caagtgatgt gtaagacgaa gaagataaaa gttgagagta agtatattat ttttaatgaa 720

tttgatcgaa catgtaagat gatatactag cattaatatt tgttttaatc ataatagtaa 780

ttctagctgg tttgatgaat taaatatcaa tgataaaata ctatagtaaa aataagaata 840

aataaattaa aataatattt ttttatgatt aatagtttat tatataatta aatatctata 900

ccattactaa atattttagt ttaaaagtta ataaatattt tgttagaaat tccaatctgc 960

ttgtaattta tcaataaaca aaatattaaa taacaagcta aagtaacaaa taatatcaaa 1020

ctaatagaaa cagtaatcta atgtaacaaa acataatcta atgctaatat aacaaagcgc 1080

aagatctatc attttatata gtattatttt caatcaacat tcttattaat ttctaaataa 1140

tacttgtagt tttattaact tctaaatgga ttgactatta attaaatgaa ttagtcgaac 1200

atgaataaac aaggtaacat gatagatcat gtcattgtgt tatcattgat cttacatttg 1260

gattgattac agtcacgttc ttgatgaaac tgccccttgc caataaccac atgttccagc 1320

ttaagcttac tgtatgctcg tttcagaact cggccctcta ttgactgagc cgtcgcaagc 1380

ctgtaaacat gaacaggttt tgtctgacca attctgtggc atctgtccat ggcttgcaag 1440

tccatttgag ggttccaatc gcttttgaaa gagtagagag tggggcaatg actagatatg 1500

gaccatccaa cccatttcct tgttggggtg ataggaaacc aatggtttga attgtctttc 1560

caagacccat ttgatcagct aaaattccat tcaaaccatt ttgccacaat gatatcagcc 1620

atttgacacc tttgagctga taagacttta actttccgcc agtcagaaga ggaacaagtt 1680

caccctgctc tttcatgact ctttcttcct ctgtcagatc tgagtcggca gattcacggc 1740

cttctttgga tcttgaaatc atagcagcaa cag 1773

<210> 96

<211> 1260

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct, targeting a DDM1 gene

of B. napus

<400> 96

ggggtgatag gaaaccaatg gtttgaattg tctttccaag acccatttga tcagctaaaa 60

ttccattcaa accattttgc cacaatgata tcagccattt gacacctttg agctgataag 120

actttaactt tccgccagtc agaagaggaa caagttcacc ctgctctttc atgactcttt 180

cttcctctgt cagatctgag tcggcagatt cacggccttc tttggatctt gaaatcatag 240

cagcaacagc tttcttagcc ttcatactgt cgccctgagg agcagccttt cttttacgtc 300

cacggccttt cttctcgggc tcaggctcag gctcaggctc ggccttttgg gtctcacctt 360

ctatcccatt tttggtgata gctgttgctg ctatgatttc aagatccaaa gaaggccgtg 420

aatctgccga ctcagatctg acagaggaag aaagagtcat gaatgagcag ggtgaacttg 480

ttcctcttct gactggcgga aagttaaagt cttatcagct caaaggtgtc aaatggctga 540

tatcattgtg gcaaaatggt ttgaatggaa ttttagctga tcaaatgggt cttggaaaga 600

caattcaaac cattggtttc ctatcacccc aacaaggaaa tgggttggat ggtccatatc 660

tagtcattgc cccactctct actctttcaa aagcgattgg aaccctcaaa tggacttgca 720

agccatggac agatgccaca gaattggtca gacaaaacct gttcatgttt acaggcttgc 780

gacggctcag tcaatagagg gccgagttct gaaacgagca tacagtaagc ttaagctgga 840

acatgtggtt attggcaagg ggcagtttca tcaagaacgt cactacggtc aagcaccctg 900

tcaagatcct cctcgcttat gtcggtttgt atcagtttat cttcagcatt ttcgtcgtcc 960

ttaagcaacg ccagtatgtc atcttcctct aacggtgttg aagacttggc acgttcttga 1020

tgaaactgcc ccttgccaat aaccacatgt tccagcttaa gcttactgta tgctcgtttc 1080

agaactcggc cctctattga ctgagccgtc gcaagcctgt aaacatgaac aggttttgtc 1140

tgaccaattc tgtggcatct gtccatggct tgcaagtcca tttgagggtt ccaatcgctt 1200

ttgaaagagt agagagtggg gcaatgacta gatatggacc atccaaccca tttccttgtt 1260

<210> 97

<211> 4421

<212> DNA

<213> Arabidopsis thaliana

<400> 97

tcttccaaaa tttgcccgcc attctctgtg tctcttcgtc taagggtttc ttccaaagaa 60

cgacgacaaa accactgaac ctaaaatccg aaatccaaaa gattcatgcg aaaaaatcgt 120

taaagagtac caatttcaga aagttacatt cttacagaga acaagtaatt ccccagaaat 180

gggatctagg gttccaatag aaaccatcga agaagacggc gaattcgatt gggaagcagc 240

agtcaaagaa atcgacttgg cttgtcttaa aaccacaaac gcttcttctt cttcgtcatc 300

ccatttcact cctttggcta atccaccaat tacggcaaat ctcactaagc cacctgcgaa 360

gagacaatct actctcgata aattcatcgg cagaaccgaa cataaaccgg agaatcatca 420

agttgtttcc gagtgtggtg ttaacgataa cgataatagt cctttagttg ggattgatcc 480

tgaggcagct aaaacttgga tttatccagt gaatgggagt gttcctttaa gagattatca 540

gtttgctata acgaagactg ctttgttttc gaatacattg gtggctttgc ctacgggact 600

tggtaaaacg cttatagctg cggttgttat gtataattac ttcagatggt ttccacaagg 660

taaaatagta tttgcggcgc cttctaggcc tcttgtgatg cagcagattg aggcgtgtca 720

taatattgtt ggaataccac aagaatggac gattgacttg acgggtcaga catgtccttc 780

gaaaagagct tttttgtgga aaagcaaacg ggttttcttt gtcactccac aagtgttaga 840

gaaggatata cagtcaggaa catgtcttac taactacttg gtttgcttgg tgatcgacga 900

ggcacatcga gctttaggga attattctta ttgtgttgta gttcgtgagt tgatggcggt 960

accgatacag ctgagaatac tggctcttac tgcaactcct ggatcaaaga cacaggccat 1020

ccagggtatc attgataatt tgcagatatc cacacttgaa tatcgaaatg agagtgacca 1080

tgatgtttgc ccttatgtcc acgacagaaa attagaagtc atcgaggttc ccttgggtca 1140

agatgcagat gatgtatcga aacgcctgtt tcatgttata cgtccatatg cagtcaggct 1200

taaaaacttt ggggttaatc taaatagaga tatacaaact ttaagtccac acgaagtact 1260

tatggcaagg gataagtttc gtcaagcacc tctaccaggc cttccccatg taaatcacgg 1320

agatgtagaa tcttgctttg cagctcttat cactctttat catattcgta agctcctttc 1380

tagtcatgga ataagaccag cgtatgagat gctagaagag aaattgaaag aagggccatt 1440

tgctaggttg atgagtaaga atgaagatat taggatgacg aagcttttga tgcagcaaag 1500

gttgtcacat ggagcaccaa gcccaaaatt gtcgaagatg ttagaaatac tggttgatca 1560

tttcaaagtg aaagatccga agacatcacg ggtcattatt ttctcaaatt tcagaggaag 1620

cgtaagagac ataatgaacg cattaagtaa tattggagat atggtcaaag caactgagtt 1680

tattggtcaa agttcaggta agacattgaa aggccagtcg caaaaaattc agcaggctgt 1740

tttggagaaa tttagagctg gggggttcaa tgttattgtc gcaacatcta ttggtgaaga 1800

aggcttggat atcatggaag ttgacctagt tatatgtttt gatgctaatg tatctcctct 1860

gaggatgatt caacggatgg gaagaactgg aaggaaaaat aatggtcgag ttgtagttct 1920

tgcttgtgaa ggatcagaaa agaacagcta tatgcgaaag caagcaagtg gacgggctat 1980

taaaaaacac atgcggaatg gaggaacaaa tagttttaat tttcatccta gtccaaggat 2040

gattccccat gtttataagc cagaagttca gcatgttgag ttttcaatca agcaattcgt 2100

tccacgtgga aagaaactac aagaggagta tgccactgag actccagctt tccagaaaaa 2160

gcttacacct gcagagacgc atatgctcgc taagtattac aacaaccccg atgaggaaaa 2220

gttgagagtg tccttaattg cgttccctca cttccagaca ttgccatcca aggtgcacaa 2280

agtaatgcat tcacgtcaaa caggcatgtt aattgacgct atgcagcact tgcaagagcc 2340

aactttttca gaacagagta aaagcttctt cactgagttt cgagctcctt tgggtgaaag 2400

agaagagctt gatacaggtc tgagggttac taatgatcca aaagatctac actctgtccg 2460

tgatttggaa gtcaacacat cacagagaaa ggcaaaacaa gttgaatctc ccacaagcac 2520

cttagagaca acagagaagg attacgaaga atcttcaccc acacaccgtt atcttttcag 2580

ttcagaatgt gcatccgttg atactctggg gaacgtcttc gtaatgccag ttcctctttt 2640

attctttcct aatgttctgg agtcagacaa tacgcctctg cctaaaacag aaaaacaaca 2700

ttcttgccgg aatacatctc acattgactt agttccagta gatacttcgg aaaaacatcg 2760

gcaagataat atctcatgca agttaaagga aagattctcg ccagacggtg ccagcgagac 2820

actagagact catagccttg tgaaaaggaa ctccaccaga gtaggtgaag atgatgtagc 2880

gaattctgtt ggagaaattg tgttatcatc ggatgaagat gactgtgagg gattggagct 2940

tagtccacgg ctcactaact tcatcaagag cggcattgtt ccagagtcac ctgtctatga 3000

ccaaggggaa gcgaacagag aagaagatct tgaatttcct cagctttctt cacccatgag 3060

gttcagtaac gaattggcag gagagtcttc tttccctgag agaaaggttc agcataagtg 3120

caacgattat aacattgtgt ctacaaccac tgaattgaga actcctcaga aggaggtagg 3180

tttggccaac ggaacagaat gcttggctgt ttctcctatt cctgaggatt ggagaactcc 3240

cttggcgaat ctgacaaaca caaacagcag cgctcgcaaa gattggcggg tgagttctgg 3300

agaaaagtta gaaactcttc gacagcctcg caagttgaag agactacgta gacttggaga 3360

ttgctcgagt gctgtaaagg agaattatcc tggtattaca gaggcagacc atatcagatc 3420

tcgttctcgc ggtaaaaagc acattagagg taagaagaag atgatcatgg atgatgatgt 3480

ccaagtcttc attgacgagg aagctgaggt ctcttcggga gcagagatgt cggctgatga 3540

gaacgaagat gtgactggcg attcatttga agatagtttc atagatgacg gaacaatgcc 3600

tacagcaaat actcaagccg agtctggtaa agttgacatg atggctgttt acaggcgttc 3660

tcttctcagc cagtcaccat taccggcgag atttcgtgat ttagccgcat caagtctgag 3720

tccttattct gctggaccct tgacgagaat aaatgagagc agaagcgact cagataaatc 3780

attgtcttct cttcgaacac caaaaacaac aaactctgag tcaaaccaag atgcaatgat 3840

gataggaaac ctttcggtag tacaaatctc gtcagatagc cggaaaagga aatttagctt 3900

atgcaactcg gcgaatgccc ccgtgattaa cttagaaagc aagtttgcag ctcatgcaca 3960

agccacggag aaggaaagcc atgaaggcgt gagaagcaat gcaggtgcgt tagagtacaa 4020

tgatgatgat gatgatgcat tctttgcgac actagacttt gatgcaatgg aagcacaagc 4080

cacattgtta ttgtcgaaac agagatccga agcaaaagag aaagaagacg caacggttat 4140

acctaatcca ggcatgcaga gaagtgatgg tatggagaaa gatgcaccat cttttgatct 4200

tggtctgtgg tgattcttct ttcatacgaa gatactaagt tatgtatata gattgacaaa 4260

ggagacagta gagcataggc atttggatgt atgttttgtg tattaagttt aggtatatcc 4320

tattgaagta cagtgcttaa ggcagtgcac atggttaaat caaggttaat gcctcaattc 4380

gttgaaccct ttaagtaatg acacaaatat gactacatcg g 4421

<210> 98

<211> 1771

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting

a FANCM gene of A. thaliana

<400> 98

gggtcaggaa catgtcttac taactacttg gtttgcttgg tgatcgacga ggcacatcga 60

gctttaggga attattctta ttgtgttgta gttcgtgagt tgatggcggt accgatacag 120

ctgagaatac tggctcttac tgcaactcct ggatcaaaga cacaggccat ccagggtatc 180

attgataatt tgcagatatc cacacttgaa tatcgaaatg agagtgacca tgatgtttgc 240

ccttatgtcc ccgacagaaa attagaagtc atcgaggttc ccttgggtca agatgcagat 300

gatgtatcga aacgcctgtt tcatgttata cgtccatatg cagtcaggct taaaaacttt 360

ggggttaatc taaatagaga tatacaaact ttaagtccac acgaagtact tatggcaagg 420

gataagtttc gtcaagcacc tctaccaggc cttccccatg taaatcacgg agatgtagaa 480

tcttgctttg cagctcttat caggtaagga aataattatt ttcttttttc cttttagtat 540

aaaatagtta agtgatgtta attagtatga ttataataat atagttgtta taattgtgaa 600

aaaataattt ataaatatat tgtttacata aacaacatag taatgtaaaa aaatatgaca 660

agtgatgtgt aagacgaaga agataaaagt tgagagtaag tatattattt ttaatgaatt 720

tgatcgaaca tgtaagatga tatactagca ttaatatttg ttttaatcat aatagtaatt 780

ctagctggtt tgatgaatta aatatcaatg ataaaatact atagtaaaaa taagaataaa 840

taaattaaaa taatattttt ttatgattaa tagtttatta tataattaaa tatctatacc 900

attactaaat attttagttt aaaagttaat aaatattttg ttagaaattc caatctgctt 960

gtaatttatc aataaacaaa atattaaata acaagctaaa gtaacaaata atatcaaact 1020

aatagaaaca gtaatctaat gtaacaaaac ataatctaat gctaatataa caaagcgcaa 1080

gatctatcat tttatatagt attattttca atcaacattc ttattaattt ctaaataata 1140

cttgtagttt tattaacttc taaatggatt gactattaat taaatgaatt agtcgaacat 1200

gaataaacaa ggtaacatga tagatcatgt cattgtgtta tcattgatct tacatttgga 1260

ttgattacag ttgataagag ctgcaaagca agattctaca tctccgtgat ttacatgggg 1320

aaggcctggt agaggtgctt gacgaaactt atcccttgcc ataagtactt cgtgtggact 1380

taaagtttgt atatctctat ttagattaac cccaaagttt ttaagcctga ctgcatatgg 1440

acgtataaca tgaaacaggc gtttcgatac atcatctgca tcttgaccca agggaacctc 1500

gatgacttct aattttctgt cggggacata agggcaaaca tcatggtcac tctcatttcg 1560

atattcaagt gtggatatct gcaaattatc aatgataccc tggatggcct gtgtctttga 1620

tccaggagtt gcagtaagag ccagtattct cagctgtatc ggtaccgcca tcaactcacg 1680

aactacaaca caataagaat aattccctaa agctcgatgt gcctcgtcga tcaccaagca 1740

aaccaagtag ttagtaagac atgttcctga c 1771

<210> 99

<211> 1771

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a hairpin RNAi (hpRNA) construct with G:U

basepairs, targeting a FANCM gene of A. thaliana

<400> 99

gggttaggaa tatgttttat taattatttg gtttgtttgg tgattgatga ggtatattga 60

gttttaggga attattttta ttgtgttgta gtttgtgagt tgatggtggt attgatatag 120

ttgagaatat tggtttttat tgtaattttt ggattaaaga tataggttat ttagggtatt 180

attgataatt tgtagatatt tatatttgaa tattgaaatg agagtgatta tgatgtttgt 240

ttttatgttt ttgatagaaa attagaagtt attgaggttt ttttgggtta agatgtagat 300

gatgtattga aatgtttgtt ttatgttata tgtttatatg tagttaggtt taaaaatttt 360

ggggttaatt taaatagaga tatataaatt ttaagtttat atgaagtatt tatggtaagg 420

gataagtttt gttaagtatt tttattaggt tttttttatg taaattatgg agatgtagaa 480

ttttgttttg tagtttttat taggtaagga aataattatt ttcttttttc cttttagtat 540

aaaatagtta agtgatgtta attagtatga ttataataat atagttgtta taattgtgaa 600

aaaataattt ataaatatat tgtttacata aacaacatag taatgtaaaa aaatatgaca 660

agtgatgtgt aagacgaaga agataaaagt tgagagtaag tatattattt ttaatgaatt 720

tgatcgaaca tgtaagatga tatactagca ttaatatttg ttttaatcat aatagtaatt 780

ctagctggtt tgatgaatta aatatcaatg ataaaatact atagtaaaaa taagaataaa 840

taaattaaaa taatattttt ttatgattaa tagtttatta tataattaaa tatctatacc 900

attactaaat attttagttt aaaagttaat aaatattttg ttagaaattc caatctgctt 960

gtaatttatc aataaacaaa atattaaata acaagctaaa gtaacaaata atatcaaact 1020

aatagaaaca gtaatctaat gtaacaaaac ataatctaat gctaatataa caaagcgcaa 1080

gatctatcat tttatatagt attattttca atcaacattc ttattaattt ctaaataata 1140

cttgtagttt tattaacttc taaatggatt gactattaat taaatgaatt agtcgaacat 1200

gaataaacaa ggtaacatga tagatcatgt cattgtgtta tcattgatct tacatttgga 1260

ttgattacag ttgataagag ctgcaaagca agattctaca tctccgtgat ttacatgggg 1320

aaggcctggt agaggtgctt gacgaaactt atcccttgcc ataagtactt cgtgtggact 1380

taaagtttgt atatctctat ttagattaac cccaaagttt ttaagcctga ctgcatatgg 1440

acgtataaca tgaaacaggc gtttcgatac atcatctgca tcttgaccca agggaacctc 1500

gatgacttct aattttctgt cggggacata agggcaaaca tcatggtcac tctcatttcg 1560

atattcaagt gtggatatct gcaaattatc aatgataccc tggatggcct gtgtctttga 1620

tccaggagtt gcagtaagag ccagtattct cagctgtatc ggtaccgcca tcaactcacg 1680

aactacaaca caataagaat aattccctaa agctcgatgt gcctcgtcga tcaccaagca 1740

aaccaagtag ttagtaagac atgttcctga c 1771

<210> 100

<211> 1259

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct, targeting a FANCM gene

of A. thaliana

<400> 100

gggacataag ggcaaacatc atggtcactc tcatttcgat attcaagtgt ggatatctgc 60

aaattatcaa tgataccctg gatggcctgt gtctttgatc caggagttgc agtaagagcc 120

agtattctca gctgtatcgg taccgccatc aactcacgaa ctacaacaca ataagaataa 180

ttccctaaag ctcgatgtgc ctcgtcgatc accaagcaaa ccaagtagtt agtaagacat 240

gttcctgact gtatatcctt ctctaacact tgtggagtga caaagaaaac ccgtttgctt 300

ttccacaaaa aagctctttt cgaaggacat gtctgacccg tcaagtcaat cgtccattct 360

tgtggtattc caacaatatg tcaggaacat gtcttactaa ctacttggtt tgcttggtga 420

tcgacgaggc acatcgagct ttagggaatt attcttattg tgttgtagtt cgtgagttga 480

tggcggtacc gatacagctg agaatactgg ctcttactgc aactcctgga tcaaagacac 540

aggccatcca gggtatcatt gataatttgc agatatccac acttgaatat cgaaatgaga 600

gtgaccatga tgtttgccct tatgtccccg acagaaaatt agaagtcatc gaggttccct 660

tgggtcaaga tgcagatgat gtatcgaaac gcctgtttca tgttatacgt ccatatgcag 720

tcaggcttaa aaactttggg gttaatctaa atagagatat acaaacttta agtccacacg 780

aagtacttat ggcaagggat aagtttcgtc aagcacctct accaggcctt ccccatgtaa 840

atcacggaga tgtagaatct tgctttgcag ctcttatcat tcgtcatcct aatatcttca 900

ttcttactca tcaacctagc aaatggccct tctttcaatt tctcttctag catctcatac 960

gctggtctta ttccatgact agaaaggagc ttacgaatat gataaagagt gataagagct 1020

gcaaagcaag attctacatc tccgtgattt acatggggaa ggcctggtag aggtgcttga 1080

cgaaacttat cccttgccat aagtacttcg tgtggactta aagtttgtat atctctattt 1140

agattaaccc caaagttttt aagcctgact gcatatggac gtataacatg aaacaggcgt 1200

ttcgatacat catctgcatc ttgacccaag ggaacctcga tgacttctaa ttttctgtc 1259

<210> 101

<211> 4228

<212> DNA

<213> Brassica napus

<400> 101

tccaaaattg gttttgcccg ccaatgtggc ttcggcgagg gtttcttcca caaaacccca 60

ctcaacctaa aatctgattc ggcgagaaac gctgtctact tatctcacgc gaaaagaaag 120

gcgtagatcc accctaaact aaaacagagc atcaagtgaa atgggacccg agtttccgat 180

cgaactcgtt gaagaagaag atggattcga ttgggaagca gcagtcagag aaatcgactt 240

ggcttgcctc aaatccttaa acccttcttc ttcttcttcg acccatttca ccaacggcaa 300

tggcactaaa cctgctaaaa gacaatctac tcttgatcga ttcatcgcaa gagccgacca 360

caagcctcct cctccgtatc ctcctgttgt ttccgacccg agtttcgagt gtggtactaa 420

cgacaacact cccagcgtcg ggattgatcc tgagacagct aaaacttgga tttatccaat 480

gaacgttcct ctaagagatt atcagtttgc tataacgaag actgctttgt tttcaaacac 540

attagttgct ttaccaacag gccttggtaa aacgctcata gctgcagttg taatgtataa 600

ttacttcaga tggtttccac aaggtaaaat tgtctttgcc gcaccttcta ggcctcttgt 660

gatgcagcag attgaggcct gccataatat cgtggggata ccacaagaat ggacgattga 720

cttgacgggt cagacttgcc cttccaaaag agcttccttg tggaaaacca aaagggtttt 780

cttcgtcact ccacaagttc ttgagaagga tatacagtca ggaacgtgtg ttaccaactg 840

cttggtttgc ttggtgatcg acgaggcaca tcgagcttta gggaattatt cttattgtgt 900

tgtagttcgt gagttgatgg cagtaccagt gcagttgaga atattggctc ttactgcaac 960

tcctggatca aagacacagg ccatacaggg tatccttgat aatttgcaga tatcaacact 1020

tgaatatcga aacgagagtg accatgatgt ctgcccttat gtccacgaca gaaaagtaga 1080

actaatcgag gttcccttgg gtaaagatgc agatgaggta tctaaacgcc tattagatgt 1140

tatacgtcca tatgctgtca ggcttaaaaa tttcggggtc attctaagca gggattatca 1200

aactttgagt ccacacgaat tacttatggc aagggataag tttcgtgaag cacctgtacc 1260

aggcattccc catataagtc acggagatgt agaatcttgc tttgcagctc ttatcacgct 1320

ttatcacatt cgcaagcttc tttctagtca tggaataagg ccagcgtatg agatgcttga 1380

agaaaaactt caggaagggc catttgctag gttgatgagt aagaatgaag atattaggat 1440

gacgaagctt ttgatgcagc aaaggttgtc gaacggagca ccaagcccga aattgtccaa 1500

gatgttggag attctagttg atcactacaa aataaaagat ccgaggacat cacgggtcat 1560

tattttctcg aatttcagag gaagcgtaag agacataatg gacgcattaa gtaatattga 1620

agatgttgtc aaagcaactg agtttattgg tcaaagttca ggtaagacac tgaagggaca 1680

gtcgcaaaaa gttcagcaag ctgttctgga gaaatttaga tctggtgggt ttaatgttat 1740

tgttgcaaca tctatcggcg aagaaggctt ggatatcatg gaagtcgact tagttatatg 1800

ttttgatgct aatgtatccc ctctgaggat gatccaacgc atgggaagaa ctggaaggaa 1860

aaataatggc cgagttgtag ttcttgcttg tgaaggatct gaaaagaata gctatatgcg 1920

aaagaaagca aatggccaag ccattaaaaa acacatgcgg aatggaggaa tgaatagttt 1980

taattttcat cctagtccaa ggatgattcc ccatgtttat aagccagaag ttcagcatgt 2040

taagttttcg atcgagcaat tcattccacg tggaaagaag ctacaagatg agcctgccac 2100

tgagactcca gctttcaaga aaaagcttac accggaagag atggatatgc tcgccaagta 2160

tttcaaaccc aacgaggaaa agtggagagt ttccttgatt gctttccctc acttccaaac 2220

attgccatcc aaagtgcaca aagtaatgca ttcacgccaa acaagcatat taattgatgc 2280

tatgcagcat ctgcaagaga caactttgac agagcaaagt aaaagtttct tcattaagta 2340

tggagctcct ttggctgaaa gagatgagct tgacgcaggt ctgagggttg gtgatgatcc 2400

gaaaggtaaa tttagtctca atgatttgga tggcaacaca tcacagagaa aggcaaaaca 2460

aattttagaa tctcccacaa gcacattaga gactacagag aaggatttcg aagcatcttc 2520

acccacacac tgttatcttt tcagttcaga atgtgcgtcc gttgatactc tggggaaggt 2580

ctttgtattg ccggttcctc tctcattctc ttctaatgta ccagggtcag actgcgtggg 2640

aagagaaaaa gaactttctt ccccgaataa gtcccacact gacgttgttc cgatagatag 2700

ttcctcaaaa catcggcaag ataatatttc atgcaagtta aagcaaggat tcttgccaga 2760

ttgtgccaac gagactttgg agtcccaaag ccttttgaaa aggcactcca ccgatgtagg 2820

taaaggagat atagagaatt gtgctggaga aattatgata tcatcggatg aagaagacga 2880

ctgtgaggat ttggagctta gtccaaggct cactaacttc atcaagagtg gcgttgttcc 2940

agattcacct gtctatgacc aaggagttgc atacgaagca aacagagaag aagaccttga 3000

tcttccaccc acgagtttaa ctaatgaatt ggcagaagag ccatcgacac ctgagaaaaa 3060

ggttcacatt gcttctacgg ccaatgaatt cagaactcct cagaaggaag aagatttagc 3120

caacgaaaca gaaagcttcg ctgtttctcc aatgcctgag gagtggagaa ctcccttggc 3180

gaatatcacc aacgcaagca gcagcgctag caaagattgg cgcgtgagtt cgggagaaaa 3240

gtcagaaact cttcgacagc ctcgcaagtt gaagagactt cgtagacttg gagattgctc 3300

gagtgctgtg aaggagaata atcctggtat tgcaaagaca gaccatatca gatctcgttc 3360

tcgcagtgta aagaacataa gaggcaagaa gaagatacgc gcggataata atgctagaat 3420

cttcattgaa gcggaagctg aggtgtcttc ggaatcagaa atgtcggttg atgagaacgt 3480

agatttgacc agcgattcat ttgaagatag cttcatagat gacggtacaa tgcctacagc 3540

aaatactcaa gccgagtgtg ctaaagttga catgatggcc gtttacagac gttctctact 3600

cagccaatca ccattaccgg caagatttcg tgatgtagct gcatcaagtc cgagtcctta 3660

ttcttctggt ctcttgaaga caataaatga gagcagaagc gactcagata aatcattgtc 3720

ttctcttaga accccacaaa caacgaacaa cgagtcaaac aaggatgcag tggccacagg 3780

agactttttg gtagcacaaa tctcaacaga cagccggaaa aggaaattca gcttatgcaa 3840

ctcagcgaat gtcccagtga ttaacttgga aaacaagttt gaagctcatg cacaagccac 3900

ggagaaggaa agccatgaag gtccgagaag caatgcaggt gcatcacagt acaaggatga 3960

ggatgaagat gatgatgcat tctacgcgac actggacttt gatgccatgg aagcgcatgc 4020

gacattgcta ttgtcgaaac aaaggtcaga aacgaaaaca aaagaagatg catcggtgaa 4080

acctcatttg ggcaatcaga ggaatgatgg tttgccgaag gatgggccat cttttgatct 4140

tggtttgtgg tgattattct cctattaagt taaagtgtat aaaggttgac atttggatgt 4200

atgttttgtg tatttagttt gtgtcata 4228

<210> 102

<211> 1769

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting

a FANCM gene of B. napus

<400> 102

gggagaaatt atgatatcat cggatgaaga agacgactgt gtggatttgg agcttagtcc 60

aaggctcact aacttcatca agagtggcgt tgttccagat tcacctgtct atgaccaagt 120

tgcatacgaa gcaaacagtg aagaagacct tgatcttcca cacacgagtt taactaatga 180

attggcagaa gagccatcga cacctgagaa aaaggttcac attgcttcta cggccaatga 240

attcagaacc ccaacgaagg aagaagattt agccaacgaa acagaaagct tcgctgtttc 300

tccaatgcct gaggagtgga gaactccctt ggcgaatatc accaacgcaa gcagcagcgc 360

tagcaaagat tggcgcgtga gttcgggaga aaagtcagaa actcttcgac agcctcgcaa 420

gttgaagaga cttcgtagac ttggagattg ctcgagtgct gtgaaggaga ataatcctgg 480

tattgcaaag acagaccata tcgtaaggaa ataattattt tcttttttcc ttttagtata 540

aaatagttaa gtgatgttaa ttagtatgat tataataata tagttgttat aattgtgaaa 600

aaataattta taaatatatt gtttacataa acaacatagt aatgtaaaaa aatatgacaa 660

gtgatgtgta agacgaagaa gataaaagtt gagagtaagt atattatttt taatgaattt 720

gatcgaacat gtaagatgat atactagcat taatatttgt tttaatcata atagtaattc 780

tagctggttt gatgaattaa atatcaatga taaaatacta tagtaaaaat aagaataaat 840

aaattaaaat aatatttttt tatgattaat agtttattat ataattaaat atctatacca 900

ttactaaata ttttagttta aaagttaata aatattttgt tagaaattcc aatctgcttg 960

taatttatca ataaacaaaa tattaaataa caagctaaag taacaaataa tatcaaacta 1020

atagaaacag taatctaatg taacaaaaca taatctaatg ctaatataac aaagcgcaag 1080

atctatcatt ttatatagta ttattttcaa tcaacattct tattaatttc taaataatac 1140

ttgtagtttt attaacttct aaatggattg actattaatt aaatgaatta gtcgaacatg 1200

aataaacaag gtaacatgat agatcatgtc attgtgttat cattgatctt acatttggat 1260

tgattacagg atatggtctg tctttgcaat accaggatta ttctccttca cagcactcga 1320

gcaatctcca agtctacgaa gtctcttcaa cttgcgaggc tgtcgaagag tttctgactt 1380

ttctcccgaa ctcacgcgcc aatctttgct agcgctgctg cttgcgttgg tgatattcgc 1440

caagggagtt ctccactcct caggcattgg agaaacagcg aagctttctg tttcgttggc 1500

taaatcttct tccttcgttg gggttctgaa ttcattggcc gtagaagcaa tgtgaacctt 1560

tttctcaggt gtcgatggct cttctgccaa ttcattagtt aaactcgtgt gtggaagatc 1620

aaggtcttct tctctgtttg cttcgtatgc aacttggtca tagacaggtg aatctggaac 1680

aacgccactc ttgatgaagt tagtgagcct tggactaagc tccaaatcct cacagtcgtc 1740

ttcttcatcc gatgatatca taatttctc 1769

<210> 103

<211> 1769

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a hairpin RNAi (hpRNA) construct with G:U

basepairs, targeting a FANCM gene of B. napus

<400> 103

gggagaaatt atgatattat tggatgaaga agatgattgt gtggatttgg agtttagttt 60

aaggtttatt aattttatta agagtggtgt tgttttagat ttatttgttt atgattaagt 120

tgtatatgaa gtaaatagtg aagaagattt tgatttttta tatatgagtt taattaatga 180

attggtagaa gagttattga tatttgagaa aaaggtttat attgttttta tggttaatga 240

atttagaatt ttaatgaagg aagaagattt agttaatgaa atagaaagtt ttgttgtttt 300

tttaatgttt gaggagtgga gaattttttt ggtgaatatt attaatgtaa gtagtagtgt 360

tagtaaagat tggtgtgtga gtttgggaga aaagttagaa attttttgat agttttgtaa 420

gttgaagaga ttttgtagat ttggagattg tttgagtgtt gtgaaggaga ataattttgg 480

tattgtaaag atagattata ttgtaaggaa ataattattt tcttttttcc ttttagtata 540

aaatagttaa gtgatgttaa ttagtatgat tataataata tagttgttat aattgtgaaa 600

aaataattta taaatatatt gtttacataa acaacatagt aatgtaaaaa aatatgacaa 660

gtgatgtgta agacgaagaa gataaaagtt gagagtaagt atattatttt taatgaattt 720

gatcgaacat gtaagatgat atactagcat taatatttgt tttaatcata atagtaattc 780

tagctggttt gatgaattaa atatcaatga taaaatacta tagtaaaaat aagaataaat 840

aaattaaaat aatatttttt tatgattaat agtttattat ataattaaat atctatacca 900

ttactaaata ttttagttta aaagttaata aatattttgt tagaaattcc aatctgcttg 960

taatttatca ataaacaaaa tattaaataa caagctaaag taacaaataa tatcaaacta 1020

atagaaacag taatctaatg taacaaaaca taatctaatg ctaatataac aaagcgcaag 1080

atctatcatt ttatatagta ttattttcaa tcaacattct tattaatttc taaataatac 1140

ttgtagtttt attaacttct aaatggattg actattaatt aaatgaatta gtcgaacatg 1200

aataaacaag gtaacatgat agatcatgtc attgtgttat cattgatctt acatttggat 1260

tgattacagg atatggtctg tctttgcaat accaggatta ttctccttca cagcactcga 1320

gcaatctcca agtctacgaa gtctcttcaa cttgcgaggc tgtcgaagag tttctgactt 1380

ttctcccgaa ctcacgcgcc aatctttgct agcgctgctg cttgcgttgg tgatattcgc 1440

caagggagtt ctccactcct caggcattgg agaaacagcg aagctttctg tttcgttggc 1500

taaatcttct tccttcgttg gggttctgaa ttcattggcc gtagaagcaa tgtgaacctt 1560

tttctcaggt gtcgatggct cttctgccaa ttcattagtt aaactcgtgt gtggaagatc 1620

aaggtcttct tctctgtttg cttcgtatgc aacttggtca tagacaggtg aatctggaac 1680

aacgccactc ttgatgaagt tagtgagcct tggactaagc tccaaatcct cacagtcgtc 1740

ttcttcatcc gatgatatca taatttctc 1769

<210> 104

<211> 1259

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct, targeting a FANCM gene

of B. napus

<400> 104

gggttctgaa ttcattggcc gtagaagcaa tgtgaacctt tttctcaggt gtcgatggct 60

cttctgccaa ttcattagtt aaactcgtgt gtggaagatc aaggtcttct tctctgtttg 120

cttcgtatgc aacttggtca tagacaggtg aatctggaac aacgccactc ttgatgaagt 180

tagtgagcct tggactaagc tccaaatcct cacagtcgtc ttcttcatcc gatgatatca 240

taatttctcc agcacaattc tctatatctc ctttacctac atcggtggag tgccttttca 300

aaaggctttg ggactccaaa gtctcgttgg cacaatctgg caagaatcct tgctttaact 360

tgcatgaaat attatcttgg agaaattatg atatcatcgg atgaagaaga cgactgtgtg 420

gatttggagc ttagtccaag gctcactaac ttcatcaaga gtggcgttgt tccagattca 480

cctgtctatg accaagttgc atacgaagca aacagtgaag aagaccttga tcttccacac 540

acgagtttaa ctaatgaatt ggcagaagag ccatcgacac ctgagaaaaa ggttcacatt 600

gcttctacgg ccaatgaatt cagaacccca acgaaggaag aagatttagc caacgaaaca 660

gaaagcttcg ctgtttctcc aatgcctgag gagtggagaa ctcccttggc gaatatcacc 720

aacgcaagca gcagcgctag caaagattgg cgcgtgagtt cgggagaaaa gtcagaaact 780

cttcgacagc ctcgcaagtt gaagagactt cgtagacttg gagattgctc gagtgctgtg 840

aaggagaata atcctggtat tgcaaagaca gaccatatcc agcttccgct tcaatgaaga 900

ttctagcatt attatccgcg cgtatcttct tcttgccttg aacacagagc aaaaggaaat 960

acagaatcat tttacctctt atgttcttta cactgcgaga acgagatctg atatggtctg 1020

tctttgcaat accaggatta ttctccttca cagcactcga gcaatctcca agtctacgaa 1080

gtctcttcaa cttgcgaggc tgtcgaagag tttctgactt ttctcccgaa ctcacgcgcc 1140

aatctttgct agcgctgctg cttgcgttgg tgatattcgc caagggagtt ctccactcct 1200

caggcattgg agaaacagcg aagctttctg tttcgttggc taaatcttct tccttcgtt 1259

<210> 105

<211> 7074

<212> DNA

<213> Nicotiana benthamiana

<400> 105

atgcgagctg tttttgaaac aaagcgtgat cgtgaaatct tggtccttgc tagtaaagtt 60

ctgggtcacc tagctagatc tggcggtgca atgactgcag atgaagtgga acgtcagata 120

aaagttgcac taggatggct tcgtggtgaa agaattgagt atcgtttctt tgctgctgtc 180

ttaatattga aggaaatggc ggaaaatgct tcaactgttt tcaatgttca tgtgccggac 240

tttgtggagg ttgtttgggt tgctctgaag gatccaacat tggctgttcg agagaaggct 300

gttgaggcat tgcgtgcctg ccttcgcgtt attgaaaagc gcgagacacg atggcgtgtt 360

cagtggtatt ataagatgtt tgaggctacc caagatggat tgaccagaac tgcgcctgtt 420

catagtatac atggctccct tctcgcagtg ggagagctgc taaggaatac aggagagttc 480

atgatgtcaa gatacaggga ggttgcagaa attgttataa gatacttgga gcaccgagat 540

cgcctagttc ggctcagcat aacttctcta cttcctcgaa ttgctcattt cctgcgtgat 600

cgatttgtga ctaactactt aacgatatgc atgaatcata tacttcatgt ccttaaaata 660

cctgcagaac gtgccagtgg gttcattgct cttggggaga tggctggtgc tctggatggt 720

gaactcatta actatttgcc gacaataacc tctcacttgc gtgatgcgat tgctccccgt 780

agaggcaggc cctcatttga ggctctggca tgtgttggaa atattgctaa agcaatggga 840

cctgcaatgg agcctcatgt tcgtggtctc ttggatgcta tgttttctgc tgggctttcc 900

ctgacactag tggaagcctt ggagcaaata actgaaagca ttccatcttt gttgccgacc 960

attcaagatc ggcttcttga atgtatttca gcaattctct ccagatctaa tcatgcactc 1020

tcaagacaat caactgctat gagtcgagga catattgcaa cagttacccc ccaagtacca 1080

gaactgagtg gtcctgcact agttcaactt gctttgcaga ctcttgctcg ttttaatttc 1140

aagggccatg atcttcttga gtttgcaagg gagtctgttg ttgtgtattt agaagatgag 1200

gatggagcta cacgaaaaga tgctgcgcta tgttgctgca aactagtagc aaattctttc 1260

ttggcgatat cttctaccca gtttagtcct agtagaatca atcgtgccag tggaaagcga 1320

cgtcgacttg ttgaagagat tgtgcaaaaa cttctcatcg ctgctgttgc cgacgctgat 1380

gttactgttc ggcattcaat tttttcttct ctgtatgctg atggaggatt cgatgagttt 1440

ctagctcagg ctgatagttt gacagctata tttgccactt taaatgatga ggattttgaa 1500

gttcgtgact atgcaatttc actagctggt agactatctg aaaagaatcc agcatatgtt 1560

cttccagcac ttcgtcgcca tcttattcag ctgttaactt acctagagca aagtgcagat 1620

aataaatgta aagaagagag tgcaaagtta ttgggttgct tgattcgcaa ttgtgaacga 1680

ttagttcttc catacattgc tcccatacac aaggctcttg ttgcgaaact ctgtgaaggc 1740

acaggagtca atgcgaatag tggcattatt agtggagttc tagtgactgt tggagatctt 1800

gccagagtgg gtggctttgc catgcggcag tatatttcag aacttatgcc attaatcgtt 1860

gaagctctac tggatggggc agctgccacc aaacgtgaag tggccgtttc aacacttggt 1920

caagttgtac agagtacagg atatgtcata actccataca atgagtatcc tcagttgctt 1980

ggtttactct tgaaactgct caatggtgaa ctggcttggt caaccagaag agaggttttg 2040

aaggttctcg gcatcatggg tgcattagat ccccatgtgc acaagcgcaa tcagcaaagc 2100

ttacccggat cccatggtga agttacccgg gtgactggtg atcctggtca acatatcaga 2160

tcaatggatg aattgcctat ggatctttgg ccctcctttg caacatctga agattattat 2220

tccactgttg ctatcaactc actcatgcgg atactcaggg atccatctct gtcaagttac 2280

caccagaaag tggttggatc tcttatgttt attttcaagt ccatgggcct tggctgtgtc 2340

ccttatttgc ctaaggtttt gcctgatctc tttcacattg tacgaacatg tgaggatggt 2400

ctaaaagaat ttataacatg gaagcttgga accttggtat ctattgtccg ccagcacatc 2460

cgtaagtatc tgccagagtt actctctctg atatcagaaa tatggtcatc tttcagcttg 2520

cctgttgcta acagacctgt tcacattgct cctattttgc atctcgtgga gcaactttgc 2580

ttggctctca acgatgaatt tagaaagtac cttgctgata tacttccctg ctgtattcaa 2640

gttcttactg atgcagagag gtttagtgac tacacatacg ttattcctat tctccacaca 2700

cttgaagttt ttggtgggac attagatgag catatgcatc tgcttcttcc tgcacttatt 2760

cggttgttta aattggatgc ttcagtagaa gtaagacgcg gtgcaatcaa aactctcaca 2820

agattgatac ctcgtgtgca ggtcactgga cacatatctt ctcttgtgca tcacttgaag 2880

cttgtcttgg acgggaacaa agaagagctc aggaaggatg ctgttgatgc actttgttgt 2940

ctagctcatg ctcttggaga ggacttcacc atttttattc attctattca caagcttatg 3000

gttaaacata ggctgcagca caaggaattt gaagaaatcc gaggacgact ggaaaaacgt 3060

gagccactga ttttggggag caccgcagct cagagattaa atcggcggtt cccagttgag 3120

gtcatcagtg atcctttgag tgatggagag aatgagcact acgaggttgg gacggacatg 3180

cataagcagc ttaaaagcca tcaggttaat gatggtagat tgcgtaccgc tggtgaggct 3240

tctcaacgaa gcactaaaga ggattgggca gagtggatga ggcatttcag cattgaactt 3300

ctgaaagaat cacctagtcc agcattgcga acttgtgcaa gactcgctca actgcagcct 3360

tttgttgggc gagagttgtt tgctgcaggt tttgttagct gctggtcaca acttaatgag 3420

gctagtcaaa ggcagctagt acgtagtcta gaaatggcat tttcgtctcc aaatatccct 3480

cctgaaattc ttgctacact tctgaacttg gcggagttta tggaacacga tgagagaccc 3540

cttcctattg atatccgtct gcttggtgct cttgcggaga agtgtcgagc atttgcaaag 3600

gccctacact acaaggaaat ggaatttgaa ggcgcacttt caaataggag ggatgcaaat 3660

cctgttgctg tagttgaagc tctaatccat ataaataatc aattacatca acatgaggca 3720

gctgttggaa tattaacata tgctcagcag catttggggg ttcaattgaa ggagtcatgg 3780

tacgagaaat tgcaacgctg ggatgatgct cttaaagcat acactgctaa ggcgtcacaa 3840

gcttcgagtc cacatcttgc tttggatgct actttagggc gtatgcgatg ccttgctgct 3900

ctagctcggt gggaggagct taacaatctt tgtaaggaat actggacacc agctgagcca 3960

gcagctcgac tggaaatggc accaatggct gctagtgcgg cctggaacat gggtgagtgg 4020

gatcagatgg cagagtatgt ttctcggctt gatgatggtg atgaaaccaa actgcgagtc 4080

ttgggaaata ccgctgccag tggcgatgga agtagtaatg gcaccttttt cagggctgtt 4140

cttctagttc ggcgagggaa gtacgatgaa gcacgtgaat atgttgaaag agcaaggaaa 4200

tgtttggcga ccgagctcgc tgcactggtt cttgagagct atgaacgtgc ttacagcaac 4260

atggtccgtg ttcagcagct ttctgaatta gaagaggtga ttgaatactg tactcttcct 4320

atgggaaacc ctgttgctga aggaagaaga gctcttgttc gcaatatgtg gaatgagcgc 4380

ataaagggta caaaaagaaa tgttgaggtt tggcaagtac ttttagctgt gagggcactt 4440

gtattgcctc ctacagaaga cattgaaaca tggatcaaat ttgcatcact ttgccggaag 4500

aatggcagaa ttagccaagc tagatctaca ttggttaaac ttttacagtt cgatccagaa 4560

tcaactcctg caactgtgcg gtatcatggt ccccctcagg tgatgctagc atacttaaag 4620

taccaatggt cacttggcga ggatcataag cgaaaggaag cctttgctag gttgcaggac 4680

cttgccatgg acctctcaag aacagcagct cttcaaccag tattgcagaa tggattagtt 4740

gcttcttctg gtgtgccact tgttgctcgt gtatatctca gactcggcac ttggaagtgg 4800

gcactttctc ctggtttgga tgatgattct attcaagaaa ttcttagtgc atttacaaat 4860

gctactcact gtgcaacgaa gtggggaaag gcatggcata cctgggcact tttcaatacc 4920

gcagtgatgt ctcattacac tctgagaggt tttgcgaata ttgcttcaca gtttgttgtt 4980

gctgccgtaa ctggttattt tcactctata gcatgcggag cacatgctaa gggtgttgat 5040

gatagtttac aggatattct tcgtcttctt actttgtggt tcaaccatgg agctacttcg 5100

gatgtccaaa tggcattgca gaaaggattc actcatgtta acatcaacac atggttggtt 5160

gttttacctc agattattgc acggatacat tcaaataacc atgctgtcag agaactgata 5220

caatccttgc tagtgcgaat tggacagagt catccacagg ctcttatgta tccgcttctt 5280

gtggcatgta agtcaattag caatttgcgc agagctgcgg ctcaagaagt ggttgataaa 5340

gttagacagc acagcggcgt actcgttgat caggcccaac ttgtctcaaa ggagcttatc 5400

agggttgcaa tactgtggca tgaaatgtgg catgaggcac tggaagaggc cagccgttta 5460

tattttggcg aacacaacat tgagggcatg ctgaaggtgt tagagcctct gcatgaaatg 5520

cttgaggaag gagcgatgag gaacaatacc actataaagg agaaagcatt catccaggca 5580

taccgtcttg agttgttgga ggcgtatgaa tgttgtatga agtatcggag aactggtaaa 5640

gatgctgaat taacgcaggc ttgggatctc tattatcatg tattcaggcg gatagataag 5700

cagcttcaaa cactcacaac cctggatttg cagtctgttt cccccgagtt actggagtgt 5760

cgaaatttgg agctagctgt tcctggaact tatatagcag atgcaccagt ggtgacaatt 5820

gcatcatttg caccccaact tgttgtaatt acatccaaac aacggcctcg aaaattgaca 5880

atccatggga gtgatggaga agactatgct tttttgctca aagggcacga agatctacgc 5940

caagatgaac gtgtcatgca gttgtttggt ctggttaata ctttgctcga gaattcaaga 6000

aagactgcag agaaagattt atcaattcaa cgatatgctg tcattccatt gtcccctaat 6060

agtggactga taggatgggt tccaaattgc gacaccttgc accagcttat tcgagaatat 6120

agggatgccc ggaagatcac cctaaatcaa gagcataaat tgatgctgag ttttgcaccg 6180

gattatgata atttgccact tattgctaag gtggaggtgt ttgaatatgc tttgcaaaat 6240

acagaaggga atgacttatc aagggttctt tggttaaaga gtcgtacttc tgaagtctgg 6300

ctggacagaa gaacaaatta tacaagaagt ttggctgtca tgagtatggt tggataccta 6360

cttggtctgg gtgatcgaca tcctagtaac ctcatgcttc accgatacag tgggaagatt 6420

ctgcatattg actttggaga ttgctttgaa gcttcaatga atcgggagaa gtttccagag 6480

aaggttccct ttcgactcac tagaatgctt gtaaaagcaa tggaggttag tggtatagag 6540

ggaaatttcc ggtcaacatg tgagaatgta atgcaagttc tccgactgca taaagatagt 6600

gttatggcta tgatggaggc ctttgttcac gatccactta taaattggcg tcttttcaac 6660

ttcaatgaag ttccgcaaat gtccgcactt gccagtgcac atgtccctcc tgttgtgaac 6720

agtgaggaat cttcttcaaa tagagagctt cttcagccac aaaggggtgc aagggagaga 6780

gaactgcttc aggcggtcaa tcaattaggt gatgccaatg aggttctaaa tgaacgtgct 6840

gtggctgtta tggctcgaat gagtaataaa ctcacaggac gtgattttgc tgctacttct 6900

acatctgcga gctctctaca acatgcactg gaccacagta cgttaatttc tggagagacg 6960

cgtgaagctg atcatggttt atcagtgaaa ctacaagtcc aaaaacttat tcaacaagcg 7020

tcgtctcatg aaaatctttg ccaaaattat gttgggtggt gtccattttg gtag 7074

<210> 106

<211> 1513

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a TOR gene of

N. benthamiana

<400> 106

tgaatttagg tgacactata gaacaaagaa gagctcagga aggatgctgt tgatgcactt 60

tgttgtctag ctcatgctct tggagaggac ttcaccattt ttattcattc tattcacaag 120

cttatggtta aacataggct gcagcacaag gaatttgaag aaatccgagg acgactggaa 180

aaacgtgagc cactgatttt ggggagcacc gcagctcaga gattaaatcg gcggttccca 240

gttgaggtca tcagtgatcc tttgagtgat ggagagaatg agcactacga ggttgggacg 300

gacatgcata agcagcttaa aagccatcag gttaatgatg gtagattgcg taccgctggt 360

gaggcttctc aacgaagcac taaagaggat tgggcagagt ggatgaggca tttcagcatt 420

gaacttctga aagaatcacc tagtccagca ttgcgaactt ttaagctgct tatgcatgtc 480

cgtcccaacc tcgtagtgct cattctctcc atcactcaaa ggatcactga tgacctcaac 540

tgggaaccgc cgatttaatc tctgagctgc ggtgctcccc aaaatcagtg gctcacgttt 600

ttccagtcgt cctcggattt cttcaaattc cttgtgctgc agcctatgtt taaccataag 660

cttgtgaata gaatgaataa aaatggtgaa gtcctctcca agagcatgag ctagacaaca 720

aagtgcatca acagcatcct tcctgagctc ttctttgttc ccgtccaaga caagcttcaa 780

gtgatgcaca agagaagata tgtgtccagt gacctgcaca cgaggtatca atcttgtgag 840

agttttgatt gcaccgcgtc ttacttctac tgaagcatcc aatttaaaca accgaataag 900

tgcaggaaga agcagatgca tatgctcatc taatgtccca ccaaaaactt caagtgtgtg 960

gagaatagga ataacgtatg tgtagtcact aaacctctct gcatcagtaa gaacttgaat 1020

acagcaggga agtatatcag caaggtactt tctaaattca cacatccgta agtatctgcc 1080

agagttactc tctctgatat cagaaatatg gtcatctttc agcttgcctg ttgctaacag 1140

acctgttcac attgctccta ttttgcatct cgtggagcaa ctttgcttgg ctctcaacga 1200

tgaatttaga aagtaccttg ctgatatact tccctgctgt attcaagttc ttactgatgc 1260

agagaggttt agtgactaca catacgttat tcctattctc cacacacttg aagtttttgg 1320

tgggacatta gatgagcata tgcatctgct tcttcctgca cttattcggt tgtttaaatt 1380

ggatgcttca gtagaagtaa gacgcggtgc aatcaaaact ctcacaagat tgatacctcg 1440

tgtgcaggtc actggacaca tatcttctct tgtgcatcac ttgaagcttg tcttggacgg 1500

cccgggactc gaa 1513

<210> 107

<211> 1941

<212> DNA

<213> Hordeum vulgare

<400> 107

atggccgcag ccacctccgc cgccgtcgca ttctcgggcg ccgccgccgc cgccgcggcc 60

ttacccaagc ccgccctcca tcctctcccg cgccaccagc ccgcctcgcg ccgcgcgctc 120

cccgcccgcg tcgtcaggtg ctgcgccgcg tcccccgccg ccaccacggc cgcgcctccc 180

cccacctctc tccggccgtg ggggccctcc gagccccgca agggcgccga catcctcgtc 240

gaggcgctcg agcgctgcgg catcgtcgac gtcttcgcct accccggcgg cgcgtccatg 300

gagatccacc aggcgctcac gcgctcgccc gtcatcacca accacctctt ccgccacgag 360

cagggggagg cgttcgcagc gtccgggtac gcacgcgcgt ccggccgcgt cggcgtctgc 420

gtcgccacct ccggccccgg ggccaccaac ctcgtctccg cgctcgccga cgctctcctc 480

gactccatcc ccatggtcgc catcacgggc caggtcccac gccgcatgat cggcacggac 540

gcgttccagg agacgcccat agtggaggtc acgcgctcca tcaccaagca caactacctg 600

gtccttgacg tggaggacat cccccgcgtc atccaggaag ccttcttcct cgcgtcctct 660

ggccgcccgg ggcctgtgct ggttgatatc cccaaggaca tccagcagca gatggccgtg 720

cctgtttggg acacgccgat gagtttgcca gggtacatcg cccgcctgcc caagccacca 780

tctactgaat cgcttgagca ggtcctgcgc ctggttggcg aggcacggcg cccgattctg 840

tatgttggtg gcggctgcgc tgcatctggc gaggagttgc gccgctttgt tgagctcact 900

ggaattccag ttacaactac tctgatgggc cttggcaact tccccagtga cgacccactg 960

tcactgcgca tgcttgggat gcatggtacc gtgtatgcaa attatgcagt agataaggct 1020

gacctgttgc ttgcatttgg tgtgcggttt gatgatcgcg tgactgggaa aattgaggct 1080

tttgcaagca ggtccaagat tgtgcacatt gacattgatc cagctgagat tggcaagaac 1140

aagcagccac atgtctccat ttgtgcagat gttaagcttg ctttacaggg gttgaatggt 1200

ctattaagtg gcagcaaagc acaacagggt ctagattttg gtccatggca caaggagttg 1260

gatcagcaga agagggagtt tcctctagga tacaagactt ttggtgaggc aatcccaccg 1320

cagtatgcta tccaggtact ggatgagctg acaaaagggg aggcgattat tgccacaggt 1380

gttgggcagc atcagatgtg ggcggctcag tattacactt acaagcggcc acgtcagtgg 1440

ctgtcttcgt ctggtttggg ggcaatggga tttgggttgc cagctgcagc tggcgcttct 1500

gtggccaacc caggtgtcac agttgttgac attgatgggg atggtagttt cctcatgaac 1560

attcaggagt tggcgttgat ccgtattgag aacctcccag tgaaggtgat gatattgaac 1620

aaccagcacc tgggaatggt ggtgcagtgg gaggataggt tttacaaggc caaccgggcg 1680

cacacatacc ttggcaaccc agaaaatgag agtgagatat atccagattt tgtgacgatt 1740

gctaaaggat tcaacgttcc ggcagttcgt gtgacaaaga agagtgaagt cagtgcagct 1800

atcaagaaga tgcttgagac cccagggccg tacctgctgg atatcattgt cccgcatcag 1860

gagcacgtgc tgcctatgat cccaagcggt ggtgctttca aggacatgat catggagggt 1920

gatggcagga cctcgtatta a 1941

<210> 108

<211> 1505

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA targeting the ALS gene of barley

(H. vulgare)

<400> 108

agatttaggt gacactatag aatggtggtg cagtgggagg ataggtttta caaggccaac 60

cgggcgcaca cataccttgg caacccagaa aatgagagtg agatatatcc agattttgtg 120

acgattgcta aaggattcaa cgttccggca gttcgtgtga caaagaagag tgaagtcagt 180

gcagctatca agaagatgct tgagacccca gggccgtacc tgctggatat cattgtcccg 240

catcaggagc acgtgctgcc tatgatccca aacggtggtg ctttcaagga catgatcatg 300

gagggtgatg gcaggacctc gtattaatct gaatttcgac ctacaagacc tacaagtgtg 360

acatgcgcaa tcagcatgat gcctgcgtgt tgtatcaact actaggggtt cgactgtgaa 420

ccatgcgttt ttctagttta cttgtttcat tcatataaga ggtcctgcca tcaccctcca 480

tgatcatgtc cttgaaagca ccaccgtttg ggatcatagg cagcacgtgc tcctgatgcg 540

ggacaatgat atccagcagg tacggccctg gggtctcaag catcttcttg atagctgcac 600

tgacttcact cttctttgtc acacgaactg ccggaacgtt gaatccttta gcaatcgtca 660

caaaatctgg atatatctca ctctcatttt ctgggttgcc aaggtatgtg tgcgcccggt 720

tggccttgta aaacctatcc tcccactgca ccaccattcc caggtgctgg ttgttcaata 780

tcatcacctt cactgggagg ttctcaatac ggatcaacgc caactcctga atgttcatga 840

ggaaactacc atccccatca atgtcaacaa ctgtgacacc tgggttggcc acagaagcgc 900

cagctgcagc tggcaaccca aatcccattg cccccaaacc agacgaagac agccactgac 960

gtggccgctt gtaagtgtaa tactgagccg cccacatctg atgctgccca acacctgtgg 1020

caataatcgc ctcccctttt gtcagctcat ccagtacctg aatggtctat taagtggcag 1080

caaagcacaa cagggtctag attttggtcc atggcacaag gagttggatc agcagaagag 1140

ggagtttcct ctaggataca agacttttgg tgaggcaatc ccaccgcagt atgctatcca 1200

ggtactggat gagctgacaa aaggggaggc gattattgcc acaggtgttg ggcagcatca 1260

gatgtgggcg gctcagtatt acacttacaa gcggccacgt cagtggctgt cttcgtctgg 1320

tttgggggca atgggatttg ggttgccagc tgcagctggc gcttctgtgg ccaacccagg 1380

tgtcacagtt gttgacattg atggggatgg tagtttcctc atgaacattc aggagttggc 1440

gttgatccgt attgagaacc tcccagtgaa ggtgatgata ttgaacaacc agcacctggc 1500

ccggg 1505

<210> 109

<211> 1824

<212> DNA

<213> Hordeum vulgare

<400> 109

atgcagactc tgtcggcgca gcccctcgcc tcctcctctt cgatacagcg ccaccatggg 60

cgccgacgcg gccccggctc cgtccggttc gctccccgcg cggccgccgc ggctgccgcc 120

acgtccacca gcacggcccg ctcgccggcg tacgtctcgt cgccgtccac gaggaaggtg 180

cccgggtacg agcagtcgtc gccgcctgcc attgcctcgc cgcagaagca ggggagcagc 240

ggcggcgagg gcgagcagag cctcaacttc ttccagcgcg cggcggccgc ggcgctcgac 300

gcgttcgagg aggggttcat caacaatgtc ctggagcggc cccacgcgct gccgcgcacg 360

gccgacccgg ccgtgcagat cgccggcaac ttcgcccccg tcggcgagca gccccccgtg 420

cgcgccctca cggtctccgg ccgcatcccg cccttcatca acggcgtcta cgcccgcaac 480

ggcgccaacc cctgcttcga gcccacggcc ggccaccacc tcttcgacgg cgacggcatg 540

gtccacgcca tccgcatccg aaacggcgcc gccgagtcct acgcctgccg cttcaccgag 600

accgcccgcc tctcccagga gcgcgccgcg gggaggcccg tcttccctaa gaccatcggc 660

gagctccacg gccactctgg catcgcgagg ctggccctct tctacgcgcg cggcgcctgc 720

ggcctcgtcg acccgtccca cggcactggt gttgccaacg ccggcctcgt ctacttcaac 780

ggccgcctcc tcgccatgtc cgaggacgac ctcccgtacc aggtccgcgt caccgccggt 840

ggcgacctcg agaccgtcgg ccgctacgac ttcgacggcc agctcgactg cgccatgatc 900

gcgcacccca agctcgaccc tgtctccggc gagctcttcg cgctcagcta cgatgtcatc 960

aagaagccgt acctcaagta cttctacttc cacgccgacg gcaccaagtc cgccgacgtc 1020

gagatcgagc tcgaccagcc caccatgatc cacgacttcg ccatcaccga gaacttcgtc 1080

gtcgtgcccg accaccagat ggtgttcaag ctcgccgaga tgttccgcgg cggctcgccg 1140

gtgatgctcg acaaggagaa gacctcccgc ttcggcgtcc tcccaaagta cgccaaggac 1200

tcgtcggaga tgatgtgggt ggacgtgccg gactgcttct gtttccacct ctggaactcg 1260

tgggaggagc cggagacgga cgaggtggtg gtgatcggct cctgcacgac ccccgcagac 1320

tccatcttca acgacacgga cgaccacctc gagagcgtgc tcaccgagat ccggctcaac 1380

acgcgcaccg gcgagtccac gcggcgggcc atcctgccgc tggagagcca ggtgaacctc 1440

gaggtcggca tggtgaaccg caacatgctg ggccggaaga cgaggtacgc ctacctggcc 1500

gtggccgagc cgtggcccaa ggtgtccggg ttcgccaagg tggacctggt gaccggcgag 1560

ctgaccaagt tcgagtacgg cgagggccgg ttcggcggcg agccgtgctt cgtgcccatg 1620

gacggcgagc acgcgcgccc cggcgccgag gacgacggct acgtgctctc cttcgtgcgc 1680

gacgaggacg ccggcacatc cgagctcctg gtcgtcaacg ccgccgacat gcggctcgag 1740

gccaccgtgc agctgccgtc ccgggtcccc tatggcttcc acggcacatt catcggcgac 1800

gccgacctcg acgcccagca ctaa 1824

<210> 110

<211> 1779

<212> DNA

<213> Hordeum vulgare

<400> 110

atgcagacac tcacagcgtc cagctcggtc tcctccatac agcggcaccg gccgcacccc 60

gcgggccgcc ggtccagctc ggtcaccttc tccgcccgcg ccgtcagctc cgcgccgcgc 120

gcgccggcac cgtcccggtt cgtgcgcggc gccgacgcgg cgcccgccaa gcccctcatt 180

gccgtcccca agccgcccgc cgtggagagg caggagaaga agctcaactt cttccagcgc 240

gccgcggtca cggcgctcga cgcgttcgag gaaggatttg tggccaacgt gctcgagcgc 300

ccgcacggcc tctccaggac ggtcgacccc gcggtgcaga tcgccggcaa cttcgcgcct 360

gtcggggaga cacctcctgt gcaggcgctg cccgtgaccg accgcatccc cccgttcatc 420

aacggcgtgt acgcccgcaa cggcgccaac ccgcacttcg accccgtcgc cgggcaccac 480

ctgttcgacg gcgacggcat ggtgcacgct ctgcgcatcc gcaacggcgt cgccgagacc 540

tacgcctccc gcttcaccga gacggagcgc ctgcagcagg agcgcgcgct ggggcgcccg 600

atgttcccca aggccattgg tgagctccat ggccactctg ggatcgcgcg ccttgctctg 660

ttctacgcgc gcgcggcctg cggcctcatc gacccctcgc gcggcaccgg cgtggccaac 720

gccggcctgg tctacttcaa cggccacctc ctcgccatgt ccgaggacga catcccgtac 780

cacgtccgcg tcaccgacga cggcgacctc cagaccgtcg gccgctacga cttcgacggg 840

cagctcgagt gccccatgat cgcgcacccc aaactcgacc ccgccaccgg ggagctccac 900

gcgctcagct acgacgtcat caagaagcct tacctgaagt acttctactt cgcggccgac 960

ggcaccaagt cggccgacgt cgagatcccg ctggaccagc ccaccatgat ccacgacttc 1020

gccatcaccg agaattacgt ggtcgtgccc gaccaccagg tggtgttcaa gctgcaggag 1080

atgctgcgcg gcggctcgcc cgtggtgctc gacaaggaga agacgtcccg cttcggcgtg 1140

ctgcccaagt gcgccgccga cgcgtcggag atggtgtggg tggacgtgcc ggactgcttc 1200

tgcttccacc tctggaacgc gtgggaggag gaggagaccg acgaggtggt ggtgatcggc 1260

tcctgcatga cccccgccga ctccatcttc aacgagtcgg acgagtgcct cgagagcgtg 1320

ctcacggaga tccgcctcaa cacccgcacc ggcgagtcca cgcggcgccc catcctggcg 1380

ctgtcagagc aggtgaacct ggaggtcggc atggtgaact ccaacctgct gggccgcaag 1440

acgcggtacg cctacctggc cgtggccgag ccgtggccca aggtgtccgg cttcgccaag 1500

gtcgacctgg ccacgggcga gctcaccaaa ttcgagtacg gcgagggccg gttcggcggc 1560

gagccctgct tcgtgcccat ggacccggcc acgtcccgcg gcgaggacga cgggtacatt 1620

ctcaccttcg tgcacgacga ggccgccggc acgtcggagc tgctggtggt caatgccgcc 1680

gacatgcggc tggaggcgac catccagctg ccgtcccgcg tgccatacgg gttccacggc 1740

accttcatca ccggcaagga gctcgaatcc caggcctga 1779

<210> 111

<211> 1500

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting the NCED1

genes of barley Hordeum vulgare and wheat Triticum aestivum

<400> 111

taatacgact cactataggg tcgacgaggc cgcaggcgcc gcgcgcgtag aagagggcca 60

gcctcgcgat gccagagtgg ccgtggagct cgccgatggt cttagggaag acgggcctcc 120

ccgcggcgcg ctcctgggag aggcgggcgg tctcggtgaa gcggcaggcg taggactcgg 180

cggcgccgtt tcggatgcgg atggcgtgga ccatgccgtc gccgtcgaag aggtggtggc 240

cggccgtggg ctcgaagcag gggttggcgc cgttgcgggc gtagacgccg ttgatgaagg 300

gcgggatgcg gccggagacc gtgagggcgc gcacgggggg ctgctcgccg acgggggcga 360

agttgccggc gatctgcacg gccgggtcgg ccgtgcgcgg cagcgcgtgg ggccgctcca 420

ggacattgtt gatgaacccc tcctcgaacg cgtcgagctc cggccgcatc ccgcccttca 480

tcaacggcgt ctacgcccgc aacggcgcca acccctgctt cgagcccacg gccggccacc 540

acctcttcga cggcgacggc atggtccacg ccatccgcat ccgaaacggc gccgccgagt 600

cctacgcctg ccgcttcacc gagaccgccc gcctctccca ggagcgcgcc gcggggaggc 660

ccgtcttccc taagaccatc ggcgagctcc acggccactc tggcatcgcg aggctggccc 720

tcttctacgc gcgcggcgcc tgcggcctcg tcgacccgta ccacggcact ggtgttgcca 780

acgccggcct cgtctacttc aacggccgcc tcctcgccat gtccgaggac gacctcccgt 840

accaggtccg cgtcaccgcc ggtggcgacc tcgagaccgt cggccgctac gacttcgacg 900

gccagctcga ctgcgccatg atcgcgcacc ccaagctcga ccctgtctcc ggcgagctct 960

tcgcgctcag ctacgatgtc atcaagaagc cgtacctcaa gtacttctac ttcacgcccg 1020

acggcaccaa gtccgccgac gtcgagatcg agctcgacga agcgggaggt cttctccttg 1080

tcgagcatca ccggcgagcc gccgcggaac atctcggcga gcttgaacac catctggtgg 1140

tcgggcacga cgacgaagtt ctcggtgatg gcgaagtcgt ggatcatggt gggctggtcg 1200

agctcgatct cgacgtcggc ggacttggtg ccgtcgggcg tgaagtagaa gtacttgagg 1260

tacggcttct tgatgacatc gtagctgagc gcgaagagct cgccggagac agggtcgagc 1320

ttggggtgcg cgatcatggc gcagtcgagc tggccgtcga agtcgtagcg gccgacggtc 1380

tcgaggtcgc caccggcggt gacgcggacc tggtacggga ggtcgtcctc ggacatggcg 1440

aggaggcggc cgttgaagta gacgaggccg gcgttggcaa caccagtgcc gtggtacgta 1500

<210> 112

<211> 1500

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting the NCED2

genes of barley Hordeum vulgare and wheat Triticum aestivum

<400> 112

taatacgact cactataggg ttggcgccgt tgcgggcgta cacgccgttg atgaacgggg 60

ggatgcggtc ggtcacgggc agcgcctgca caggaggtgt ctccccgaca ggcgcgaagt 120

tgccggcgat ctgcaccgcg gggtcgaccg tcctggagag gccgtgcggg cgctcgagca 180

cgttggccac aaatccttcc tcgaacgcgt cgagcgccgt gaccgcggcg cgctggaaga 240

agttgagctt cttctcctgc ctctccacgg cgggcggctt ggggacggca atgaggggct 300

tggcgggcgc cgcgtcggcg ccgcgcacga accgggacgg tgccggcgcg cgcggcgcgg 360

agctgacggc gcgggcggag aaggtgaccg agctggaccg gcggcccgcg gggtgcggcc 420

ggtgccgctg tatggaggag accgagctgg acgctgtcga cgcggcgccc gccaagcccc 480

tcattgccgt ccccaagccg cccgccgtgg agaggcagga gaagaagctc aacttcttcc 540

agcgcgccgc ggtcacggcg ctcgacgcgt tcgaggaagg atttgtggcc aacgtgctcg 600

agcgcccgca cggcctctcc aggacggtcg accccgcggt gcagatcgcc ggcaacttcg 660

cgcctgtcgg ggagacacct cctgtgcagg cgctgcccgt gaccgaccgc atccccccgt 720

tcatcaacgg cgtgtacgcc cgcaacggcg ccaacccgta cttcgacccc gtcgccgggc 780

accacctgtt cgacggcgac ggcatggtgc acgctctgcg catccgcaac ggcgtcgccg 840

agacctacgc ctcccgcttc accgagacgg agcgcctgca gcaggagcgc gcgctggggc 900

gcccgatgtt ccccaaggcc attggtgagc tccatggcca ctctgggatc gcgcgccttg 960

ctctgttcta cgcgcgcgcg gcctgcggcc tcatcgaccc ctcgcgcggc accggcgtgg 1020

ccaacgccgg cctggtctac ttcaacggcc acctcctccc cggtggcggg gtcgagtttg 1080

gggtgcgcga tcatggggca ctcgagctgc ccgtcgaagt cgtagcggcc gacggtctgg 1140

aggtcgccgt cgtcggtgac gcggacgtgg tacgggatgt cgtcctcgga catggcgagg 1200

aggtggccgt tgaagtagac caggccggcg ttggccacgc cggtgccgcg cgaggggtcg 1260

atgaggccgc aggccgcgcg cgcgtagaac agagcaaggc gcgcgatccc agagtggcca 1320

tggagctcac caatggcctt ggggaacatc gggcgcccca gcgcgcgctc ctgctgcagg 1380

cgctccgtct cggtgaagcg ggaggcgtag gtctcggcga cgccgttgcg gatgcgcaga 1440

gcgtgcacca tgccgtcgcc gtcgaacagg tggtgcccgg cgacggggtc gaagtacgta 1500

<210> 113

<211> 1521

<212> DNA

<213> Hordeum vulgare

<400> 113

atggccttct tcctcctcct gtgcatcctc gtctctgtgg ccatcgtgtc ctacgcccac 60

cacgcaatcc ggcggaggcg ccagggctgc gctcatggcc gtcatgagca ggccgccctc 120

aagctgcccc ccggctccat gggcctgcct tacgtcggcg agaccctgca gctctactcc 180

caggacccca gcgtcttcct ctcctccaag cagaagcggt acggcgagat cttcaagacg 240

cacctcctgg ggtgcccgtg cgtgatgctg gcgagcccgg aggcggcgcg cttcgtgctg 300

gtgtcgcggg cccacctctt caagccgacg tacccgcgga gcaaggagcg cctcatcggc 360

ccgtcggcgc tcttcttcca ccagggcgac taccacctcc gcctccgccg gctcgtccag 420

ggcccgctcg gccccgaggc cctgcgcaag ctcgtgccgg acatcgaggc cgccgttcgc 480

tccacgctcg ccgcctgggc ggacggcgac gtcgccagca ctttccacgc catgaagagg 540

ctctcgttcg acgtcggcat cgtgacgatc ttcggcgggc ggctggacga gcggcggaag 600

gaggagctca ggcggaacta cgccgtcgtg gagaaaggct acaactcctt ccccaacagc 660

ttccccggga cgctatacta caaggcgatc caggcgaggc ggcggctgaa cggcgtgctg 720

agcgacgtcg tgcacgagcg tagggagcgg ggcgagcacg gcgacgacct cctcggctgc 780

ctcatgcggt cgcgggccgg cggcgacgac gccgacgacg agggcgcgct gctgacggac 840

gagcaggtcg ccgacaacgt catcggcgtg ctgttcgcgg cgcaggacac gacggccagc 900

gtgctcacct ggatcgtcaa gtacctccac gaccgcccga agctgctcga ggccgtcagg 960

gcggagcacg cggcgatcca cgaggccaac gacggcggga ggcggccgct gacatgggcg 1020

cagacgagga gcatgacgct gacgcacagg gtgattttgg agagcctaag gatggccagc 1080

atcatctcct tcacgttcag ggaggccgtg gccgacgtgg agtacaaagg gtttcttatc 1140

cccaaggggt ggaaggtgat gccgctcttc aggaacatcc atcacagccc ggactacttc 1200

caggatccac acaagttcga cccttcgcga ttcaaggtgg cgccgcggcc gaacaccttc 1260

acsccgttcg ggagcggggt gcacgcgtgc ccggggaacg agctggccaa gctcgagatg 1320

ctggtgctca tccaccacct ggtcaccggc tacaggtggg aggttgttgg atcgagcgac 1380

gacgtcgagt acagcccatt ccccgttccc cgccatggcc tgctcgccag ggtacggcga 1440

gatgacggcg tctgcgcggg taggaagggg tgcccgactg atgaagatga caactacgac 1500

gacgacgaag tgatagtgtg a 1521

<210> 114

<211> 1506

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting the ABA-OH-2

genes of barley Hordeum vulgare and wheat Triticum aestivum

<400> 114

taatacgact cactataggg cggtcgtgga ggtacttgac gatccaggtg agcacgctgg 60

ccgtcgtgtc ctgcgccgcg aacagcacgc cgatgacgtt gtcggcgacc tgctcgtccg 120

tcagcagcgc gccctcgtcg tcggcgtcgt cgccgccggc ccgcgaccgc atgaggcagc 180

cgaggaggtc gtcgccgtgc tcgccccgct ccctacgctc gtgcacgacg tcgctcagca 240

cgccgttcag ccgccgcctc gcctggatcg ccttgtagta tagcgtcccg gggaagctgt 300

tggggaagga gttgtagcct ttctccacga cggcgtagtt ccgcctgagc tcctccttcc 360

gccgctcgtc cagccgcccg ccgaagatcg tcacgatgcc gacgtcgaac gagagcctct 420

tcatggcgtg gaaagtgctg gcgacgtcgc cgtccgccta caactccttc cccaacagct 480

tccccgggac gctatactac aaggcgatcc aggcgaggcg gcggctgaac ggcgtgctga 540

gcgacgtcgt gcacgagcgt agggagcggg gcgagcacgg cgacgacctc ctcggctgcc 600

tcatgcggtc gcgggccggc ggcgacgacg ccgacgacga gggcgcgctg ctgacggacg 660

agcaggtcgc cgacaacgtc atcggcgtgc tgttcgcggc gcaggacacg acggccagcg 720

tgctcacctg gatcgtcaag tacctccacg accgcccgta gctgctcgag gccgtcaggg 780

cggagcacgc ggcgatccac gaggccaacg acggcgggag gcggccgctg acatgggcgc 840

agacgaggag catgacgctg acgcacaggg tgattttgga gagcctaagg atggccagca 900

tcatctcctt cacgttcagg gaggccgtgg ccgacgtgga gtacaaaggg tttcttatcc 960

ccaaggggtg gaaggtgatg ccgctcttca ggaacatcca tcacagcccg gactacttcc 1020

aggatccaca caagttcgac ccttcgcgat tcaaggtcgc tcgatccaac aacctcccac 1080

ctgtagccgg tgaccaggtg gtggatgagc accagcatct cgagcttggc cagctcgttc 1140

cccgggcacg cgtgcacccc gctcccgaac ggagtgaagg tgttcggccg cggcgccacc 1200

ttgaatcgcg aagggtcgaa cttgtgtgga tcctggaagt agtccgggct gtgatggatg 1260

ttcctgaaga gcggcatcac cttccacccc ttggggataa gaaacccttt gtactccacg 1320

tcggccacgg cctccctgaa cgtgaaggag atgatgctgg ccatccttag gctctccaaa 1380

atcaccctgt gcgtcagcgt catgctcctc gtctgcgccc atgtcagcgg ccgcctcccg 1440

ccgtcgttgg cctcgtggat cgccgcgtgc tccgccctga cggcctcgag cagctacgta 1500

ggtacc 1506

<210> 115

<211> 3885

<212> DNA

<213> Arabidopsis thaliana

<400> 115

atggaagctg aaattgtgaa tgtgagacct cagctagggt ttatccagag aatggttcct 60

gctctacttc ctgtcctttt ggtttctgtc ggatatattg atcccgggaa atgggttgca 120

aatatcgaag gaggtgctcg tttcgggtat gacttggtgg caattactct gcttttcaat 180

tttgccgcca tcttatgcca atatgttgca gctcgcataa gcgttgtgac tggtaaacac 240

ttggctcaga tctgcaatga agaatatgac aagtggacgt gcatgttctt gggcattcag 300

gcggagttct cagcaattct gctcgacctt accatggttg tgggagttgc gcatgcactt 360

aaccttttgt ttggggtgga gttatccact ggagtgtttt tggccgccat ggatgcgttt 420

ttatttcctg ttttcgcctc tttccttgaa aatggtatgg caaatacagt atccatttac 480

tctgcaggcc tggtattact tctctatgta tctggcgtct tgctgagtca gtctgagatc 540

ccactctcta tgaatggagt gttaactcgg ttaaatggag agagcgcatt cgcactgatg 600

ggtcttcttg gcgcaagcat cgtccctcac aatttttata tccattctta ttttgctggg 660

gaaagtacat cttcgtctga tgtcgacaag agcagcttgt gtcaagacca tttgttcgcc 720

atctttggtg tcttcagcgg actgtcactt gtaaattatg tattgatgaa tgcagcagct 780

aatgtgtttc acagtactgg ccttgtggta ctgacttttc acgatgcctt gtcactaatg 840

gagcaggtat ttatgagtcc gctcattcca gtggtctttt tgatgctctt gttcttctct 900

agtcaaatta ccgcactagc ttgggctttc ggtggagagg tcgtcctgca tgacttcctg 960

aagatagaaa tacccgcttg gcttcatcgt gctacaatca gaattcttgc agttgctcct 1020

gcgctttatt gtgtatggac atctggtgca gacggaatat accagttact tatattcacc 1080

caggtcttgg tggcaatgat gcttccttgc tcggtaatac cgcttttccg cattgcttcg 1140

tcgagacaaa tcatgggtgt ccataaaatc cctcaggttg gcgagttcct cgcacttaca 1200

acgtttttgg gatttctggg gttgaatgtt gtttttgttg ttgagatggt atttgggagc 1260

agtgactggg ctggtggttt gagatggaat accgtgatgg gcacctcgat tcagtacacc 1320

actctgcttg tatcgtcatg tgcatcctta tgcctgatac tctggctggc agccacgccg 1380

ctgaaatctg cgagtaacag agcggaagct caaatatgga acatggatgc tcaaaatgct 1440

ttatcttatc catctgttca agaagaggaa attgaaagaa cagaaacaag gaggaacgaa 1500

gacgaatcaa tagtgcggtt ggaaagcagg gtaaaggatc agttggatac tacgtctgtt 1560

actagctcgg tctatgattt gccagagaac attctaatga cggatcaaga aatccgttcg 1620

agccctccag aggaaagaga gttggatgta aagtactcta cctctcaagt tagtagtctt 1680

aaggaagact ctgatgtaaa ggaacagtct gtattgcagt caacagtggt taatgaggtc 1740

agtgataagg atctgattgt tgaaacaaag atggcgaaaa ttgaaccaat gagtcctgtg 1800

gagaagattg ttagcatgga gaataacagc aagtttattg aaaaggatgt tgaaggggtt 1860

tcatgggaaa cagaagaagc taccaaagct gctcctacaa gcaactttac tgtcggatct 1920

gatggtcctc cttcattccg cagcttaagt ggggaagggg gaagtgggac tggaagcctt 1980

tcacggttgc aaggtttggg acgtgctgcc cggagacact tatctgcgat ccttgatgaa 2040

ttttggggac atttatatga ttttcatggg caattggttg ctgaagccag ggcaaagaaa 2100

ctagatcagc tgtttggcac tgatcaaaag tcagcctctt ctatgaaagc agattcgttt 2160

ggaaaagaca ttagcagtgg atattgcatg tcaccaactg cgaagggaat ggattcacag 2220

atgacttcaa gtttatatga ttcactgaag cagcagagga caccgggaag tatcgattcg 2280

ttgtatggat tacaaagagg ttcgtcaccg tcaccgttgg tcaaccgtat gcagatgttg 2340

ggtgcatatg gtaacaccac taataataat aatgcttacg aattgagtga gagaagatac 2400

tctagcctgc gtgctccatc atcttcagag ggttgggaac accaacaacc agctacagtt 2460

cacggatacc agatgaagtc atatgtagac aatttggcaa aagaaaggct tgaagcctta 2520

caatcccgtg gagagatccc gacatcgaga tctatggcgc ttggtacatt gagctataca 2580

cagcaacttg ctttagcctt gaaacagaag tcccagaatg gtctaacccc tggaccagct 2640

cctgggtttg agaattttgc tgggtctaga agcatatcgc gacaatctga aagatcttat 2700

tacggtgttc catcttctgg caatactgat actgttggcg cagcagtagc caatgagaaa 2760

aaatatagta gcatgccaga tatctcagga ttgtctatgt ccgcaaggaa catgcattta 2820

ccaaacaaca agagtggata ctgggatccg tcaagtggag gaggagggta tggtgcgtct 2880

tatggtcggt taagcaatga atcatcgtta tattctaatt tggggtcacg ggtgggagta 2940

ccctcgactt atgatgacat ttctcaatca agaggaggct acagagatgc ctacagtttg 3000

ccacagagtg caacaacagg gaccggatcg ctttggtcca gacagccctt tgagcagttt 3060

ggtgtagcgg agaggaatgg tgctgttggt gaggagctca ggaatagatc gaatccgatc 3120

aatatagaca acaacgcttc ttctaatgtt gatgcagagg ctaagcttct tcagtcgttc 3180

aggcactgta ttctaaagct tattaaactt gaaggatccg agtggttgtt tggacaaagc 3240

gatggagttg atgaagaact gattgaccgg gtagctgcac gagagaagtt tatctatgaa 3300

gctgaagctc gagaaataaa ccaggtgggt cacatggggg agccactaat ttcatcggtt 3360

cctaactgtg gagatggttg cgtttggaga gctgatttga ttgtgagctt tggagtttgg 3420

tgcattcacc gtgtccttga cttgtctctc atggagagtc ggcctgagct ttggggaaag 3480

tacacttacg ttctcaaccg cctacaggga gtgattgatc cggcgttctc aaagctgcgg 3540

acaccaatga caccgtgctt ttgccttcag attccagcga gccaccagag agcgagtccg 3600

acttcagcta acggaatgtt acctccggct gcaaaaccgg ctaaaggcaa atgcacaacc 3660

gcagtcacac ttcttgatct aatcaaagac gttgaaatgg caatctcttg tagaaaaggc 3720

cgaaccggta cagctgcagg tgatgtggct ttcccaaagg ggaaagagaa tttggcttcg 3780

gttttgaagc ggtataaacg tcggttatcg aataaaccag taggtatgaa tcaggatgga 3840

cccggttcaa gaaaaaacgt gactgcgtac ggatcattgg gttga 3885

<210> 116

<211> 1080

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting the EIN2 gene

of A. thaliana

<400> 116

taatacgact cactataggg agctgcaaca tattggcata agatggcggc aaaattgaaa 60

agcagagtaa ttgccaccaa gtcatacccg aaacgagcac ctccttcgat atttgcaacc 120

catttcccgg gatcaatata tccgacagaa accaaaagga caggaagtag agcaggaacc 180

attctctgga taaaccctag ctgaggtctc acattcacaa tttcagcttc catcctaaat 240

ctatctgata atataattac tcagagtagg attcaaggta aactctacag tcgtggttca 300

ctaaaagcct cagttgagta aaattcacag atttgatcta aaacacctga atgtgagacc 360

tcagctaggg tttatccaga gaatggttcc tgctctactt cctgtccttt tggtttctgt 420

cggatatatt gatcccggga aatgggttgc aaatatcgaa ggaggtgctc gtttcgggta 480

tgacttggtg gcaattactc tgcttttcaa ttttgccgcc atcttatgcc aatatgttgc 540

agctcccaac agcgttgtga ctggtaaaca cttggctcag atctgcaatg aagaatatga 600

caagtggacg tgcatgttct tgggcattca ggcggagttc tcagcaattc tgctcgacct 660

taccatggtt gtgggagttg cgcatgcact taaccttttg tttggggtgg agttatccac 720

tggagtgttt ttggccgcca tggatgcgag tgggatctca gactgactca gcaagacgcc 780

agatacatag agaagtaata ccaggcctgc agagtaaatg gatactgtat ttgccatacc 840

attttcaagg aaagaggcga aaacaggaaa taaaaacgca tccatggcgg ccaaaaacac 900

tccagtggat aactccaccc caaacaaaag gttaagtgca tgcgcaactc ccacaaccat 960

ggtaaggtcg agcagaattg ctgagaactc cgcctgaatg cccaagaaca tgcacgtcca 1020

cttgtcatat tcttcattgc agatctgagc caagtgttta ccagtcacaa cgctgttgac 1080

<210> 117

<211> 1188

<212> DNA

<213> Arabidopsis thaliana

<400> 117

atggtgatgg ctggtgcttc ttctttggat gagatcagac aggctcagag agctgatgga 60

cctgcaggca tcttggctat tggcactgct aaccctgaga accatgtgct tcaggcggag 120

tatcctgact actacttccg catcaccaac agtgaacaca tgaccgacct caaggagaag 180

ttcaagcgca tgtgcgacaa gtcgacaatt cggaaacgtc acatgcatct gacggaggaa 240

ttcctcaagg aaaacccaca catgtgtgct tacatggctc cttctctgga caccagacag 300

gacatcgtgg tggtcgaagt ccctaagcta ggcaaagaag cggcagtgaa ggccatcaag 360

gagtggggcc agcccaagtc aaagatcact catgtcgtct tctgcactac ctccggcgtc 420

gacatgcctg gtgctgacta ccagctcacc aagcttcttg gtctccgtcc ttccgtcaag 480

cgtctcatga tgtaccagca aggttgcttc gccggcggta ctgtcctccg tatcgctaag 540

gatctcgccg agaacaatcg tggagcacgt gtcctcgttg tctgctctga gatcacagcc 600

gttaccttcc gtggtccctc tgacacccac cttgactccc tcgtcggtca ggctcttttc 660

agtgatggcg ccgccgcact cattgtgggg tcggaccctg acacatctgt cggagagaaa 720

cccatctttg agatggtgtc tgccgctcag accatccttc cagactctga tggtgccata 780

gacggacatt tgagggaagt tggtctcacc ttccatctcc tcaaggatgt tcccggcctc 840

atctccaaga acattgtgaa gagtctagac gaagcgttta aacctttggg gataagtgac 900

tggaactccc tcttctggat agcccaccct ggaggtccag cgatcctaga ccaggtggag 960

ataaagctag gactaaagga agagaagatg agggcgacac gtcacgtgtt gagcgagtat 1020

ggaaacatgt cgagcgcgtg cgttctcttc atactagacg agatgaggag gaagtcagct 1080

aaggatggtg tggccacgac aggagaaggg ttggagtggg gtgtcttgtt tggtttcgga 1140

ccaggtctca ctgttgagac agtcgtcttg cacagcgttc ctctctaa 1188

<210> 118

<211> 1080

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting the CHS gene

of A. thaliana

<400> 118

taatacgact cactataggg gccacaccat ccttagctga cttcctcctc atctcgtcta 60

gtatgaagag aacgcacgcg ctcgacatgt ttccatactc gctcaacacg tgacgtgtcg 120

ccctcatctt ctcttccttt agtcctagct ttatctccac ctggtctagg atcgctggac 180

ctccagggtg ggctatccag aagagggagt tccagtcact tatccccaaa ggtttaaacg 240

cttcgtctag actcttcaca atgttcttgg agatgaggcc gggaacatcc ttgaggagat 300

ggaaggtgag accaacttcc ctcaaatgtc cgtctatggc accatcagac tggaactccc 360

tcttctggat agcccaccct ggaggtccag cgatcctaga ccaggtggag ataaagctag 420

gactaaagga agagaagatg agggcgacac gtcacgtgtt gagcgagtat ggaaacatgt 480

cgagcgcgtg cgttctcttc atactagacg agatgaggag gaagtcagct aaggatggtg 540

tggccccgac aggagaaggg ttggagtggg gtgtcttgtt tggtttcgga ccaggtctca 600

ctgttgagac agtcgtcttg cacagcgttc ctctctaaac agaacgcttg ccttctatct 660

gcctacctac ctacgcaaaa ctttaatcct gtcttatgtt ttatataata taatcattat 720

atgtttacgc aataattaag gaagaatgac atttccaaac aaagatttga tgtcattcaa 780

gacccataga tttaatattg taaaaagaca caaaaaagag agtacaaaaa cagtcgaata 840

gacctgtcca gcacatatca catatcacat caaatgcatt cttccttaat tattgcgtaa 900

acatataatg attatattat ataaaacata agacaggatt aaagttttgc gtaggtaggt 960

aggcagatag aaggcaagcg ttctgtttag agaggaacgc tgtgcaagac gactgtctca 1020

acagtgagac ctggtccgaa accaaacaag acaccccact ccaacccttc tcctgtcaac 1080

<210> 119

<211> 3456

<212> DNA

<213> Lupinus angustifolius

<400> 119

atgttgactc ttcaacccac acatgagtca agtagtcaat accctcctca tacacttata 60

gctgagacct gtcactttga ttatctgtac tatactaatc aaagttctct aattatgtca 120

cttggagaat catccctgca atggaaatac catgttttct tgagttttag gggaggtgac 180

acccgcttaa gcttcactaa tcacttatat gctgcgttgg tgcgaaaagg aatcattact 240

ttccgagatg acaaacaact tcacaaagga gatgccattt ctcaacatct gcatcaatca 300

atccaacagt ctctagctgc cattgttgtt atctcggaga actatgcttc ttccacttgg 360

tgtttggatg agctaaaact aattcttgaa tcgagaatag atgtttttcc agtcttttat 420

ggtgtcactc cttctgatgt tcgataccag aaaaatagtt ttgctgaggc tttcaataaa 480

catgttgtaa gatttgaaca agatgaagag aaagtgcaaa aatggagaga ttgcttgaaa 540

gaagttgctg atttttctgg atgggagtcc aaggacatgg ctgaagcaga actcattgaa 600

gatgttattg aaaaggtatg gataaaacta caaccaaaat tgccatccta caatgaagga 660

gtggttggat ttgattcaag ggtgaagaaa atgatttcac ttttaagcat aggatcacaa 720

gatattcggt ttatcgggat atggggtatg gctggaactg gaaaaacaat tcttgctaga 780

gtaatctacg aaacaataag tagccaattt gagattaaat gtttccttct taatgttaga 840

gaggtttctc aaacatctga tggattggtt tccttacaaa gaaaacttct ttctaccctt 900

aagataagca acctagaaat tgatgatttg tatgatggaa agaagaaaat tatgaacctt 960

ttgtgcaaca aaagtgttct tcttgtcctt gatgacatta gtcatttaag tcagctagag 1020

aatttggcta aaactaaagg ttggtttggt ccatgcagca gagtgataat aacaaccaaa 1080

gatatgcact tactagtatc acatggtgcg tgtgagaagt atgagatgag aatcttaaat 1140

gaaagttctt cctttcaact cttcagccag aaagcattca gaagagataa acctccagag 1200

ggttatttag aaataactaa aagtatggtc aaatatgctg gaggtcttcc tttggcactt 1260

aaagtgttgg gttcttttgt ttgtggaaga agtctcagtc agtggaagga tgctttggat 1320

aagataaaac aagttctgcc gaaagacatt ttgaacacac taataatagg ttatgatgga 1380

ctagaagatg cagaaaagac tttgttttta gatattgctt tcttctttac aggacggtcg 1440

aaaattgaag tgatacaggt attggcagat tgtggcctta atccaacaat tggaataagt 1500

cttcttattg aaagatctct agtaagttgt tgtggaggaa ttttggaaat gcatgattta 1560

cttcaagaaa tgggtagaaa tattgtatat caagaatctc cggatgatgc aagcagacgc 1620

agtaggttat gctctttaga agatattaac cgagtattca gaaaaaacaa gggaaccaat 1680

atcattcaag gaatagttct gaaatcaagt gacccatgtg aagcatattg gcatcctgaa 1740

gccttctcaa aaatggataa tcttagagta ctcatcattt tgtgtgattt gcaccttccc 1800

ctcggcctca aatgtctctc tagttcatta aaacttcttg aatggaaggg atatcctttg 1860

gaatatctac catttggcct gcaactgcta gaacttgttc acttgaaaat gcattgcagc 1920

aaacttaaac aactttggaa tggaactcaa attttcagag agctaaaatc aattgatctc 1980

agtgattcca gagatctaat tcaaactcca gatatttctg aggttccatg tcttgagagt 2040

ttagttttga aaggttgtaa aaaccttgtt gaggttcatc aatctgttgc aaagcacaag 2100

aatgttgcta tactagacct ggaaggttgc atcagtctta agaccctgcc aagaaaattg 2160

gagatgaatg ctttggaaaa gttcattctc tccggctgct cacaaattaa aaaccttccc 2220

gaatttgggg agagtatgga atgtctatct atgcttaatt taagagattg cacaagtctt 2280

gtttctcttc cacagagtgt tcgaaacatg aaatccttta gagatctcaa tatccatggt 2340

tgctcaaaat tgtttaagct gacaaacaat tcaaatgaaa ataatgtcgt ggaagaaatt 2400

gatgagactg aaacaggtag gagagaagtg cattcatcat ggagcttttc tctccttact 2460

gagaaagtgt ttgatttcgt aaagtatcca gttagcatgg actcgaagtt gccttctctc 2520

tcaagtttcc ctcggttgaa gaaattagat atgggcaact gtaatctcag tgatggacca 2580

attatagatc atattggaca tttaacatca ctggaagtgt tatatttagc tgggaacaac 2640

tttgttgacc ttacagcaag cattggtaac ctttctcggc tacaacgcct tggtttatat 2700

aaatgccgaa gacttaggac attgcctgag cttccaccca gtgtatgcca gttacttatg 2760

aacgactgca ctcaactgga acctatgtta tttgacacac aaataatttt gaaaatattt 2820

gaggcaaata gatggagcct gacacgcgaa ttgtggttcc tgattccagg gagtgaaatc 2880

ccagcatggt ttgagcatca agattatttt agcctgaaac caagtttagc gcctttcgat 2940

tatcacgagg agtatgcttt tattgtttca acaatagtaa acatccctga ctattgcctt 3000

tcaagtgatt ggataggaat tattgtatgc tttttactgg aaagtggttt aaaggcagac 3060

ctacacagac atattcgtag aagtccggtc acgatcggat ggtcttttaa agatcccgat 3120

gcagaaacgg tttacccctt acgcttcact aaacgtcgtt ggacacattt caaaggcaat 3180

cacctattga ttactacttt tggaagtgat catagaatat acaagcacta cttaacttgt 3240

ggcaaaagca aagtgcaatt gatattttgt ggtgagaata tttgcaagtg cgggaagcta 3300

aagctgaaaa actgtgggat ccgtgtgatt tgtaaggaag atggtgtatc gcgtagaggc 3360

gaggaaacga gtgaagttga ggtgccttcc acttcagttg aatctgatgt tcacaaacaa 3420

tcacgaataa ctgaaattac agatgaatat gaataa 3456

<210> 120

<211> 1280

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting the L.

angustifolius N-like gene

<400> 120

taatacgact cactataggg tatcgaacat cagaaggagt gacaccataa aagactggaa 60

aaacatctat tctcgattca agaattagtt ttagctcatc caaacaccaa gtggaagagt 120

atttccattg cagggatgat tctccaagtg acataattag agaactttga ttagtatagt 180

acagataatc aaagtgacag gtctcagcta taagtgtatg aggagggtat tgactacttg 240

actcatgtgt gggttgaaga gtcaacatat agcttcacgg atcccacagt ttttcagctt 300

tagcttcccg cacttgcaaa tattctcacc acaaaatatc aattgcactt tgcttttgcc 360

acaagttaag tagtgcttgt atattctatg atcaccctgt tgactcttca acccacacat 420

gagtcaagta gtcaataccc tcctcataca cttatagctg agacctgtca ctttgattat 480

ctgtactata ctaatcaaag ttctctaatt atgtcacttg gagaatcatc cctgcaatgg 540

aaatactctt ccacttggtg tttggatgag ctaaaactaa ttcttgaatc gagaatagat 600

gtttttccag tcttttatgg tgtcactcct tctgatgttc gatacccgta gttttgctga 660

ggctttcaat aaacatgttg taagatttga acaagatgaa gagaaagtgc agtttgagca 720

tcaagattat tttagcctga aaccaagttt agcgcctttc gattatcacg aggagtatgc 780

ttttattgtt tcaacaatag taaacatccc tgactattgc ctttcaagtg attggatagg 840

aattattgta tgctttttac tggaaagtgg tttaaaggca gacctacaca gacatattcc 900

aaaagtagta atcaataggt gattgccttt gaaatgtgtc caacgacgtt tagtgaagcg 960

taaggggtaa accgtttctg catcgggatc tttaaaagac catccgatcg tgaccggact 1020

tctacgaata tgtctgtgta ggtctgcctt taaaccactt tccagtaaaa agcatacaat 1080

aattcctatc caatcacttg aaaggcaata gtcagggatg tttactattg ttgaaacaat 1140

aaaagcatac tcctcgtgat aatcgaaagg cgctaaactt ggtttcaggc taaaataatc 1200

ttgatgctca aactgcactt tctcttcatc ttgttcaaat cttacaacat gtttattgaa 1260

agcctcagca aaactacgta 1280

<210> 121

<211> 1527

<212> DNA

<213> Vitis pseudoreticulata

<400> 121

atggctggcg acgaggagac gacgacgacg gcagcaacac ttgaaacaac gtccacttgg 60

gctgttgcct ctgtttgctt tattttgatt gcactctcca tacttattga gcatgccctc 120

catctcttag ccaagtactt caacaagaag cggaggaggt ctctcattca tgctcttaac 180

aacgtcaaat cggagttgat gctcttgggg ttcgtctctt tgttgctgac tgtgtgccaa 240

aagtatattg cgaagatttg tatcccaagg agcgtaggtg aaacttttct tccctgcaag 300

accttgacag aaagtgattc agaagaagaa accaaatgcg aagagcaggg aaagatgtct 360

ttgctgtcta gacaaggcgt ggaggaacta caatacttaa ttttcgtgct ggccttcttc 420

cattccctct actgcgtcct cacattcggt cttgggatgg ccaagatgaa gaaatgggag 480

tcctgggagg cagaaacaag aacactggaa tatcagttta caaatgatcc acggaggttc 540

aggctcatcc atcagacatc atttggaaag caacatctga gatattggag tgagcatcag 600

atacttcgtt ggccggcttg ttttattcgg cagttctatc catccgtctc caaagtggat 660

tacttgactc ttagacatgg gttcattatg gcccattttg cagaaggaag caactatgac 720

ttccaaaagt atataaaaag agctttggaa aaagactttg gagtggtggt gggaggaagt 780

ttctgggttt ggagtttctc catgcttttt gtgttcttca atgctcaagt attttacaac 840

tatttatggc taccctttat tccattggtg atgctgttgt tggttggaac aaagctacag 900

ggcattataa ctaagatgtg cttagatagc catgataaag ctctcgttgt tagaggaact 960

ttgcttgtca ggcccagtga tcacttcttc tggtttggaa aaccggaatt gctcctacat 1020

cttatgcact ttatattgtt tcagaactct tttcaactgg cgttctttac atggacttgg 1080

tacaaatttg gattcagatc atgcttccat gatacaactg aggatatcgt cataaggctt 1140

gtcatgggtg tgttagtaca actcctttgt ggctacgtga cactgcctct gtatgccctg 1200

gtcacgcaga tggggacatc aatgaggaca attgtcttta ctgagggagt cgttgaaggt 1260

ctgaacagat ggagaaggaa agccaagaaa aacatagcac gcaggaacaa ccactcagct 1320

cgtccctccc tggatgcttc actcgacaat tcaccttctt ttaacactct ggatacttct 1380

ttctctgtag acctcgatca gccatcatca gatgctggtt atttgactgt tgaaatatca 1440

gatgaagaga cggtcgctac taaacggcca gaaccgcgtc agaagttggg atcttttgag 1500

ggtttcgact cgtgcaaaac atcataa 1527

<210> 122

<211> 1480

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a first ledRNA construct targeting a Vitis

MLO gene

<400> 122

taatacgact cactataggg tagccataaa tagttgtaaa atacttgagc attgaagaac 60

acaaaaagca tggagaaact ccaaacccag aaacttcctc ccaccaccac tccaaagtct 120

ttttccaaag ctctttttat atacttttgg aagtcatagt tgcttccttc tgcaaaatgg 180

gccataatga acccatgtct aagagtcaag taatccactt tggagacgga tggatagaac 240

tgctgaataa aacaaaccgg ccaacgaagt atccgatgct cactccaata tctcagatgt 300

tgctttccaa atgatgtctg atggatgagc gtgaacctcc gtggatcatt tgtaaactga 360

tattccagtg ttcttgtttc tgcctcccag gactcccatt tcttcatctt ggccatccca 420

agaccgaatg tgaggacgca gtagagcaca tcatttggaa agcaacatct gagatattgg 480

agtgagcatc ggatacttcg ttggccggtt tgttttattc agcagttcta tccatccgtc 540

tccaaagtgg attacttgac tcttagacat gggttcatta tggcccattt tgcagaagga 600

agcaactatg acttccaaaa gtatataaaa agagctttgg aaaaagactt tggagtggtg 660

gtgggaggaa gtttctgggt ttggagtttc tccatgcttt ttgtgttctt caatgctcaa 720

gtattttaca actatttatg gctacccgta attccattgg tgatgctgtt gttggttgga 780

acaaagctac agggcattat aactaagatg tgcctagata gccatgataa agctctcgtt 840

gttagaggaa ctttgcttgt caggcccagt gatcacttct tctggtttgg aaaaccggaa 900

ttgctcctac atcttatgca ctttatattg tttcagaact cttttcaact ggcgttcttt 960

acatggactt ggtacaaatt tggattcaga tcatgcttcc atgatacaac tgaggatatc 1020

gtcataaggc ttgtcatggg tgtgttatgg ctttccttct ccatctgttc agaccttcaa 1080

cgactccctc agtaaagaca attgtcctca ttgatgtccc catctgcgtg accagggcat 1140

acagaggcag tgtcacgtag ccacaaagga gttgtactaa cacacccatg acaagcctta 1200

tgacgatatc ctcagttgta tcatggaagc atgatctgaa tccaaatttg taccaagtcc 1260

atgtaaagaa cgccagttga aaagagttct gaaacaatat aaagtgcata agatgtagga 1320

gcaattccgg ttttccaaac cagaagaagt gatcactggg cctgacaagc aaagttcctc 1380

taacaacgag agctttatca tggctatcta ggcacatctt agttataatg ccctgtagct 1440

ttgttccaac caacaacagc atcaccaatg gaattacgta 1480

<210> 123

<211> 894

<212> DNA

<213> Myzus persicae

<400> 123

atgttcaaac acttgtgcaa taccgtttca caaagtataa aacctagtag ttttttatca 60

aaagtttgtt caaacaaata tctcgtcgtg ccgtaccgga tagcgatttt taacaacatg 120

ggaagttaca aattgtacct ggccgtcatg gcaatagctg tcatagctgc agttcaggaa 180

attagttgca aggttcagac ttccgaacag gacgatgatc aggaaggata ttacgatgat 240

gagggaggag tgaacgataa tcagggagaa gagaacgata atcagggaga agagaacgat 300

aatcagggag aagagaacga taatcaggga gaagagaagg aagaagtttc cgaaccagag 360

atggagcacc atcagtgcga agaatacaaa tcgaagatct ggaacgatgc atttagcaac 420

ccgaaggcta tgaacctgat gaaactgacg tttaatacag ctaaggaatt gggctccaac 480

gaagtgtgct cggacacgac ccgggcctta tttaacttcg tcgatgtgat ggccaccagc 540

ccgtacgccc acttctcgct aggtatgttt aacaagatgg tggcgtttat tttgagggag 600

gtggacacga catcggacaa atttaaagag acgaagcagg tggtcgaccg tatctcgaaa 660

actccagaga tccgtgacta tatcaggaac tcggccgcca agaccgtcga cttgctcaag 720

gaacccaaga ttagagcacg actgttcaga gtgatgaaag ccttcgagag tctgataaaa 780

ccaaacgaaa acgaagcatt aatcaaacag aagattaagg ggttaaccaa tgctcccgtc 840

aagttagcca agggtgccat gaaaacggtt ggacgtttct ttagacattt ttaa 894

<210> 124

<211> 960

<212> DNA

<213> Myzus persicae

<400> 124

atgactgaga caatgcaact ccgtggtacc cttcgtgggc ataatggttg ggttacgcag 60

atcgccacca atccgatcca cactgacatg attctgtctt gttcacgaga caagaccttg 120

attgtttggg atctgacacg tgatgagctc aactatggta tccccaagaa acgtttgtac 180

ggacattcgc acttcgtcag cgacgtcgtt ctttcatcag atggtaacta cgctctttcc 240

ggttcttggg ataagactct tcgtctgtgg gatttggctg ctggacgtac cactcgtcgt 300

tttgaagacc acaccaagga tgtattgagc gttgccttct ctgctgacaa ccgtcaaatc 360

gtttctggaa gtcgggacaa gactatcaag ttgtggaata ctttggctga gtgcaaatac 420

actattcagg atgatggaca tagcgattgg gtatcatgtg tacggttctc tcctaatatc 480

cataacccaa tcattgtgag tgctggttgg gacaaggttg tcaaggtatg gaacttaact 540

aactgccgca tcaagaccaa ccattatgga cacactggat accttaacac cgttactgtt 600

tcacctgatg gttctttgtg tgcttcagga ggaaaagatt gcaaagctat gttatgggat 660

cttaatgacg gcaaacactt gcacacactg gaccataacg atatcattga agctttgtgc 720

tttagcccca accgttactg gttgtgcgct gcatttggac catcaatcaa aatttgggat 780

ttggaaagca aagaaatggt tgaggaactt cgcccagaag ttgtatctca atcacagaat 840

agcaataccg aaccacccag atgtctgtca cttgcatggt caactgatgg acaaacattg 900

tttgctggat actcagacaa taacattaga gtttggcaag tgtctgtcag tgctcgttaa 960

<210> 125

<211> 1401

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric construct encoding the ledRNA targeting M.persicae C002

gene

<400> 125

gaattctaat acgactcact atagggtgct ccatctctgg ttcggaaact tcttccttct 60

cttctccctg attatcgttc tcttctccct gattatcgtt ctcttctccc tgattatcgt 120

tctcttctcc ctgattatcg ttcactcctc cctcatcatc gtaatatcct tcctgatcat 180

cgtcctgttc ggaagtctga accttgcaac taatttcctg aactgcagct atgacagcta 240

ttgccatgac ggccaggtac aatttgtaac ttcccatgtt gttaaaaatc gctatccggt 300

acggcacgac gagatatttg tttgaacaaa cttttgataa aaaactacta ggttttatac 360

tttgtgaaac ggtattgcac aagtgtttga acataagaga gttggaagtt acaaattgta 420

cctggccgtc atggcaatag ctgtcatagc tgcagttcag gaaattagtt gcaaggttca 480

gacttccgaa caggacgatg atcaggaagg atattacgat gatgagggag gagtgaacga 540

taatcaggga gaagagaacg ataatcaggg agaagagaac gataatcagg gagaagagaa 600

cgataatcag ggagaagaga aggaagaagt ttccgaacca gagatggagc acccaacagt 660

gcgaagaata caaatcgaag atctggaacg atgcatttag caacccgaag gctatgaacc 720

tgatgaaact gacgtttaat acagctaagg aattgggctc caacgaagtg tgctcggaca 780

cgacccgggc cttatttaac ttcgtcgatg tgatggccac cagcccgtac gcccacttct 840

cgctaggtat gtttaacaag atggtggcgt ttattttgag ggaggtggac acgacatcgg 900

acaatctgaa cagtcgtgct ctaatcttgg gttccttgag caagtggacg gtcttggcgg 960

ccgagttcct gatatagtca cggatctctg gagttttcga gatacgggcg accacctgct 1020

tcgtctcttt aaatttgtcc gatgtcgtgt ccacctccct caaaataaac gccaccatct 1080

tgttaaacat acctagcgag aagtgggcgt acgggctggt ggccatcaca tcgacgaagt 1140

taaataaggc ccgggtcgtg tccgagcaca cttcgttgga gcccaattcc ttagctgtat 1200

taaacgtcag tttcatcagg ttcatagcct tcgggttgct aaatgcatcg ttccagatct 1260

tcgatttgta ttcttcgcac tgttaacaag cttagcatat ccatgatatc tgttagtttt 1320

tttcctgaaa gagcggccgc cctagcataa ccccgcgggg cctcttcggg ggtctcgcgg 1380

ggttttttgc tgaaaggatc c 1401

<210> 126

<211> 1401

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric construct encoding the ledRNA targeting M.persicae

Rack-1 gene

<400> 126

gaattctaat acgactcact atagggttat ggatattagg agagaaccgt acacatgata 60

cccaatcgct atgtccatca tcctgaatag tgtatttgca ctcagccaaa gtattccaca 120

acttgatagt cttgtcccga cttccagaaa cgatttgacg gttgtcagca gagaaggcaa 180

cgctcaatac atccttggtg tggtcttcaa aacgacgagt ggtacgtcca gcagccaaat 240

cccacagacg aagagtctta tcccaagaac cggaaagagc gtagttacca tctgatgaaa 300

gaacgacgtc gctgacgaag tgcgaatgtc cgtacaaacg tttcttgggg ataccatagt 360

tgagctcatc acgtgtcaga tcccaaacaa tcaaggtctt gtcccggttc ttgggataag 420

actcttcgtc tgtgggattt ggctgctgga cgtaccactc gtcgttttga agaccacacc 480

aaggatgtat tgagcgttgc cttctctgct gacaaccgtc aaatcgtttc tggaagtcgg 540

gacaagacta tcaagttgtg gaatactttg gctgagtgca aatacactat tcaggatgat 600

ggacatagcg attgggtatc atgtgtacgg ttctctccta atatccataa cccaaccatt 660

gtgagtgctg gttgggacaa ggttgtcaag gtatggaact taactaactg ccgcatcaag 720

accaaccatt atggacacac tggatacctt aacaccgtta ctgtttcacc tgatggttct 780

ttgtgtgctt caggaggaaa agattgcaaa gctatgttat gggatcttaa tgacggcaaa 840

cacttgcaca cactggacca taacgatatc attgaagctt tgtgctttag ccccaaccgt 900

tacacagaca tctgggtggt tcggtattgc tattctgtga ttgagataca acttctgggc 960

gaagttcctc aaccatttct ttgctttcca aatcccaaat tttgattgat ggtccaaatg 1020

cagcgcacaa ccagtaacgg ttggggctaa agcacaaagc ttcaatgata tcgttatggt 1080

ccagtgtgtg caagtgtttg ccgtcattaa gatcccataa catagctttg caatcttttc 1140

ctcctgaagc acacaaagaa ccatcaggtg aaacagtaac ggtgttaagg tatccagtgt 1200

gtccataatg gttggtcttg atgcggcagt tagttaagtt ccataccttg acaaccttgt 1260

cccaaccagc actcacaatg gttaacccgg gtagcatatc catgatatct gttagttttt 1320

ttcctgaaag agcggccgcc ctagcataac cccgcggggc ctcttcgggg gtctcgcggg 1380

gttttttgct gaaaggatcc c 1401

<210> 127

<211> 2396

<212> DNA

<213> Helicoverpa armigera

<400> 127

agacattgat tagtgagctc caaactccgt acgtacgttc ttagtttagt ttgttcgttc 60

gtattgtcgc agtcacatcg ctccggtgcc cgcttcgaca tttcccgcca aaagtgacgt 120

aacatatccg tgatctgtgt gaatatgtca gtgacttttt taaattaatt ttttaatagc 180

aaaattgtga tcgaaggaat ttttacaaga tgacggctgg gaatgaagag catgagcctc 240

taattacatc gtctgtcgac aatcagcgtg tggcctacag taattcacca ccggatgacc 300

gcacaccaga atcttcttcc ccacgcggca gtggcggaga agtaacgcta gccataccat 360

cacaccgcaa ctatggagcc atcggaggcg tggagaaggt cacatacacc tgggcagaca 420

tcaatgcctt tgctactgaa tccaggtcta ggtcccgaag gatttggaac ttctggaagc 480

cctccgccag tggcatgttc cagcaaagga aacagttgtt gaggaatgta aatggagccg 540

cctacccagg cgaactgctc gccatcatgg gatcctccgg tgccgggaag accacactcc 600

tcaacactct gaccttccgc actccaagcg gggtgctgtc cagtggcact cgagcactga 660

acggccagcc tgctacccct gaggcgttat cagcactgtc tgcgtatgtt cagcagcagg 720

atctgttcat tggcacgctg actgtgaagg agcatttagt attccaggct atggtgcgga 780

tggaccgaca tataccgtat gcgcagcgca tgaggagagt tcaagaggtt attactgagt 840

tggcgctaac aaaatgccag aacacagtga taggcatccc tgggcggctg aagggtatct 900

ccggcgggga gatgaagagg ctgtccttcg ccagcgaggt gctcacggat ccaccgctca 960

tgttctgcga tgaacccacc tctggactcg attcttttat ggcgcagaat gttatacagg 1020

tactgaaagg tctcgcacaa aaaggcaaga cagtcgtatg cacgatccac cagccgtctt 1080

cggagctgta cgcgatgttc gataagctgc tcatcatggc agacgggaag gtcgccttcc 1140

tcggctcccc tgatcaggct aatgatttct ttaaagacct aggagcagcg tgtcctccta 1200

actacaaccc agcggaccac ttcatccaac tcctggcggg agtgccgggc agggaggaga 1260

ccacgcgcac cactatcgat actgtctgca cggcattcgc gcgctctgag gtcggctgca 1320

agattgctgc agaagctgaa aatgcactct actttgagcg caagatatcg cagggctggg 1380

cggacccggc gtggtctgaa gccacggcta tccgcgcgcg ccgctcgccg tacaaggcgt 1440

cgtggtgcgc gcagttccgc gcggtgctgt ggcgctcgtg gctgtccgtc actaaggagc 1500

ccatgctcat caaagtgcgc ttcctacaga ctattatggt atcgatcctg atcggcgtga 1560

tctacttcgg gcagcacctg gaccaggacg gcgtgatgaa catcaacggc gccatcttca 1620

tgttcctcac caacatgacc ttccagaaca tcttcgctgt tattaacgta ttctgctcag 1680

aactgccaat attcatacga gaacaccact ccgggatgta tcgagctgac gtgtacttcc 1740

tatcgaagac gttagccgaa gcacctgtgt tcgccaccat accacttgtg ttcaccacca 1800

tagcatacta catgataggg ctgaaccctg aacctaagcg gttctttata gcgtccggtt 1860

tggctgccct gattactaac gttgctacgt cgtttggcta cctgatatcg tgtgccagca 1920

acagcgtgag catggcagcg tcagtgggac ctcccatcat catccccttc atgttgttcg 1980

gaggcttctt cctcaacact ggctccgtac caccatacct gggctggata tcgtacctgt 2040

cctggttcca ctacggcaac gaagcgctgc tggtcaacca gtggtctgga gtggaaacca 2100

tcgcctgcac ccgggagaac ttcacctgtc ccgcctctgg gcaggtcgtc ttggatactc 2160

ttagcttttc tgaggatgac ttcacaatgg acgtggtgaa catgatccta cttttcatcg 2220

gcttcagatt tttggcgtat ctcgctctct tgtaccgcgc tcgccgaggc aagtgagtct 2280

taggtacaaa atgctgcgag aatgggccat atgaaggaag aatgttgaat aaatagtgta 2340

attatttagg atgtaaggag tcaatggaga tttgataaat aaaacaattt ataccg 2396

<210> 128

<211> 1480

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a ABC

transporter white gene of Helicoverpa armigera

<400> 128

taatacgact cactataggg tatatgtcgg tccatccgca ccatagcctg gaatactaaa 60

tgctccttca cagtcagcgt gccaatgaac agatcctgct gctgaacata cgcagacagt 120

gctgataacg cctcaggggt agcaggctgg ccgttcagtg ctcgagtgcc actggacagc 180

accccgcttg gagtgcggaa ggtcagagtg ttgaggagtg tggtcttccc ggcaccggag 240

gatcccatga tggcgagcag ttcgcctggg taggcggctc catttacatt cctcaacaac 300

tgtttccttt gctggaacat gccactggcg gagggcttcc agaagttcca aatccttcgg 360

gacctagacc tggattcagt agcaaaggca ttgatgtctg cccaggtgta tgtgaccttc 420

tccacgcctc cgatggctcc atagttgttc cagcaaagga aacagttgtt gaggaatgta 480

aatggagccg cctacccagg cgaactgctc gccatcatgg gatcctccgg tgccgggaag 540

accacactcc tcaacactct gaccttccgc actccaagcg gggtgctgtc cagtggcact 600

cgagcactga acggccagcc tgctacccct gaggcgttat cagcactgtc tgcgtatgtt 660

cagcagcagg atctgttcat tggcacgctg actgtgaagg agcatttagt attccaggct 720

atggtgcgga tggaccgaca tatacccgta tgcgcagcgc atgaggagag ttcaagaggt 780

tattactgag ttggcgctaa caaaatgcca gaacacagtg ataggcatcc ctgggcggct 840

gaagggtatc tccggcgggg agatgaagag gctgtccttc gccagcgagg tgctcacgga 900

tccaccgctc atgttctgcg atgaacccac ctctggactc gattctttta tggcgcagaa 960

tgttatacag gtactgaaag gtctcgcaca aaaaggcaag acagtcgtat gcacgatcca 1020

ccagccgtct tcggagctgt acgcgatgat gaagtggtcc gctgggttgt agttaggagg 1080

acacgctgct cctaggtctt taaagaaatc attagcctga tcaggggagc cgaggaaggc 1140

gaccttcccg tctgccatga tgagcagctt atcgaacatc gcgtacagct ccgaagacgg 1200

ctggtggatc gtgcatacga ctgtcttgcc tttttgtgcg agacctttca gtacctgtat 1260

aacattctgc gccataaaag aatcgagtcc agaggtgggt tcatcgcaga acatgagcgg 1320

tggatccgtg agcacctcgc tggcgaagga cagcctcttc atctccccgc cggagatacc 1380

cttcagccgc ccagggatgc ctatcactgt gttctggcat tttgttagcg ccaactcagt 1440

aataacctct tgaactctcc tcatgcgctg cgcatacgta 1480

<210> 129

<211> 811

<212> DNA

<213> Linepithema humile

<400> 129

agagagaacg atgaggacaa tgagatggaa aaaacaacaa cgtcccaacg tcccttcgac 60

gacgccattc caccagccct ataaaacccc gaggatcatc ggcgtcccaa cattactcgg 120

tcagagtctc gaggaacgcc gtgtccgaga tgatcatcac caggaaccgc atcaaccgcg 180

caactctaat ctgcgttctg gcgtcgtggc tttgcttggc gtctcgcgct tccgccgaat 240

acgaatcgcg ggagatgtcg aacggcggac cgggcgtcga cgcctcgtgc atcgagggca 300

agtgcatgaa gcgcaccgcc acgcaggatg ctaccgccag catgtggttc ggcccgcgtt 360

tgggaagacg gcgcagatcg gacgagaagc aggaagtgaa ttccgagata caggctctgg 420

cggaagcctt ggatagcggg cgtttggccc tatttgccat tccagctaac gacaagagac 480

aaccgactca atttacaccg cgactggggc gaggatcaga cgaggaccta tcctcctacg 540

gagacgcgat tgagaggaac gagatcgacg atcgtatatt acccgcgtta ttcgcgccgc 600

gtttaggacg acgaattcct tggtcaccgt cgccgagact tggacgccaa ttacgcagca 660

ttttgcgaaa aatgtaggcg ccgtcgaaag attattatca aaagttacaa atgaagagtg 720

atctcgtaga cctgcgcgtg aagatgaaat aacaactaaa attatagcac tattaagaca 780

taaagaaata aagtactgat gtttatttgt a 811

<210> 130

<211> 1360

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a PBAN gene in

Argentine ants

<400> 130

taatacgact cactataggg aattcacttc ctgcttctcg tccgatctgc gccgtcttcc 60

caaacgcggg ccgaaccaca tgctggcggt agcatcctgc gtggcggtgc gcttcatgca 120

cttgccctcg atgcacgagg cgtcgacgcc cggtccgccg ttcgacatct cccgcgattc 180

gtattcggcg gaagcgcgag acgccaagca aagccacgac gccagaacgc agattagagt 240

tgcgcggttg atgcggttcc tggtgatgat catctcggac acggcgttcc tcgagactct 300

gaccgagtaa tgttgggacg ccgatgatcc tcggggtttt atagggctgg tggaatggcg 360

tcgtcgaagg gacgttggga cgttgttgtt ttttccatct cattgtcctc atcgttcacg 420

ccgtgtccga gatgatcatc accaggaacc gcatcaaccg cgcaactcta atctgcgttc 480

tggcgtcgtg gctttgcttg gcgtctcgcg cttccgccga atacgaatcg cgggagatgt 540

cgaacggcgg accgggcgtc gacgcctcgt gcatcgaggg caagtgcatg aagcgcaccg 600

ccacgcagga tgctaccgcc agcatgtggt tcggcccgcg tttgggaaga cggcgcagat 660

cggacgagaa gcaggaagtg aattcccgta atacaggctc tggcggaagc cttggatagc 720

gggcgtttgg ccctatttgc cattccagct aacgacaaga gacaaccgac tcaatttaca 780

ccgcgactgg ggcgaggatc agacgaggac ctatcctcct acggagacgc gattgagagg 840

aacgagatcg acgatcgtat attacccgcg ttattcgcgc cgcgtttagg acgacgaatt 900

ccttggtcac cgtcgccgag acttggacgc caattacgca gcattttgcg aaaaatgaaa 960

catcagtact ttatttcttt atgtcttaat agtgctataa ttttagttgt tatttcatct 1020

tcacgcgcag gtctacgaga tcactcttca tttgtaactt ttgataataa tctttcgacg 1080

gcgcctacat ttttcgcaaa atgctgcgta attggcgtcc aagtctcggc gacggtgacc 1140

aaggaattcg tcgtcctaaa cgcggcgcga ataacgcggg taatatacga tcgtcgatct 1200

cgttcctctc aatcgcgtct ccgtaggagg ataggtcctc gtctgatcct cgccccagtc 1260

gcggtgtaaa ttgagtcggt tgtctcttgt cgttagctgg aatggcaaat agggccaaac 1320

gcccgctatc caaggcttcc gccagagcct gtattacgta 1360

<210> 131

<211> 1480

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a gene

encoding V-type proton ATPase catalytic subunit A of L. cuprina

<400> 131

taatacgact cactataggg aagttcttgt catagaaatc atccaaagca cgcatgtatt 60

tggagtagga aatcaaccaa ttgatggagg ggaaatgttt acgttgggcc aatttcttgt 120

ccaaacccca gaacacttgt acgataccca aagtggcaga agtaacggga tcagagaaat 180

caccaccagg aggagataca gcaccgacaa tggaaacgga accttcacgt tcagggttac 240

ccaaacactt gacacgacca gcacgttcgt agaaggaggc caaacgggca cccaagtagg 300

ctgggtaacc ggaatcggca ggcatttcag ccaaacgacc agaaatttca cgaagagctt 360

cggcccaacg ggaggtagaa tcagccatca tagatacgtt gtaacccata tcacggaagt 420

attcagacaa ggtaataccg gtataaacga ttccggttac ccagcctact tgggtgcccg 480

tttggcctcc ttctacgaac gtgctggtcg tgtcaagtgt ttgggtaacc ctgaacgtga 540

aggttccgtt tccattgtcg gtgctgtatc tcctcctggt ggtgatttct ctgatcccgt 600

tacttctgcc actttgggta tcgtacaagt gttctggggt ttggacaaga aattggccca 660

acgtaaacat ttcccctcca tcaattggtt gatttcctac tccaaataca tgcgtgcttt 720

ggatgatttc tatgacaaga acttcccgta attcgtacca ttgcgtacca aggtcaagga 780

aatcttgcaa gaagaagaag atttgtccga aattgtacaa ttggtcggta aggcttcatt 840

ggccgaaact gacaagatca ccttggaagt cgccaaattg cttaaggacg atttcttgca 900

acagaactcc tactcatcat acgacagatt ctgccccttc tacaagagtg tgggtatgtt 960

gaagaacatc attgccttct acgacttggc tcgtcactcc gtcgaatcca ccgctcaatc 1020

tgaaaacaaa atcacctgga atgtcattct gaaagcctgt tgtaaatctt cgtgtaattg 1080

ttcaaagtca gccttgatct tggcttcacc gtccttaacg ggatccttga atttcatgga 1140

agacaattgg tacataatgt tacccatagc ttcacggatg acattccagg tgattttgtt 1200

ttcagattga gcggtggatt cgacggagtg acgagccaag tcgtagaagg caatgatgtt 1260

cttcaacata cccacactct tgtagaaggg gcagaatctg tcgtatgatg agtaggagtt 1320

ctgttgcaag aaatcgtcct taagcaattt ggcgacttcc aaggtgatct tgtcagtttc 1380

ggccaatgaa gccttaccga ccaattgtac aatttcggac aaatcttctt cttcttgcaa 1440

gatttccttg accttggtac gcaatggtac gaattacgta 1480

<210> 132

<211> 1480

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a gene

encoding RNAse 1/2 of L. cuprina

<400> 132

taatacgact cactataggg aataatttgt ggtagacata gcgggttact tcctcatgtt 60

cgttaaagca gacctggtat tgtctcatga aacgggaaga tgacaattca aaaccaacat 120

tgacgagagt agtgccacca ttgcagctgc tgcccgactt cttagctaca aaagcaggcc 180

aactggtgca aacaagactg ggcagggagt gctgaacacc attgacttta aaagtggtgc 240

cactaacaca ggtagcgata tgggtcttgc ctgataaagg atgagcaaag ccactggtac 300

aatggatctc aatattcttg ccagcagcca catcaattct tccagtatca gaaaaagggt 360

agagttcagt agtgccaggt ttgatataca agggttgttt agccttaaga ccaccgcgaa 420

tgggtatgga acaaccacca ctgcgtggaa tattgagatc cattgtacca gtggctttgc 480

tcatccttta tcaggcaaga cccatatcgc tacctgtgtt agtggcacca cttttaaagt 540

caatggtgtt cagcactccc tgcccagtct tgtttgcacc agttggcctg cttttgtagc 600

taagaagtcg ggcagcagct gcaatggtgg cactactctc gtcaatgttg gttttgaatt 660

gtcatcttcc cgtttcatga gacaatacca ggtctgcttt aacgaacatg aggaagtaac 720

ccgctatgtc taccacaaat tattcccgta cccaacagcg tgccactttc ctattcatta 780

atgcagctcc ccagtggcaa gttttcaatg ccggtaattg ggctcgtgta gaggatggtg 840

tacgcgcctg ggtgtccaaa aataaaatca atgttcgatg ctataccggt gtttatggtg 900

tcaccactct acccaacaaa gagggacgtg agactcctct atatttgtct cgtgatgcca 960

ataataatgg tttgattcct gttcccaaat tatacttccg tgtggttata caacctgcca 1020

ccaataaggg tattgttttc gttggtgtca caggcataag aataaccagc agtgatatca 1080

gttttcttcc aactaatata gttaacctta tcactgacat ctttgcaaat aatatagtcc 1140

tttttgattt gttccaaagt caaatgggga ttgttgacac caacgaaaac aataccctta 1200

ttggtggcag gttgtataac cacacggaag tataatttgg gaacaggaat caaaccatta 1260

ttattggcat cacgagacaa atatagagga gtctcacgtc cctctttgtt gggtagagtg 1320

gtgacaccat aaacaccggt atagcatcga acattgattt tatttttgga cacccaggcg 1380

cgtacaccat cctctacacg agcccaatta ccggcattga aaacttgcca ctggggagct 1440

gcattaatga ataggaaagt ggcacgctgt tgggtacgta 1480

<210> 133

<211> 1480

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a gene

encoding chitin synthase of L. cuprina

<400> 133

taatacgact cactataggg tggcttgatt tttatattaa caccatacac ctcaaatgca 60

gctttttcaa tgctggtaat caatattttt acatattcat tgagaggagg atttttcgga 120

ttctccacat atttttcatc cagtataaag gcatcatcaa agaaaatatt agctaaaatg 180

taaaaaaaag aaaacttaat tgatagatac tcttcattcc aatccacaat tcgtctattt 240

aatgttatac attgttcaac caaaaaacca caataccacg gacaaatgaa cagtttttcg 300

gtgggtaaat ttttatcatt ttttggacgc catatatgat ttgttatcca caattgtgac 360

agccaccaca acaaccatat ccaaagataa tctttagcga ccacattaaa aaaactccat 420

gtatcgtgac cgaagcccag tgacgttgat aaaaatttac ccaccgaaaa actgttcatt 480

tgtccgtggt attgtggttt tttggttgaa caatgtataa cattaaatag acgaattgtg 540

gattggaatg aagagtatct atcaattaag ttttcttttt tttacatttt agctaatatt 600

ttctttgatg atgcctttat actggatgaa aaatatgtgg agaatccgaa aaatcctcct 660

ctcaatgaat atgtaaaaat attgattacc agcattgaaa aagctgcatt tgaggtgtat 720

ggtgttaata taaaaatcaa gccacccgta aaaattgaaa caccttatgg cggtcgtttg 780

gtgtggacac tgcctggtcg ctcaaagatg attgcccatt taaaaaacaa agataaaata 840

cgacataaga aacgctggtc acaggttatg tacatgtact atttgttggg ttttcgtata 900

atggaattgg aatcagtatc ggccaagcgt aaggcagtga tagcagaaaa tacatttttg 960

ctggctcttg atggtgatat tgactttcaa ccgcaggcag tgcaactgtt aatagaccgt 1020

atgaaggcca tagatgaatt aggtgctagc caggactaca taaaacacaa ccaataacat 1080

gctctgttgc tttttgcaac caatgaccta tagcgtattc gaagatttga taccaaacca 1140

tagggcctct accaactgga tgaatacgac cacaggcagc acctaattca tctatggcct 1200

tcatacggtc tattaacagt tgcactgcct gcggttgaaa gtcaatatca ccatcaagag 1260

ccagcaaaaa tgtattttct gctatcactg ccttacgctt ggccgatact gattccaatt 1320

ccattatacg aaaacccaac aaatagtaca tgtacataac ctgtgaccag cgtttcttat 1380

gtcgtatttt atctttgttt tttaaatggg caatcatctt tgagcgacca ggcagtgtcc 1440

acaccaaacg accgccataa ggtgtttcaa tttttacgta 1480

<210> 134

<211> 1481

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a gene

encoding ecdysone receptor (EcR) of L. cuprina

<400> 134

taatacgact cactataggg tgaaagatca tcacgacctg atgatatgga attagctgat 60

tcagatctgg gtgtatgatg catcatacta ctgttattca tatgatgatg gtgatgatga 120

tttaatccat tactgatgat actgtgaatg ccaatattgg catgaataac ttggccattt 180

tgtgaggcct gtaacgagtt taactgttgg gcattgctaa cgatattggg accattaata 240

ttgaccgata agccaccacc accgccgcca atacccatgt ggccatttgt gtggtgggaa 300

ctgctattac tgtgattact gttgctgttg tggtgtaaat gattgtggct gtgattgtga 360

ttattcactt gactgccacc accaccaccc agaccattga gtgaagtcat accgggtaca 420

ccaccaccac ctccacctcc tccaacaaat cacagtaata gcagttccca ccacacaaat 480

ggccacatgg gtattggcgg cggtggtggt ggcttatcgg tcaatattaa tggtcccaat 540

atcgttagca atgcccaaca gttaaactcg ttacaggcct cacaaaatgg ccaagttatt 600

catgccaata ttggcattca cagtatcatc agtaatggat taaatcatca tcaccatcat 660

catatgaata acagtagtat gatgcatcat acacccagat ctgaatcagc taattccata 720

tcatcaggtc gtgatgatct ttcacccgta tccaccaaat caccccctta gtggttcgaa 780

acacttgtgt tccatttgtg gagaccgcgc cagtggaaaa cattatgggg tctacagttg 840

tgagggttgt aaagggttct tcaaacgtac cgtacgcaag gacttgacat atgcttgtcg 900

tgaggacaga aattgcatta tagataaacg acaaagaaat cgttgccagt attgtcgtta 960

tcaaaagtgt ttagcttgtg gcatgaaacg cgaagcggtc caagaggaac gacaacgtgg 1020

tactcgtgct gctaacgcta gagctgcctt ttgctcggct tcaatgatgc gttctatagt 1080

gagatcacgt aatgaactgc tgggtttaaa gtcttctccg ccagcaccaa ccacattgct 1140

taccccacca ccacctcctc caccaccgcc agcaccagca gctctagcgt tagcagcacg 1200

agtaccacgt tgtcgttcct cttggaccgc ttcgcgtttc atgccacaag ctaaacactt 1260

ttgataacga caatactggc aacgatttct ttgtcgttta tctataatgc aatttctgtc 1320

ctcacgacaa gcatatgtca agtccttgcg tacggtacgt ttgaagaacc ctttacaacc 1380

ctcacaactg tagaccccat aatgttttcc actggcgcgg tctccacaaa tggaacacaa 1440

gtgtttcgaa ccactaaggg ggtgatttgg tggatacgta g 1481

<210> 135

<211> 1481

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a gene

encoding gamma-tubulin 1/1-like of L. cuprina

<400> 135

taatacgact cactataggg aaaacgctgt aggtttgtat aagtttctta ggaaaacgat 60

cagacaaacg ttccataata taagagccca tgccggaacc agtaccaccg gctatagaat 120

ggcatagaac aaatccctcc aaggaatcac tgccatctgc ctcacgatca ataatgtcaa 180

aaatttcctc ttgtaatttt tcaccttgac tatagccgga agcccaattg ttgccggcac 240

caccaccatg tttagacaag taaacatttt cgggattata taacttggca tagggtgaac 300

tcataatggt gtgtataact cgcggctcca aatccaaaag tacggcacgt ggtatatagt 360

gatcatcgtc agcctgataa aagaatacat ccttgcgatc tactccatct gtagcaaaat 420

cctctaacac tccactaggt gaaatgctat acacaccatt atgagttcac cctatgccaa 480

gttatataat cccgaaaatg tttacttgtc taaacatggt ggtggtgccg gcaacaattg 540

ggcttccggc tatagtcaag gtgaaaaatt acaagaggaa atttttgaca ttattgatcg 600

tgaggcagat ggcagtgatt ccttggaggg atttgttcta tgccattcta tagccggtgg 660

tactggttcc ggcatgggct cttatattat ggaacgtttg tctgatcgtt ttcctaagaa 720

acttatacaa acctacagcg ttttcccgta ccacaaccct acgttatcct tcatatatga 780

ataataattt gataggattg acggcacctt tgatacctac cccccaatta cattttctaa 840

tgaccggtta tactcctcta actacagata gtgatcccaa tttgaatata cgcaaaacta 900

cggtactaga tgttatgaga cgtttattgc aacccaaaaa tatgatggtt tcatcgggtc 960

cggataaagc aaatattcat tgttatattt ccatattaaa tattatacag ggtgaagtag 1020

atcccactca agtccacaaa tctctactga ttggccatca ttaggcccga aactttatga 1080

ttactttgta tatatggaga acttctggac aaggctactt gtatactggc cggaccccag 1140

ggtatgaatt gagctaattt gcgttcacgt atacgttgta gagatttgtg gacttgagtg 1200

ggatctactt caccctgtat aatatttaat atggaaatat aacaatgaat atttgcttta 1260

tccggacccg atgaaaccat catatttttg ggttgcaata aacgtctcat aacatctagt 1320

accgtagttt tgcgtatatt caaattggga tcactatctg tagttagagg agtataaccg 1380

gtcattagaa aatgtaattg gggggtaggt atcaaaggtg ccgtcaatcc tatcaaatta 1440

ttattcatat atgaaggata acgtagggtt gtggtacgta g 1481

<210> 136

<211> 1605

<212> DNA

<213> Triticum aestivum

<400> 136

atggcaaagg acgacgggta ccccccggcg cggacgctgc cggagacgcc gtcctgggcg 60

gtggcgctgg tcttcgccgt catgatcatc gtctccgtcc tcctggagca cgcgctccac 120

aagctcggcc attggttcca caagcggcac aagaacgcgc tggcggaggc gctggagaag 180

atgaaggcgg agctgatgct ggtgggattc atctcgctgc tgctcgccgt cacgcaggac 240

ccaatctccg ggatatgcat ctcccagaag gccgccagca tcatgcgccc ctgcaaggtg 300

gaacccggtt ccgtcaagag caagtacaag gactactact gcgccaaaga gggcaaggtg 360

gcgctcatgt ccacgggcag cctgcaccag ctccacatat tcatcttcgt gctagccgtc 420

ttccatgtca cctacagcgt catcatcatg gctctaagcc gtctcaagat gagaacatgg 480

aagaaatggg agacagagac cgcctccttg gaataccagt tcgcaaatga tcctgcgcgg 540

ttccgcttca cgcaccagac gtcgttcgtg aagcggcacc tgggcctgtc cagcaccccc 600

ggcgtcagat gggtggtggc cttcttcagg cagttcttca ggtcggtcac caaggtggac 660

tacctcatct tgagggcagg cttcatcaac gcgcacttgt cgcagaacag caagttcgac 720

ttccacaagt acatcaagag gtccatggag gacgacttca aagtcgtcgt tggcatcagc 780

ctcccgctgt gggctgtggc gatcctcacc ctcttccttg atatcgacgg gatcggcaca 840

ctcacctggg tttctttcat ccctctcatc atcctcttgt gtgttggaac caagctagag 900

atgatcatca tggagatggc cctggagatc caggaccggt cgagcgtcat caagggggca 960

cccgtggtcg agcccagcaa caagttcttc tggttccacc gccccgactg ggtcctcttc 1020

ttcatacacc tgacgctgtt ccagaacgcg tttcagatgg cacatttcgt gtggacagtg 1080

gccacgcccg gcttgaagga ctgcttccat atgaacatcg ggctgagcat catgaaggtc 1140

gtgctggggc tggctctcca gttcctgtgc agctacatca ccttccccct ctacgcgcta 1200

gtcacacaga tgggatcaaa catgaagagg tccatctttg acgagcagac agccaaggcg 1260

ctgaccaact ggcggaacac ggccaaggag aagaagaagg tccgagacac ggacatgctg 1320

atggcgcaga tgatcggcga cgcaacaccc agccgaggca cgtccccgat gcctagccgg 1380

ggctcatcgc cggtgcacct gcttcagaag ggcatgggac ggtctgacga tccccagagc 1440

gcaccgacct cgccaaggac catggaggag gctagggaca tgtacccggt tgtggtggcg 1500

catcctgtac acagactaaa tcctgctgac aggcggaggt cggtctcttc atcagccctc 1560

gatgccgaca tccccagcgc agatttttcc ttcagccagg gatga 1605

<210> 137

<211> 1277

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct, targeting Mlo from

Triticum aestivum

<400> 137

taatacgact cactataggg tgcccccttg atgacgctcg accggtcctg gatctccagg 60

gccatctcca tgatgatcat ctctagcttg gttccaacac acaagaggat gatgagaggg 120

atgaaagaaa cccaggtgag tgtgccgatc ccgtcgatat caaggaagag ggtgaggatc 180

gccacagccc acagcgggag gctgatgcca acgacgactt tgaagtcgtc ctccatggac 240

ctcttgatgt acttgtggaa gtcgaacttg ctgttatgcg acaaatgcgc gttgatgaag 300

cctgccctca aggtgaggta gtccaccttg gtgaccgacc tgaagaactg cctgaagaag 360

gccaccaccc atctgacgcc gggggtgctg gagaggggtt cgacttccac aagtacatca 420

agaggtccat ggaggacgac ttcaaagtcg tcgttggcat cagcctcccg ctgtgggctg 480

tggcgatcct caccctcttc cttgatatcg acgggatcgg cacactcacc tgggtttctt 540

tcatccctct catcatcctc ttgtgtgttg gaaccaagct agagatgatc atcatggaga 600

tggccctgga gatccaggac cggtcgagcg tcatcaaggg ggcacccgac gtcgagccca 660

gcaacaagtt cttctggttc caccgccccg actgggtcct cttcttcata cacctgacgc 720

tgttccagaa gtcacacaga tgggatcaaa catgaagagg tccatcttcg acgagcagac 780

agccaaggcg ctgaccaact ggcggaacac ggccaaggag aagaagaagg tccgagacac 840

ggacatgctg atggcgcaga tgatcggcga cgcgacgccc agccgaggca cgtcccccac 900

cacaaccggg tacatgtccc tagcctcctc catggtcctt ggcgaggtcg gtgcgctctg 960

gggatcgtca gaccgtccca tgcccttctg aagcaggtgc accggcgatg agccccggct 1020

aggcatcggg gacgtgcctc ggctgggcgt cgcgtcgccg atcatctgcg ccatcagcat 1080

gtccgtgtct cggaccttct tcttctcctt ggccgtgttc cgccagttgg tcagcgcctt 1140

ggctgtctgc tcgtcgaaga tggacctctt catgtttgat cccatctgtg tgacttctgg 1200

aacagcgtca ggtgtatgaa gaagaggacc cagtcggggc ggtggaacca gaagaacttg 1260

ttgctgggct cgacgtc 1277

<210> 138

<211> 1527

<212> DNA

<213> Vitis pseudoreticulata

<400> 138

atggctggcg acgaggagac gacgacgacg gcagcaacac ttgaaacaac gtccacttgg 60

gctgttgcct ctgtttgctt tattttgatt gcactctcca tacttattga gcatgccctc 120

catctcttag ccaagtactt caacaagaag cggaggaggt ctctcattca tgctcttaac 180

aacgtcaaat cggagttgat gctcttgggg ttcgtctctt tgttgctgac tgtgtgccaa 240

aagtatattg cgaagatttg tatcccaagg agcgtaggtg aaacttttct tccctgcaag 300

accttgacag aaagtgattc agaagaagaa accaaatgcg aagagcaggg aaagatgtct 360

ttgctgtcta gacaaggcgt ggaggaacta caatacttaa ttttcgtgct ggccttcttc 420

cattccctct actgcgtcct cacattcggt cttgggatgg ccaagatgaa gaaatgggag 480

tcctgggagg cagaaacaag aacactggaa tatcagttta caaatgatcc acggaggttc 540

aggctcatcc atcagacatc atttggaaag caacatctga gatattggag tgagcatcag 600

atacttcgtt ggccggcttg ttttattcgg cagttctatc catccgtctc caaagtggat 660

tacttgactc ttagacatgg gttcattatg gcccattttg cagaaggaag caactatgac 720

ttccaaaagt atataaaaag agctttggaa aaagactttg gagtggtggt gggaggaagt 780

ttctgggttt ggagtttctc catgcttttt gtgttcttca atgctcaagt attttacaac 840

tatttatggc taccctttat tccattggtg atgctgttgt tggttggaac aaagctacag 900

ggcattataa ctaagatgtg cttagatagc catgataaag ctctcgttgt tagaggaact 960

ttgcttgtca ggcccagtga tcacttcttc tggtttggaa aaccggaatt gctcctacat 1020

cttatgcact ttatattgtt tcagaactct tttcaactgg cgttctttac atggacttgg 1080

tacaaatttg gattcagatc atgcttccat gatacaactg aggatatcgt cataaggctt 1140

gtcatgggtg tgttagtaca actcctttgt ggctacgtga cactgcctct gtatgccctg 1200

gtcacgcaga tggggacatc aatgaggaca attgtcttta ctgagggagt cgttgaaggt 1260

ctgaacagat ggagaaggaa agccaagaaa aacatagcac gcaggaacaa ccactcagct 1320

cgtccctccc tggatgcttc actcgacaat tcaccttctt ttaacactct ggatacttct 1380

ttctctgtag acctcgatca gccatcatca gatgctggtt atttgactgt tgaaatatca 1440

gatgaagaga cggtcgctac taaacggcca gaaccgcgtc agaagttggg atcttttgag 1500

ggtttcgact cgtgcaaaac atcataa 1527

<210> 139

<211> 1480

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA encoding a ledRNA construct targeting a Vitis MLO

gene

<400> 139

taatacgact cactataggg tagccataaa tagttgtaaa atacttgagc attgaagaac 60

acaaaaagca tggagaaact ccaaacccag aaacttcctc ccaccaccac tccaaagtct 120

ttttccaaag ctctttttat atacttttgg aagtcatagt tgcttccttc tgcaaaatgg 180

gccataatga acccatgtct aagagtcaag taatccactt tggagacgga tggatagaac 240

tgctgaataa aacaaaccgg ccaacgaagt atccgatgct cactccaata tctcagatgt 300

tgctttccaa atgatgtctg atggatgagc gtgaacctcc gtggatcatt tgtaaactga 360

tattccagtg ttcttgtttc tgcctcccag gactcccatt tcttcatctt ggccatccca 420

agaccgaatg tgaggacgca gtagagcaca tcatttggaa agcaacatct gagatattgg 480

agtgagcatc ggatacttcg ttggccggtt tgttttattc agcagttcta tccatccgtc 540

tccaaagtgg attacttgac tcttagacat gggttcatta tggcccattt tgcagaagga 600

agcaactatg acttccaaaa gtatataaaa agagctttgg aaaaagactt tggagtggtg 660

gtgggaggaa gtttctgggt ttggagtttc tccatgcttt ttgtgttctt caatgctcaa 720

gtattttaca actatttatg gctacccgta attccattgg tgatgctgtt gttggttgga 780

acaaagctac agggcattat aactaagatg tgcctagata gccatgataa agctctcgtt 840

gttagaggaa ctttgcttgt caggcccagt gatcacttct tctggtttgg aaaaccggaa 900

ttgctcctac atcttatgca ctttatattg tttcagaact cttttcaact ggcgttcttt 960

acatggactt ggtacaaatt tggattcaga tcatgcttcc atgatacaac tgaggatatc 1020

gtcataaggc ttgtcatggg tgtgttatgg ctttccttct ccatctgttc agaccttcaa 1080

cgactccctc agtaaagaca attgtcctca ttgatgtccc catctgcgtg accagggcat 1140

acagaggcag tgtcacgtag ccacaaagga gttgtactaa cacacccatg acaagcctta 1200

tgacgatatc ctcagttgta tcatggaagc atgatctgaa tccaaatttg taccaagtcc 1260

atgtaaagaa cgccagttga aaagagttct gaaacaatat aaagtgcata agatgtagga 1320

gcaattccgg ttttccaaac cagaagaagt gatcactggg cctgacaagc aaagttcctc 1380

taacaacgag agctttatca tggctatcta ggcacatctt agttataatg ccctgtagct 1440

ttgttccaac caacaacagc atcaccaatg gaattacgta 1480

<210> 140

<211> 1531

<212> DNA

<213> Rhizoctonia solani

<400> 140

cattccatcg ctgctacagt tgataccccg cgacgcgtcc ttaccgcctc ttgtgttcca 60

ctgggttcca attattggct ccgctatcgc gtacggtgat gaccctcttg catttctctt 120

ttcgtgccgc gaaaagtacg gcgacctgtt caccttcgtt cttctcggcc gcaaaatgac 180

cgtcgcgttg ggcccaaagg gtagtaattt tatcctggga ggaaaacttt cccaagtctc 240

agccgaggaa gcctacaccc accttaccac tccagtcttt ggcaaggatg ttgtctatga 300

cgtcccgaac catgtactca tggagcaaaa gaagtttgtc aagttcggac ttaccaccga 360

gaacttccga gcctacgtcg atatgatcgt agacgagacc gtgaacaacc tcattcgtaa 420

ggagctctcc cctgaaaact gcccacgcga ctcccagggc tgggggtgct tccatgcatt 480

caaaaagctg gccgagctca cgattctcac cgcctcgcgc acgctgcaag gcaacgaagt 540

tcgctccaac cttgacaaaa gcttcgcaga attgtatcag gacctcgatg gcggcttcac 600

ccctatcaac ttcctcttcc ccaaccttcc gctccctagt tactggcgtc gagaccgtgc 660

tcaaaagaag atgagcgact tttacgtgaa cattattgag aaacgtaagg cacagagtca 720

aggggatgag catgatatga ttgctgcttt gttgaatcag acctacaaag atggccgcgc 780

gctcagcgac cgtgaggtag ctcatatcat gattgcactc ctcatggccg gtcagcacac 840

tagttctgca acttcttcct ggacgcttct tcacctcgcc gatcgccctg atattgccga 900

gaaattgtac gaggaacaag tcaaagtctt tggtaatgcc gatggttcga tgcgcccctt 960

gaactacgag gagttgaagg atttgccagt actgagcgca gttatccgtg agaccttgcg 1020

catgcatccg cctatccaca gtatcatgcg caaatgcatt aacgatatgg ttgttccggc 1080

gtccctggct gcccccaccg gcaaggctaa cgagggccga acctacgttg ttcccaaggg 1140

ccactatctt ctggcatctc ccgccgttgc acaagtcgac cctcgtgtat ggcgtgacgc 1200

cgacaagtgg gatcctctgc gctggttgga cccaacggga gctgctgctc aggccggttc 1260

tttgtacaac gacgagcacg gtgaaacggt tgactatggt tggggtgccg ttagtaaggg 1320

taccgagagc ccttatcagc catttggtgc tggtaggcat aggtgcattg gtgaacaatt 1380

cgcaaatata caactcggcg ccattttgtc tactataatc cggaacatgg agatgcgtat 1440

cgaaaagcac gttcctgacc acaattacca tactatgatc gtcctgccta aagatccctg 1500

tggtatcagg ttcaagctcc gcaccaaggc g 1531

<210> 141

<211> 1617

<212> DNA

<213> Rhizoctonia solani

<400> 141

atgaaccagt tcgcatcttg cccatggctc gaatctgcca ccttcattcc tttactgggc 60

gcttcatgcg tgatcttgat ggccacctgt gcgtgcatcc ttttgaatgt catcgcccaa 120

ttggttatac cccctgatcc gtcattgcca cctcaggttt tctacgttct accgtatatt 180

ggctcggcca ttgaatacgg taaggatcca ataggtttct tatcatctgg caggagaaag 240

tatggggacg tttttacctt cgttctcttg ggacgccgag tgaccgtcgc gcttggtccc 300

aagggaagta atttcgtcct tgggggaaag ttgtcacagg tctcggccga agaagcgtac 360

acacacttga ctacacctgt cttcggcaag ggcgtgattt tcgatgttcc aaatcatgtc 420

tttatggaac agaagaagtt catcaaatcc ggcctcacaa ctgaaaatct acgcgcctac 480

gtgaacatga tatccgagga gactaccaca ttcctgaata aagacttggc tgatacctgt 540

cgtggaaagg aatgggggag gtttcatgta cttgacactc tggctgggct tacgatcctg 600

accgcctcga ggacgctcca aggcagagaa gtgcggtcgt ctctggataa gaccttttct 660

caggtttata aggatttgga tgggggattc acacccttga accttatgtt cgccaatctc 720

cctctgccca gttactggag gagggaccgt gctcaacgga aaatgagcga tttttatgtg 780

gacattatca ggaatcgcca agaggaacat cgggattctg aacatgacat gatctctgca 840

ttagcatcga gagagtacaa ggacggttct cctctaggcg accgcgagat cgctcacttg 900

atgatagcct tactcatggc tgggcagcac accagctctt cgaccggttc ttgggcattg 960

ctacacttag ccgatcgccc agatgttgta aaacaactac ttgcagagca agaagaagtg 1020

cttggtaacg aagatggaaa cttacgacct ctaaccttcg agggcctcca aaaactcccc 1080

gttctcaact cggttattcg cgaaacttta cgtattcatc cgcccattca tagtatcatg 1140

cgcaaatgca tagacgatat tgttgtcccg gctactctcg cctccccaag ttcggactcg 1200

acttacatcg taccaaaagg acattttctc ctcgcctctc cggctcactc gcaagtcgac 1260

ccagacgttt ggttcagcgc gagcgaatgg gaccactcac gatggctaga tccaaacgga 1320

gtggccgctc aagccgagtc actctacctg ggtgaccaag gtgaaaaagt cgactatggg 1380

tggggtgtgg taagcaaagg gaccgagagc ccataccagc cattcggtgc tggaaggcat 1440

cgatgtatcg gtgagaagtt cgcttatgta caacttggga cgattctgtc gactgttgtg 1500

agaacaattg agatggggtt ggactcgggt gttccggcgc acaactatca taccatgatt 1560

gttcagccga aggagccctg catgattcag ttcaggttcc gggataggca aagggag 1617

<210> 142

<211> 1823

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of a chimeric DNA encoding a ledRNA construct

targeting a gene encoding Cyp51

<400> 142

aagaattcta atacgactca ctatagggtc caaccagcgc agaggatccc acttgtcggc 60

gtcacgccat acacgagggt cgacttgtgc aacggcggga gatgccagaa gatagtggcc 120

cttgggaaca acgtaggttc ggccctcgtt agccttgccg gtgggggcag ccagggacgc 180

cggaacaacc atatcgttaa tgcatttgcg catgatactg tggataggcg gatgcatgcg 240

caaggtctca cggataactg cgctcagtac tggcaaatcc ttcaactcct cgtagttcaa 300

ggggcgcatc gaaccatcgg cattaccaaa gactttgact tgttcctcgt acaatttctc 360

ggcaatatca gggcgatcgg cgaggtgaag aagcgtccag gaagaagttg cagaactagt 420

gtgctgaccg gccatgagga gtgcaatcat gatatgagct acctcacggt cgctgagcgc 480

gcggccatct ttgtaggtct gattcaacaa agcagcgccc tgatattgcc gagaaattgt 540

acgaggaaca agtcaaagtc tttggtaatg ccgatggttc gatgcgcccc ttgaactacg 600

aggagttgaa ggatttgcca gtactgagcg cagttatccg tgagaccttg cgcatgcatc 660

cgcctatcca cagtatcatg cgcaaatgca ttaacgatat ggttgttccg gcgtccctgg 720

ctgcccccac cggcaaggct aacgagggcc gaacctacgt tgttcccaag ggccactatc 780

ttctggcatc tcccgccgtt gcacaagtcg accctcgtgt atggcgtgac gccgacaagt 840

gggatcctct gcgctggttg gacccgtata ttggctcggc cattgaatac ggtaaggatc 900

caataggttt cttatcatct ggcaggagaa agtatgggga cgtttttacc ttcgttctct 960

tgggacgccg agtgaccgtc gcgcttggtc ccaagggaag taatttcgtc cttgggggaa 1020

agttgtcaca ggtctcggcc gaagaagcgt acacacactt gactacacct gtcttcggca 1080

agggcgtgat tttcgatgtt ccaaatcatg tctttatgga acagaagaag ttcatcaaat 1140

ccggcctcac aactgaaaat ctacgcgcct acgtgaacat gatatccgag gagactacca 1200

cattcctgaa taaagctgag aaaaggtctt atccagagac gaccgcactt ctctgccttg 1260

gagcgtcctc gaggcggtca ggatcgtaag cccagccaga gtgtcaagta catgaaacct 1320

cccccattcc tttccacgac aggtatcagc caagtcttta ttcaggaatg tggtagtctc 1380

ctcggatatc atgttcacgt aggcgcgtag attttcagtt gtgaggccgg atttgatgaa 1440

cttcttctgt tccataaaga catgatttgg aacatcgaaa atcacgccct tgccgaagac 1500

aggtgtagtc aagtgtgtgt acgcttcttc ggccgagacc tgtgacaact ttcccccaag 1560

gacgaaatta cttcccttgg gaccaagcgc gacggtcact cggcgtccca agagaacgaa 1620

ggtaaaaacg tccccatact ttctcctgcc agatgataag aaacctattg gatccttacc 1680

gtattcaatg gccgagccaa tatacgtagg tacccgggta gcatatccat gatatctgtt 1740

agtttttttc ctgaaagagc ggccgcccta gcataacccc gcggggcctc ttcgggggtc 1800

tcgcggggtt ttttgctgaa aga 1823

<210> 143

<211> 3554

<212> DNA

<213> Phytophthora cinnamomi

<400> 143

atggggctca ccggcgcggg catcatcgcc tccgtcgtgg gcatcctggg cggcgtgtcg 60

ctgtcctgcg gcggctggtc gtcgctgtcc ctcggcgctc gctcgctctt cgtgacgacg 120

cagttcctct cggccttcgc catgtacgtg ccgcactgac catcactcct gctccaattc 180

tgaaacgagg gcgcactatt gggtgtcgtt tcggttctaa ttttggaacg ttccagacac 240

taatttgatt ttccgctgtt gtgatattcc acctcagggg attcgtggtc gccttcaccg 300

ccatctcgtc gctgacaagc accaacgagt ggatcgccgt ggcggccggc ggcggcgcgg 360

gcttcgtgat cgccctcatc gtgggcttca tgacggtctt cggcccgtac atcctgatcc 420

tcgtcacagg cggcatcatc gcctgctacc tgctgctcgt ggacgcgtac gacggcgtga 480

aactcttccc gtcagacaay cagctggcgc gccaggagtt cgtcatcgcc ttcatgatca 540

tcttcgagct cgtgtgctgc tcgtcgtcca agacgtcgga gctggagaac caccgcttca 600

agtacatcat cttctcggcc atcacgggcg gctggatggc ctcggacggc gtgtcccgcc 660

tcatcgactc gacggctgtt ctgtcggacg tggcctacac ctccatccag gacggcggct 720

cggccgcgct caagggcatc gacagcagcg cgcagacgct catgttcctc atctggggcg 780

ccgtcttcgt ggtcggcggc ctcaaccagc tctccatgcg ctggggactc atgtgctaca 840

accgcgttgg tgcgcacgcc cagctcggcc ccgtcgagga gcagatgccg gagctgccca 900

cgggcgccac gctgcctgcc cagaccatga acgagcgcgt gcgcctcgtg tgcgagaact 960

gtttcgccac ggtgcccagc ggcacggcct tctgtaccga gtgtggtgag gcaatgccct 1020

cggacgacgc cgacccgaac gtcagcatct cgcaggcgca gatgccgtcg gtcacgatga 1080

acaacaagtc tcaagtgccc gaccgctggc agcaggtgcc gcaccgcacg tacctcagca 1140

cgacgtcgtt tgtcgacccc aagcacgcca aggagggcgg cgtgagcatg aaggacaaca 1200

gccgcagcat ccgcttcatg gactcgggtg tgcagggccc cgacggcaag atgagccagt 1260

acaacgactc gatcgccggc gtgcgcaact attacgagcc ttcattccgg tcgttcgcca 1320

tgtcgaccta ctcgatcgcc aaccgcgccg ctgagcccgt tgacacgccc aacatccgca 1380

agtacaagat gtctggtagt ggcatgttcc acgtcttcta cttcggtacg gctgccaccg 1440

gtatcttctg gctgtactac ttgactacga tgtacccgca gcagtacttc tgcgaccacg 1500

cccgccccac gcttccctgc agtgagctgc ccagcagcga gatttcgggc tgctacagtt 1560

caacggtcaa cttcgactcc tcttccggcg agggttactg catcaagaac gtgccgttca 1620

tgtcgtggct catgtacgcg atgatgatct ttagcgagtt cctcaactac ttcctaggtc 1680

tgctgttcaa cttcagtatg tggcgtccga ttcgtcgtgg cgcccgttac atgaacgact 1740

tcaaaccgcc tatcccgaaa gagcagtggc cgaccgtcga catcttcctg tgtcactaca 1800

tggaacctgt gacggactcc atggctacgc tgaagaactg tcttgcgatg cagtaccctc 1860

cggagctgct gcacattttc atccttgatg atggttacgc caagtctgtg tgggacgcca 1920

acaaccactt caaggttacg gtcaacacca aggtgattga gatttgtggt gacctgcgtg 1980

gcgacgtcgc tcgcatcatg cacgagcgcg tggtcggccc tgtgcaggac gatcagtccc 2040

tgaagacgtg gcgtcgccag cacagctctg tgcgtgagct ccgcaaagag ggaagcaagg 2100

gcgtgcagcg tcgcgactgt gctgttggtt cactgtcgga cgactacgac taccgtgacc 2160

gcggtatccc gcgtgtgact ttcatcggtc gcatgaagcc cgaaacgcac cactccaagg 2220

ctggtaacat caacaacgcc ctcttcaacg aaggtgccga tggcaagtac ttgctgattc 2280

tggataatga tatgaagccg cacccgaagt tcttgcttgc cgtgctgccg ttcttcttct 2340

cggagggcga ggctgtggac ggcggaggcc gccagtacag tgacgacatt tcctggaacc 2400

aggtgtcgta cgtgcagact cctcagtact tcgaggacac gccccagctg accatcatgg 2460

gagacccgtg tggacacaag aacaccattt tcttcgacgc tgtacagtgt ggtcgtgatg 2520

gtttcgactc ggcagctttc gccggtacca acgccgtttt ccgtcgccag gctttcgact 2580

ccatcggtgg cattcagtac ggtacccaga cggaagatgc ctacacgggt aacgtgctgc 2640

acacttctgg ctgggactcg gtgtacttcc gcaaggattt cgagggtgat gccaaggacc 2700

gcattcgtct gtgcgaaggt gccgtgcccg aaacggtcgc tgctgccatg ggtcagaaga 2760

agcgttgggc caagggtgcc gtgcagattc tgctcatgaa gaatgagagc gaggtcgacc 2820

cggactggcg tccgccgcgt gtgcctgccc cggacccgaa gccggcgctt gcgttcccgc 2880

gcaagatgtt cttctacgac tcggtgctct acccgttcgg ttccattccc gctctgtgtt 2940

acgtggcgat cgctatttac tacctgtgta cgggtgacgc tcccatctac gctcgtggta 3000

ccaagttcct gtactctttc ttgcccgtga cgttctgccg ttgggtactc aacctgctgg 3060

ccaaccgcgc cgtcgacaac aacgatgtgt ggcgtgccca gcagacctgg ttctccttct 3120

ccttcatcac gatgatggct attgtggagg ctattcaggc gcgtgtgacg ggcaaagaca 3180

agtcgtgggc caacacgggt gccggtcaga agacgtcgtg gacagagatc cccaacgtgc 3240

tcttcttctt cacgctgctc tttagtcaac tggtggcgct gattcggttc tttgagtacg 3300

agaacgccac gaacccgtgg aactacgtgt ctgctatgtt cttcggcttc ttcgtgatga 3360

gtcagttcta ccccatggtc aagatgagta tcacggagta ctgtggttgg gaccacacgg 3420

ccgcgacctt tacggccaac gtgttcggct cgctgctggt ggtgtacatc gtggtgttcg 3480

tgcagctgtg gcaggtctac tacgagggca acctgcagac ggcccagggt acatcaggtt 3540

ccacttcgtc ttag 3554

<210> 144

<211> 1409

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of a chimeric DNA encoding a ledRNA construct

targeting a gene encoding CesA3

<400> 144

aagaattcta atacgactca ctatagggtc ggcgtcgtcc gagggcattg cctcaccaca 60

ctcggtacag aaggccgtgc cgctgggcac cgtggcgaaa cagttctcgc acacgaggcg 120

cacgcgctcg ttcatggtct gggcaggcag cgtggcgccc gtgggcagct ccggcatctg 180

ctcctcgacg gggccgagct gggcgtgcgc accaacgcgg ttgtagcaca tgagtcccca 240

gcgcatggag agctggttga ggccgccgac cacgaagacg gcgccccaga tgaggaacat 300

gagcgtctgc gcgctgctgt cgatgccctt gagcgcggcc gagccgccgt cctggatgga 360

ggtgtaggcc acgtccgaca gaacagccgt cgagtcgatg aggcgtcgtg gtcggcggcc 420

tcaaccagct ctccatgcgc tggggactca tgtgctacaa ccgcgttggt gcgcacgccc 480

agctcggccc cgtcgaggag cagatgccgg agctgcccac gggcgccacg ctgcctgccc 540

agaccatgaa cgagcgcgtg cgcctcgtgt gcgagaactg tttcgccacg gtgcccagcg 600

gcacggcctt ctgtaccgag tgtggtgagg caatgccctc ggacgacgcc gacccgtacg 660

tcagcatctc gcaggcgcag atgccgtcgg tcacgatgaa caacaagtct caagtgcccg 720

accgctggca gcaggtgccg caccgcacgt acctcagcac gacgtcgttt gtcgacccca 780

agcacgccaa ggagggcggc gtgagcatga aggacaacag ccgcagcatc cgcttcatgg 840

actcgggtgt gcagggcccc gacggcaaga tgagccagta caacgactcg atcgccggcg 900

tgcgcagacg tggaacatgc cactaccaga catcttgtac ttgcggatgt tgggcgtgtc 960

aacgggctca gcggcgcggt tggcgatcga gtaggtcgac atggcgaacg accggaatga 1020

aggctcgtaa tagttgcgca cgccggcgat cgagtcgttg tactggctca tcttgccgtc 1080

ggggccctgc acacccgagt ccatgaagcg gatgctgcgg ctgttgtcct tcatgctcac 1140

gccgccctcc ttggcgtgct tggggtcgac aaacgacgtc gtgctgaggt acgtgcggtg 1200

cggcacctgc tgccagcggt cgggcacttg agacttgttg ttcatcgtga ccgacggcat 1260

ctgcgcctgc gagatgctga cgtacgtagg tacccgggta gcatatccat gatatctgtt 1320

agtttttttc ctgaaagagc ggccgcccta gcataacccc gcggggcctc ttcgggggtc 1380

tcgcggggtt ttttgctgaa agaagctta 1409