Methods for increasing efficiency of genome engineering

文档序号：90752 发布日期：2021-10-08 浏览：16次中文

阅读说明：本技术 用于提高基因组工程化效率的方法 (Methods for increasing efficiency of genome engineering ) 是由 J·F·古铁雷斯-马科斯于 2020-01-28 设计创作，主要内容包括：本发明涉及用于真核细胞中基因组工程化的方法和材料,并且特别涉及经由递送一个或多个RKD2和RKD4基因以及基因组工程化组分来提高基因组工程化(即转化或基因组编辑)效率的方法。(The present invention relates to methods and materials for genome engineering in eukaryotic cells, and in particular to methods for increasing the efficiency of genome engineering (i.e., transformation or genome editing) via delivery of one or more RKD2 and RKD4 genes and genome engineering components.)

1.A method for making a genetic modification in a plant cell, the method comprising

(a) Introducing into said plant cell

(i) A nucleic acid comprising a polynucleotide sequence encoding RKD2 polypeptide or RKD4 polypeptide, a recombinant gene comprising said nucleic acid, or a DNA construct comprising said nucleic acid; and

(ii) a transgene and/or genome engineered component of interest;

(b) optionally, culturing said plant cell under conditions that allow synthesis of RKD2 polypeptide or RKD4 polypeptide from said nucleic acid following chemical induction; and

(c) optionally, culturing said plant cell under conditions that allow genetic modification of the genome of said plant cell by integration of the activity of said transgene of interest and said genome engineering component in the presence of RKD2 polypeptide or RKD4 polypeptide;

wherein said polynucleotide sequence encoding RKD2 polypeptide or RKD4 polypeptide is operably linked to a heterologous promoter that is directly chemically inducible or indirectly chemically inducible, preferably indirectly chemically inducible via an intermediate transcription factor.

2. The method of claim 1, wherein the method is effective to increase the efficiency of plant regeneration.

3. The method of claim 1 or 2, wherein said method is effective to increase the regenerative capacity of said plant cell.

4. Method according to any one of claims 1 to 3, wherein in step (b) RKD2 polypeptide or RKD4 polypeptide is synthesized from said nucleic acid after direct or indirect chemical induction of said heterologous promoter, preferably after addition of β -estradiol or a glucocorticoid such as dexamethasone to said plant cells.

5. The method of claim 1, wherein the heterologous promoter is the XVE/OLExA system for chemical β -estradiol inducibility.

6. The method of claim 5, wherein the XVE/OLexA system comprises the amino acid sequence of SEQ ID NO:22, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 22.

7. The method of claim 1, wherein the heterologous promoter is an inducible bidirectional promoter.

8. The method of claim 7, wherein the inducible bi-directional promoter is operably linked to a second nucleic acid sequence encoding a desired polypeptide.

9. The method of claim 8, wherein the second nucleic acid sequence comprises a reporter gene and a polynucleotide encoding a reporter protein.

10. The method of claim 9, wherein the reporter is GUS or tdTomato.

11. The method of claim 7, wherein said inducible bidirectional promoter is a dexamethasone inducible promoter.

12. The method of claim 11, wherein the promoter is pOp1, pOp2, pOp4, or pOp 6.

13. The method of claim 12, wherein said pOp6 comprises SEQ ID NO:15, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 15.

14. The method of claim 12 or 13, wherein in step (a) (i), another nucleic acid comprising a polynucleotide sequence encoding a transcription factor operably linked to a strong constitutive promoter is introduced into the plant cell, wherein the transcription factor activates pOp6 upon binding to dexamethasone.

15. The method of claim 14, wherein the transcription factor is LhGR or LhG 4.

16. The method of claim 15, wherein the LhGR has the sequence of SEQ ID NO: 17, or an amino acid sequence identical to SEQ ID NO: 17, amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; or wherein said nucleic acid encoding LhGR comprises SEQ ID NO: 16, or a sequence identical to SEQ ID NO: 16, or a coding sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

17. The method of claim 14, wherein said strong constitutive promoter is a ubiquitin promoter or a double 35S promoter.

18. The method of claim 17, wherein the double 35S promoter comprises SEQ ID NO:21, or a nucleotide sequence identical to SEQ ID NO:21, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

19. The method of claim 17, wherein the ubiquitin promoter comprises SEQ ID NO:23, or a nucleotide sequence identical to SEQ ID NO:23 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

20. The method of claim 19, wherein the ubiquitin promoter additionally comprises a ubiquitin intron comprising the amino acid sequence of SEQ ID NO:20, or a nucleotide sequence identical to SEQ ID NO:20, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto.

21. The method of any one of claims 1 to 20, wherein said plant cell is cultured under conditions that allow genetic modification of the genome of said plant cell by integration of the activity of said transgene of interest and said genome engineering component in the presence of RKD2 polypeptide or RKD4 polypeptide.

22. The method of claim 21, wherein the cultured plant cells do not express the reporter gene in the absence of a chemical agent effective to induce a chemically inducible promoter.

23. The method of claim 21 or 22, wherein the plant cell is cultured to produce an embryonic structure.

24. The method of claim 23, wherein said embryogenic structure is cultured to produce a regenerated plant.

25. Method according to any one of claims 1 to 24, wherein said RKD2 polypeptide or RKD4 polypeptide is transiently present, has transient activity and/or is transiently expressed in said plant cell and/or wherein a nucleic acid encoding an RKD2 polypeptide is transiently present, has transient activity and/or is transiently expressed in said plant cell or a nucleic acid encoding an RKD4 polypeptide is transiently present, has transient activity and/or is transiently expressed in said plant cell.

26. The method of any one of claims 1-25, wherein said RKD2 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence identical to SEQ ID NO: 5, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; or the nucleic acid encoding RKD2 polypeptide encodes the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence identical to SEQ ID NO: 5, or a variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

27. The method of any one of claims 1-25, wherein said RKD4 polypeptide comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence identical to SEQ ID NO:2, an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; or the nucleic acid encoding RKD4 polypeptide encodes the amino acid sequence of SEQ ID NO:2, or an amino acid sequence identical to SEQ ID NO:2, or a variant thereof, 2 having an amino acid sequence which is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical.

28. Method according to any one of claims 1 to 25, wherein said nucleic acid encoding RKD2 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of SEQ ID NO:

d) comprises the amino acid sequence of SEQ ID NO: 3 or 4;

e) comprises a nucleotide sequence substantially identical to SEQ ID NO: 3 or 4, a nucleic acid having a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical; and

f) a nucleic acid which hybridizes under stringent hybridization conditions with the complementary strand of a nucleic acid as defined in a) or b).

29. Method according to any one of claims 1 to 25, wherein said nucleic acid encoding RKD4 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of SEQ ID NO:

a) comprises the amino acid sequence of SEQ ID NO: 1;

b) comprises a nucleotide sequence substantially identical to SEQ ID NO:1, a nucleic acid having a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical; and

c) a nucleic acid which hybridizes with the complementary strand of the nucleic acid as defined in (1) or (2) under stringent hybridization conditions.

30. The method of any one of claims 1 to 29, wherein the genome engineering component comprises

e) An enzyme that induces a Double Strand Break (DSB) or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, wherein the DSB-inducing enzyme preferably recognizes a predetermined site in the genome of the cell;

f) an enzyme that induces Single Strand Breaks (SSBs), or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, wherein the SSB-inducing enzyme preferably recognizes a predetermined site in the genome of the cell;

g) a base-editing enzyme, optionally fused to a disarmed DSB or SSB inducing enzyme, wherein said base-editing enzyme preferentially recognizes a predetermined site in the genome of said cell; or

h) An enzyme that effects DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ribosylation, or histone citrullination, optionally fused to a disarmed DSB or SSB-inducing enzyme, wherein said enzyme preferably recognizes a predetermined site in the genome of said cell.

31. The method of any one of claims 1 to 30, wherein the genome engineered component comprising a DSB or SSB-inducing enzyme or variant thereof is a CRISPR/Cas endonuclease, a CRISPR/Cas9 endonuclease, a CRISPR/Cpf1 endonuclease, a CRISPR/Csm1 endonuclease, a CRISPR/MAD7 endonuclease, a CRISPR/CasX endonuclease, a CRISPR/CasY endonuclease, a Zinc Finger Nuclease (ZFN), a homing endonuclease, a meganuclease, or a TAL effector nuclease.

32. The method of any one of claims 1 to 31, wherein the activity of the genome-engineered component in step (c) comprises inducing one or more double-strand breaks in the genome of the plant cell, one or more single-strand breaks in the genome of the plant cell, one or more base editing events in the genome of the plant cell, or one or more of DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ubiquitination, histone ribosylation, or histone citrullination in the genome of the plant cell.

33. The method of claim 32, wherein after inducing the one or more double-strand breaks or the one or more single-strand breaks, non-homologous end joining (NHEJ) and/or Homologous Directed Repair (HDR) of the breaks by a homologous recombination mechanism is performed.

34. The method of any one of claims 1 to 33, wherein the transgene in step (a) (ii) is selected from the group consisting of: genes encoding resistance or tolerance to abiotic stress, including drought stress, osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, nitrogen deficiency, phosphate deficiency, salt stress or water immersion, herbicide resistance, including resistance to glyphosate, glufosinate/glufosinate, hygromycin, protoporphyrinogen oxidase (PPO) inhibitors, ALS inhibitors, and dicamba; genes encoding resistance or tolerance to biotic stress, including viral resistance genes, fungal resistance genes, bacterial resistance genes, insect resistance genes; or a gene encoding a yield-related trait comprising lodging resistance, flowering time, shattering resistance, seed color, endosperm composition or nutrient content.

35. The method of any one of claims 1 to 34, wherein in step (c) the modification of the genome is selected from

i) (ii) substitution of at least one nucleotide;

ii) deletion of at least one nucleotide;

iii) insertion of at least one nucleotide;

iv) changes in DNA methylation;

v) changes in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ubiquitination, histone ribosylation, or histone citrullination; and

vi) any combination of i) -v).

36. The method of any one of claims 1 to 35, wherein the method is effective in promoting cell proliferation or cell regeneration, preferably after genetic modification.

37. The method of any one of claims 1 to 36, wherein the method is effective to induce embryogenesis from a single cell, preferably facilitated after genetic modification.

38. The method of any one of claims 1 to 37, wherein said method is effective to increase the stable transformation efficiency of said transgene into said plant cell.

39. The method of any one of claims 1 to 38, wherein the method is effective to increase the efficiency of editing the plant cell genome by the genome engineering component.

40. A genetically modified plant cell obtained or obtainable by a method according to any one of claims 1 to 39.

41. A plant or plant part comprising the genetically modified plant cell of claim 40.

42. A plant cell comprising

a) A polynucleotide encoding RKD2 polypeptide; or a polynucleotide encoding RKD4 polypeptide; and

b) a transgene and/or genome engineered component of interest;

wherein said polynucleotide encoding RKD2 polypeptide or RKD4 polypeptide is operably linked to a heterologous promoter that is directly chemically inducible or indirectly chemically inducible, preferably indirectly chemically inducible via an intermediate transcription factor.

43. A plant cell comprising a DNA construct, wherein the DNA construct comprises

a) A nucleic acid comprising a polynucleotide encoding RKD2 or RKD4 polypeptide operably linked to a chemically inducible bi-directional promoter,

b) a reporter gene operably linked to the bidirectional promoter, and

c) a third recombinant gene encoding a transcription factor operably linked to a strong constitutive promoter.

44. A method for producing a genetically modified plant comprising the steps of:

(d) regenerating a plant from the modified plant cell of step (a).

45. The method of claim 44, wherein the plant produced is free of any genome-engineered components stably integrated into the genome of the plant.

46. A genetically modified plant or part thereof, or progeny plant thereof obtained or obtainable by the method of claim 44 or claim 45.

47. Use of a nucleic acid comprising a polynucleotide encoding RKD2 polypeptide or RKD4 polypeptide, a recombinant gene comprising said nucleic acid, a DNA construct comprising said nucleic acid for improving the efficiency of plant regeneration or increasing the regeneration capacity of a plant cell following chemical induction, wherein said polynucleotide encoding said RKD2 polypeptide or RKD4 polypeptide is operably linked to a heterologous promoter which is directly chemically inducible or indirectly chemically inducible, preferably indirectly chemically inducible via an intermediate transcription factor.

Technical Field

Described herein are novel methods and materials for genome engineering in eukaryotic cells, particularly methods for increasing the efficiency of genome engineering (i.e., transformation or genome editing) by delivering nucleic acids encoding RKD2 and/or RKD4 and genome engineering components.

Sequence listing

This application contains a sequence listing in ASCII format submitted electronically and is incorporated by reference herein in its entirety. The ASCII copy was created at 25.11.2018, entitled KWS0304_ SELIST _20181123_ ST25.txt, size 73,166 bytes.

Background

Traditional breeding provides domesticated plants and animals, while modern biotechnology, particularly genomic engineering, is expanding breeding capacity and enabling improvements that are not possible with traditional closely related species crosses alone. Various characteristics such as high yield, herbicide tolerance, and pest resistance have been introduced into crops using biotechnology, making great progress in global agriculture and food safety. However, the presence of exogenous DNA in such biotechnological products can raise biosafety and environmental concerns.

By isolating any integrated DNA, genome editing techniques can be used to generate site-specific modifications of the target genome in the absence of exogenous DNA in the terminal plant. Furthermore, by transient expression, genome editing can have transient editing activity to create site-specific modifications without the need for DNA integration at any point in the process. Genome-edited plants, particularly plants derived from transient activities, are very different from traditional genome-modified plants and may not be amenable to plant regulation as Genetic Modification (GM). Thus, genome editing techniques, particularly via transient editing methods, can provide highly accurate, safe, and powerful tools for plant breeding and development for agriculture.

However, genome engineering based on transient activities faces more challenges. Transient engineering typically results in fewer modified cells than stable transformation. Without an integrated selectable marker, it is extremely challenging to identify engineered cells and achieve homologous modification in regenerated plants. These challenges prevent the conventional implementation of transient gene editing as a breeding tool for plant improvement. Therefore, there is an urgent need for novel methods and materials that improve the efficiency of genome engineering.

Disclosure of Invention

In one aspect, a method for genetic modification in a plant cell is provided, the method comprising

(a) Introducing into said plant cell

(ii) a transgene and/or genome engineered component of interest;

(b) optionally, culturing the plant cell under conditions that allow synthesis of RKD2 polypeptide or RKD4 polypeptide from the nucleic acid after chemical induction; and

(c) optionally, culturing the plant cell under conditions that allow genetic modification of the genome of the plant cell by integration of the activity of the transgene of interest and the genome engineering component in the presence of RKD2 polypeptide or RKD4 polypeptide;

wherein the polynucleotide sequence encoding RKD2 polypeptide or RKD4 polypeptide is operably linked to a heterologous promoter that is directly chemically inducible or indirectly chemically inducible, preferably indirectly chemically inducible via an intermediate transcription factor.

In some embodiments, the methods are effective in increasing the efficiency of plant regeneration. In some embodiments, the method is effective in increasing the regenerative capacity of a plant cell. In some embodiments, in step (b), RKD2 polypeptide or RKD4 polypeptide is synthesized from the nucleic acid after direct or indirect chemical induction of the heterologous promoter, preferably after addition of β -estradiol or a glucocorticoid such as dexamethasone to the plant cell.

In some embodiments, the heterologous promoter is the XVE/OLexA system for chemical inducibility of chemical β -estradiol. In various embodiments, the XVE/OLexA system comprises the amino acid sequence of SEQ ID NO:22, or a nucleotide sequence identical to SEQ ID NO:22, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto.

In some embodiments, the heterologous promoter is an inducible bidirectional promoter. In various embodiments, the inducible bidirectional promoter is operably linked to a second nucleic acid sequence encoding a desired polypeptide. In various embodiments, the second nucleic acid sequence comprises a reporter gene and a polynucleotide encoding a reporter protein. In some embodiments, the reporter is GUS or tdTomato.

In various embodiments, the inducible bidirectional promoter is a dexamethasone-inducible promoter. In some particular embodiments, the promoter is pOp1, pOp2, pOp4, or pOp 6. In some particular embodiments, pOp6 includes SEQ ID NO:15, or a nucleotide sequence identical to SEQ ID NO:15, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto.

In some embodiments, in step (a) (i), another nucleic acid comprising a polynucleotide sequence encoding a transcription factor operably linked to a strong constitutive promoter is introduced into the plant cell, wherein the transcription factor activates pOp6 upon binding to dexamethasone. In a particular embodiment, the transcription factor is LhGR or LhG 4. In some embodiments, LhGR has the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence identical to SEQ ID NO: 17, amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; or wherein the nucleic acid encoding LhGR comprises SEQ ID NO: 16, or a sequence identical to SEQ ID NO: 16, or a coding sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

In some embodiments, the strong constitutive promoter is a ubiquitin promoter or a double 35S promoter. In some specific embodiments, the double 35S promoter comprises the nucleotide sequence of SEQ ID NO. 21, or a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO. 21. In some particular embodiments, the ubiquitin promoter comprises the nucleotide sequence of SEQ ID NO. 23, or a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO. 23. In some particular embodiments, the ubiquitin promoter additionally comprises a ubiquitin intron comprising the nucleotide sequence of SEQ ID No. 20, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 20.

In some embodiments, the plant cell is cultured under conditions that allow genetic modification of the genome of the plant cell by integration of the activity of the transgene of interest and the genome engineering component in the presence of RKD2 polypeptide or RKD4 polypeptide; in some particular embodiments, the cultured plant cells do not express the reporter gene in the absence of a chemical agent effective to induce a chemically inducible promoter. In some embodiments, the plant cell is cultured to produce an embryonic structure. In some embodiments, the embryogenic structure is cultured to produce a regenerated plant.

In various embodiments, RKD2 polypeptide or RKD4 polypeptide is transiently present, has transient activity and/or is transiently expressed in a plant cell and/or nucleic acid encoding RKD2 polypeptide is transiently present, has transient activity and/or is transiently expressed in a plant cell or nucleic acid encoding RKD4 polypeptide is transiently present, has transient activity and/or is transiently expressed in a plant cell.

In various embodiments, RKD2 polypeptide comprises SEQ ID NO: 5, or an amino acid sequence identical to SEQ ID NO: 5, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; or the nucleic acid encoding RKD2 polypeptide encodes the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence identical to SEQ ID NO: 5, or a variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

In various embodiments, RKD4 polypeptide comprises SEQ ID NO:2, or an amino acid sequence identical to SEQ ID NO:2, an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; or the nucleic acid encoding RKD4 polypeptide encodes the amino acid sequence of SEQ ID NO:2, or an amino acid sequence identical to SEQ ID NO:2, or a variant thereof, 2 having an amino acid sequence which is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical.

In some embodiments, the nucleic acid encoding RKD2 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 3 or 4;

b) comprises a nucleotide sequence substantially identical to SEQ ID NO: 3 or 4, a nucleic acid having a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical;

c) and nucleic acids which hybridize under stringent hybridization conditions with the complementary strand of the nucleic acids defined under a) or b).

In some embodiments, the nucleic acid encoding RKD4 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 1;

c) a nucleic acid which hybridizes with the complementary strand of the nucleic acid as defined in (1) or (2) under stringent hybridization conditions.

In some embodiments, the genome engineering component comprises:

a) an enzyme that induces a Double Strand Break (DSB) or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, wherein the DSB-inducing enzyme preferably recognizes a predetermined site in the genome of the cell;

b) an enzyme that induces Single Strand Breaks (SSBs), or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, wherein the SSB-inducing enzyme preferably recognizes a predetermined site in the genome of the cell;

c) a base-editing enzyme, optionally fused to a disarmed DSB or SSB inducing enzyme, wherein said base-editing enzyme preferentially recognizes a predetermined site in the genome of said cell; or

d) An enzyme that effects DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ribosylation, or histone citrullination, optionally fused to a disarmed DSB or SSB-inducing enzyme, wherein said enzyme preferably recognizes a predetermined site in the genome of said cell.

In some embodiments, the genome-engineered component comprising a DSB or SSB-inducing enzyme or variant thereof is a CRISPR/Cas endonuclease, CRISPR/Cas9 endonuclease, CRISPR/Cpf1 endonuclease, CRISPR/Csm1 endonuclease, CRISPR/MAD7 endonuclease, CRISPR/CasX endonuclease, CRISPR/CasY endonuclease, Zinc Finger Nuclease (ZFN), homing endonuclease, meganuclease, or TAL effector nuclease.

In some embodiments, the activity of the genome engineering component in step (c) comprises inducing one or more of a double-strand break in the genome of the plant cell, a single-strand break in the genome of the plant cell, a base editing event in the genome of the plant cell, or DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ubiquitination, histone ribosylation, or histone citrullination in the genome of the plant cell. In particular embodiments, after induction of one or more double-stranded breaks or one or more single-stranded breaks, non-homologous end joining (NHEJ) and/or Homologous Directed Repair (HDR) of the break by homologous recombination mechanisms is performed.

In some embodiments, the transgene in step (a) (ii) is selected from: genes encoding resistance or tolerance to abiotic stress, including drought stress, osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, nitrogen deficiency, phosphate deficiency, salt stress or water immersion, herbicide resistance, including resistance to glyphosate, glufosinate/glufosinate, hygromycin, protoporphyrinogen oxidase (PPO) inhibitors, ALS inhibitors, and dicamba; genes encoding resistance or tolerance to biotic stress, including viral resistance genes, fungal resistance genes, bacterial resistance genes, insect resistance genes; or a gene encoding a yield-related trait comprising lodging resistance, flowering time, shattering resistance, seed color, endosperm composition or nutrient content.

In some embodiments, in step (c), the modification of the genome is selected from

i) (ii) substitution of at least one nucleotide;

ii) deletion of at least one nucleotide;

iii) insertion of at least one nucleotide;

iv) changes in DNA methylation;

v) changes in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ubiquitination, histone ribosylation, or histone citrullination; and

vi) any combination of i) -v).

In various embodiments, the methods are effective in promoting cell proliferation or cell regeneration, preferably after genetic modification. In various embodiments, the methods are effective to induce embryogenesis from a single cell, preferably facilitated after genetic modification. In various embodiments, the methods are effective in increasing the stable transformation efficiency of a transgene into a plant cell. In various embodiments, the methods are effective to increase the efficiency of genome engineering components to edit the genome of a plant cell.

In another aspect, there is provided a genetically modified plant cell obtained or obtainable by a method according to any one of the above aspects and embodiments. Plants or plant parts comprising the genetically modified plant cells are also provided.

In another aspect, there is provided a plant cell comprising

a) A polynucleotide encoding RKD2 polypeptide; or a polynucleotide encoding RKD4 polypeptide; and

b) a transgene and/or genome engineered component of interest;

In another aspect, a plant cell comprising a DNA construct is provided, wherein the DNA construct comprises

a) A nucleic acid comprising a polynucleotide encoding RKD2 or RKD4 polypeptide operably linked to a chemically inducible bi-directional promoter.

b) A reporter gene operably linked to the bidirectional promoter, and

c) a third recombinant gene encoding a transcription factor operably linked to a strong constitutive promoter.

In another aspect, there is provided a method for producing a genetically modified plant, comprising the steps of:

(a) genetically modifying a plant cell according to any of the above methods, and

(b) regenerating a plant from the modified plant cell of step (a).

In some embodiments, the plant produced does not contain any of the genome-engineered components stably integrated into the plant genome. Also provided are genetically modified plants or parts thereof obtained or obtainable by the methods.

In another aspect, there is provided a use of a nucleic acid comprising a polynucleotide encoding RKD2 polypeptide or RKD4 polypeptide, a recombinant gene comprising said nucleic acid, a DNA construct comprising said nucleic acid for increasing the efficiency of plant regeneration or increasing the regeneration capacity of a plant cell upon chemical induction, wherein said polynucleotide encoding RKD2 polypeptide or RKD4 polypeptide is operably linked to a heterologous promoter which is directly chemically inducible or indirectly chemically inducible, preferably indirectly chemically inducible via an intermediate transcription factor.

Drawings

FIG. 1 shows a map of the gene expression vector pZY ZZ-TOP carrying the pOp6/LHGR system chemically induced by dexamethasone (Dex) for GUS expression and the developmental genes of interest. The plasmid contains pOp6 a chemically inducible promoter. Both the developmental gene and the GUS reporter gene were expressed following dexamethasone exposure. pZY ZZ-TOP further comprises the gene LhGR operably linked to the ubiquitin promoter. In the presence of dexamethasone, LhGR enters the nucleus and activates pOp 6.

FIG. 2 is a graph showing a comparison of regeneration efficiencies mediated by various developmental genes under the control of a chemically inducible promoter system after induction. Plants were transformed with the pZY-ZZ-TOP plasmid, into which different developmental genes (i.e. LEC2, WUS, BBM, AGL15 and RKD4) were inserted. Some plants were treated with dexamethasone to induce expression of developmental genes (as indicated by "+" on the X-axis), while others were not so treated ("-"). The efficiency of regeneration on the Y-axis refers to the frequency of somatic embryo formation.

FIG. 3 shows the somatic embryo formation induced in Arabidopsis (Arabidopsis thaliana) plants expressing RKD4 in the pOp6/LhGR transactivation system described above. Somatic embryo formation was observed in roots (left panel), leaves (middle panel), or both roots and leaves (right panel).

FIG. 4 shows AtRKD 4-mediated regeneration of Phalaenopsis amabilis plants after induction of AtRKD4 expression with dexamethasone at the base of the leaves. The presence of somatic embryos (arrows) was determined on days 0 (left), 7 (middle), and 14 (right) after removal of dexamethasone from the medium.

FIG. 5 shows estradiol-induced expression of TaRKD2 in Triticum aestivum (Triticum aestivum) plants. Induction and development of embryonic structure was observed: callus formation (left panel, arrow), greening (middle panel) and emergence (right panel, arrow).

FIG. 6 shows estradiol-induced expression of TaRKD2 in barley (Hordeum vulgare) plants. Induction and development of embryonic structure was observed: callus formation (top left), shoot and root development (top right, arrow), green change (bottom left) and seedling formation (bottom right).

FIG. 7 shows dexamethasone-induced expression of AtRKD4 and TaRKD2 in maize (Zea mays). The dexamethasone-inducible construct was co-bombarded with the red fluorescent tdTomato gene in the presence of a constitutive promoter (see example 1). After exposure to dexamethasone, induction of massive callus structures stably integrated with tdTomato was observed on AtRKD4 (left panel) and TaRKD2 (33 days after right panel bombardment).

FIG. 8 shows a plasmid map of pERV1-hygro carrying the XVE/OLexA system for the chemoinducibility of β -estradiol for expression of Developmental genes of interest (Borghi, L. (2010). indicator gene expression systems for plants in Plant development Biology (pp.65-75). Humana Press, Totowa, NJ.).

Detailed Description

Definition of

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "about" as used in the context of this application means +/-10% of the stated value, preferably +/-5% of the stated value. For example, about 100 nucleotides (nt) is understood to mean a value of 90 to 110nt, preferably a value of 95 to 105 nt.

Regeneration of transformed or genetically edited plant cells may include a somatic embryogenesis process, which is an artificial process by which plants or embryos are derived from individual somatic cells or populations of somatic cells. Somatic embryos are formed from plant cells that are not normally involved in embryonic development, i.e., shoots, leaves, shoots, and like plant tissues. Applications of this process may include: clonal propagation of genetically identical plant material; eliminating viruses; providing source tissues for gene transformation; generating whole plants from single cells such as protoplasts; the development of synthetic seed technology. Cells derived from competent source tissue can be cultured to form callus. Plant growth regulators such as auxins or cytokinins in the tissue culture medium can be manipulated to induce callus formation and subsequently altered to induce callus formation into embryos. Somatic embryos are described as occurring in two ways: either directly or indirectly. Direct embryogenesis occurs when embryos produce identical clones starting from explant tissue. Indirect embryogenesis occurs when an explant produces undifferentiated or partially differentiated cells (i.e., callus) which then maintain or differentiate into plant tissue such as leaves, stems or roots.

The term "transgene" as used according to the present invention refers to a plant, plant cell, tissue, organ or material comprising a gene or gene construct comprising a "transgene" transferred into said plant, plant cell, tissue, organ or material by natural means or by a transformation technique from another organism. The term "transgene" includes nucleic acid sequences, including DNA or RNA, or amino acid sequences, or combinations or mixtures thereof. Thus, the term "transgene" is not limited to sequences that are commonly identified as "genes", i.e., sequences that encode proteins. It may also refer to, for example, non-protein coding DNA or RNA sequences. Thus, the term "transgenic" generally means that the corresponding nucleic acid or amino acid sequence does not naturally occur in the corresponding target cell, which includes a plant, plant cell, tissue, organ, or material. Thus, the term "transgene" or "transgenic" as used herein refers to a nucleic acid sequence or amino acid sequence that is extracted or synthetically produced from the genome of one organism and then introduced into another organism in a transient or stable manner by artificial techniques such as molecular biology, genetics, and the like. As used herein, "plant material" refers to any material that can be obtained from a plant at any stage of development. The plant material may be obtained by in situ (in planta) or in vitro culture of the plant or a tissue or organ thereof. Thus, the term includes plant cells, tissues and organs and developed plant structures, as well as subcellular components such as nucleic acids, polypeptides and all chemical plant substances or metabolites that may be found in the plant cell or compartment and/or may be produced by the plant, or may be obtained from any plant cell, tissue or extract of a plant at any stage of development. The term also includes derivatives of plant material, such as protoplasts, which are derived from at least one plant cell comprised by the plant material. Thus, the term also includes meristematic cells or meristems of plants.

The term "genome engineering" as used herein refers to strategies and techniques for genetic modification of any genetic information or genome of a plant cell, including genome transformation, genome editing, and the like. Likewise, "genome editing" refers to a technique for targeted specific modification of any genetic information or genome of a plant cell. Thus, "genome engineering" and "genome editing" include editing of a gene coding region, as well as editing of regions of the genome other than the gene coding region. These also include the editing or engineering of the nucleus (if present) and other genetic information of the plant cell. In addition, "genome engineering" also includes epigenetic editing or engineering, i.e., targeted modifications, such as methylation, histone modifications, or non-coding RNAs that may result in genetic changes in gene expression.

The term "genome editing" as used herein refers to strategies and techniques for targeted specific modification of any genetic information or genome of a plant cell, including editing regions of the genome other than the gene-coding regions (e.g., intron sequences, non-coding RNAs, mirnas, sequences of regulatory elements such as promoters, terminators, transcription activator binding sites, cis-or trans-acting elements, etc.). In addition, "genome editing" may include base editing for targeted replacement of a single nucleobase. It may also include editing the nuclear genome and other genetic information of the plant cell, i.e. the mitochondrial genome or chloroplast genome and the miRNA, pre-mRNA or mRNA. Furthermore, "genome editing" may include epigenetic editing or engineering, i.e. targeted modification, e.g. DNA methylation or histone modification, such as histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone threonyl, histone ribosylation or histone citrullination, which may result in a heritable change in gene expression. "genome editing" may also include epigenetic editing or engineering of non-coding RNAs that may result in genetic changes in gene expression.

As used herein, "base editor" refers to a protein or fragment thereof having the same catalytic activity as the protein from which it is derived, which protein or fragment thereof, when provided alone or as a molecular complex, is referred to herein as a base editing complex. The base editor has the ability to mediate targeted base modifications, i.e. to switch the base of interest resulting in a point mutation of interest, which in turn may result in a targeted mutation if the base switch does not cause a silent mutation but switches the amino acid encoded by the codon comprising the position to be switched with the base editor.

As used herein, "regulatory element" refers to a nucleotide sequence that is not part of a nucleotide sequence encoding a protein, but mediates expression of the nucleotide sequence encoding the protein. Regulatory elements include, for example, promoters, cis-regulatory elements, enhancers, introns, or terminators. Depending on the type of regulatory element, it is located on the nucleic acid molecule either before (i.e., 5 'to) or after (i.e., 3' to) the nucleotide sequence encoding the protein. Regulatory elements function in living plant cells. The term "operably linked" means that the regulatory element is linked to the nucleotide sequence encoding the protein in such a way that it is positioned relative to the nucleotide sequence encoding the protein, e.g., a nucleic acid molecule, in such a way that expression of the nucleotide sequence encoding the protein can take place in a living cell under the control of the regulatory element.

As used herein, "upstream" refers to a location on a nucleic acid molecule that is near the 5' end of the nucleic acid molecule. Likewise, the term "downstream" refers to a location on a nucleic acid molecule that is near the 3' end of the nucleic acid molecule. For the avoidance of doubt, nucleic acid molecules and their sequences are generally represented in the 5 'to 3' direction (left to right).

As used herein, a "flanking region" is a region of a repair nucleic acid molecule having a nucleotide sequence that is homologous to the nucleotide sequence of the DNA region flanking (i.e., upstream or downstream) the preselected site.

As used herein, "transient expression" refers to the phenomenon whereby the transferred protein/polypeptide and nucleic acid fragments encoding the protein/polypeptide are transiently expressed and/or active in a cell and are rapidly shut down and/or degraded as the cell grows.

As used herein, a "double-stranded DNA break inducing enzyme", "double-stranded break inducing enzyme", or "DSBI enzyme" is an enzyme capable of inducing a double-stranded DNA break at a specific nucleotide sequence called a "recognition site" or a "predetermined site". Accordingly, a "single-stranded DNA break inducing enzyme", "single-stranded break inducing enzyme", or "SSBI enzyme" is an enzyme capable of inducing single-stranded DNA or RNA breaks at a specific nucleotide sequence called a "recognition site" or a "predetermined site".

As used herein, a "repair nucleic acid molecule" is a single-or double-stranded DNA molecule or RNA molecule that serves as a template for modifying genomic DNA or RNA at a preselected site that is near or at the cleavage site. As used herein, "used as a template for modifying genomic DNA" refers to replication or integration of a repair nucleic acid molecule at a preselected site by homologous recombination between the flanking regions and corresponding homologous regions in the genome of interest flanking the preselected site, optionally in combination with non-homologous end joining (NHEJ) at one of the two ends of the repair nucleic acid molecule (e.g., in the case of only one flanking region).

As used herein, "modification of a genome" refers to a genome that has been altered in at least one nucleotide or by at least one epigenetic edit.

As used herein, "preselected site," "predefined site," or "predefined site" refers to a particular nucleotide sequence in a genome (e.g., a nuclear genome or chloroplast genome) at which one or more nucleotides are desired to be inserted, substituted, and/or deleted.

As used herein, "plant hormone" or "plant growth regulator" refers to any naturally occurring or synthetic material and chemical substance that promotes plant cell division and/or plant morphogenesis. As used herein, "regeneration" refers to the process by which a single or multiple cells proliferate and develop into tissues, organs, and ultimately, whole plants.

As used herein, the term "vector" or "plasmid (vector)" refers to a construct for introduction or transformation, transfection or transduction into any eukaryotic cell, including a plant, plant cell, tissue, organ or material according to the present invention, including in particular plasmids or (plasmid) vectors, cosmids, artificial yeast or bacterial artificial chromosomes (YACs and BACs), phagemids, bacteriophage-based vectors, expression cassettes, isolated single-or double-stranded nucleic acid sequences, including linear or circular sequences, or amino acid sequences, viral vectors, including modified viruses, and combinations or mixtures thereof.

"recombination" in the context of a recombinant gene may include regulatory sequences and/or localization sequences. The recombinant construct or DNA construct according to the invention may be integrated into a vector, or may be a vector (including a plasmid vector), and/or it may be present in isolation from the vector structure, e.g.in the form of a single-or double-stranded nucleic acid. After introduction of the recombinant gene or DNA construct, for example by biological or physical transformation or transfection, it may persist extrachromosomally, i.e. not integrated into the genome of the target cell, for example in the form of double-stranded or single-stranded DNA. Alternatively, the recombinant gene or DNA construct may be stably integrated into the genome of the target cell, including the nuclear genome or other genetic elements of the target cell, including the genome of a plastid such as a mitochondrion or chloroplast.

The inventors show that nucleic acids encoding RKD2 and/or RKD4 mediate strong effects especially early in regeneration after delivery of transgenic and/or genome engineered components. This effect does not affect the development of the plant, and the regenerated plant exhibits good plant growth and fertility during adulthood. Thus, integration of RKD2 and RKD4 genes can be isolated in the progeny by hybridization and selection.

RKD2 and RKD4 are plant-specific RWP-RK transcription factors. RKD2 and RKD4 are found in Arabidopsis and other plant species such as wheat and maize. RKD2 and RKD4, alone or in combination, can improve genome engineering and/or improve plant regeneration of transformed or gene-edited plant cells. RKD2 and RKD4, either alone or together, may increase the performance or capacity of plant cells (preferably derived from somatic tissue, embryonic tissue, callus or protoplasts) to regenerate throughout a plant, preferably a fertile plant. Thus, RKD2 and RKD4 either individually or together may modulate somatic embryo formation (somatic embryo formation) and/or may increase the rate of proliferation of plant cells. The combination of RKD2 and RKD4 together may have a synergistic effect. In the various methods disclosed herein, RKD2, RKD4, or a combination of RKD2 and RKD4 may be transiently co-expressed. Polynucleotides encoding RKD2 or RKD4 may be introduced into plant cells.

Also provided is a nucleic acid encoding RKD2 or RKD4 comprising SEQ ID NO:2 or 5. Further provided is a nucleic acid encoding RKD2 or RKD4 comprising a nucleotide sequence identical to SEQ ID NO:2 or 5, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

Comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence substantially identical to SEQ ID NO:2, a nucleic acid encoding RKD4 having an amino acid sequence at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 84%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto can include a nucleic acid encoding RKD4 comprising an amino acid sequence of SEQ ID NO: 1. The nucleic acid may comprise a nucleotide sequence identical to SEQ ID NO:1, nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. Alternatively, the nucleic acid may hybridize under stringent hybridization conditions to a nucleic acid comprising SEQ ID NO:1 or a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO:1, having a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical.

Comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence substantially identical to SEQ ID NO: 5 nucleic acid encoding RKD2 having an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto may further comprise a nucleic acid encoding RKD2 comprising SEQ ID NO: 3 or 4. The nucleic acid may comprise a nucleotide sequence identical to SEQ ID NO: 3, a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. The nucleic acid may comprise a nucleotide sequence identical to SEQ ID NO: 4, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical. Alternatively, the nucleic acid may hybridize under stringent hybridization conditions to a nucleic acid comprising SEQ ID NO: 3 or a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO: 3, having a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical. Alternatively, the nucleic acid may hybridize under stringent hybridization conditions to a nucleic acid comprising SEQ ID NO: 4 or a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO: 4, having a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical.

A recombinant gene is provided comprising a nucleic acid encoding RKD2 or RKD4, the nucleic acid of RKD2 or RKD4 comprising SEQ ID NO:2 or 5, or an amino acid sequence substantially identical to SEQ ID NO:2 or 5, or an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. The nucleic acid may be operably linked to one or more regulatory elements. The regulatory element may be a promoter, cis-regulatory element, enhancer, intron, or terminator. The regulatory element may be 5' to the nucleic acid sequence. The regulatory element may be 3' to the nucleic acid sequence. The regulatory element may be a directional promoter. The nucleic acid may comprise a nucleic acid comprising SEQ ID NO: 1. 3 or 4. The nucleic acid may comprise a nucleotide sequence identical to SEQ ID NO: 1. 3 or 4, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical. Under stringent hybridization conditions, the nucleic acid can hybridize to a nucleic acid comprising SEQ ID NO: 1. 3 or 4 or a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO: 1. 3 or 4, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

In some embodiments, the nucleic acid is operably linked to a heterologous promoter. The heterologous promoter may be an inducible promoter.

The heterologous promoter may be a bi-directional inducible promoter, such as chemically inducible promoter pOp 6. The pOp6 promoter contains six copies of an optimized lac operator sequence, as described in Samalova et al, Plant J., 2005, 41(6): 919-35. Dexamethasone mobilized LhGR localization to the nucleus, as part of the pOp6/LhGR expression system, where it activated pOp 6. The LhGR may be operably linked to a strong constitutive promoter (e.g. ubiquitin promoter or double 35S promoter). The LhGR coding sequence may also include a ubiquitin intron. Alternatively, LhGR and a strong constitutive promoter may be expressed on separate DNA constructs.

In some embodiments, the strong constitutive promoter is a ubiquitin promoter or a double 35S promoter. In some particular embodiments, the double 35S promoter comprises SEQ ID NO:21, or a nucleotide sequence identical to SEQ ID NO:21, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. In some particular embodiments, the ubiquitin promoter comprises SEQ ID NO:23, or a nucleotide sequence identical to SEQ ID NO:23 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. In some particular embodiments, the ubiquitin promoter further comprises a ubiquitin intron comprising the amino acid sequence of SEQ ID NO:20, or a nucleotide sequence identical to SEQ ID NO:20, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto.

Activated pOp6 is capable of directing transcription of a gene operably linked to pOp 6. Due to the bidirectional property, pOp6 can express both the target gene (such as developmental gene) and the reporter gene (such as GUS or tdTomato). Alternatively, pOp1, pOp2, or pOp4 may be used.

Alternatively, the heterologous promoter may be XVE/OlexA, for example, as part of the β -estradiol inducible XVE/OlexA system. Upon exposure to β -estradiol, XVE/OlexA is activated and directs the transcription of a gene to which XVE/OlexA is operably linked.

The GUS reporter system is suitable for most plants (GUS is β -glucuronidase) due to the lower level of activity of β -glucuronidase in plants. the tDTomato gene (tDT) encodes an exceptionally bright red fluorescent protein with a maximum excitation at 554nm and a maximum emission at 581 nm. In addition, other reporter enzymes such as luciferase, β -galactosidase, Chloramphenicol Acetyltransferase (CAT), and alkaline phosphatase may also be used.

Also provided are DNA constructs, preferably vectors, comprising any of the nucleic acids or recombinant genes described above. The nucleic acid may comprise a nucleic acid comprising SEQ ID NO: 1. 3 or 4. The nucleic acid may comprise a nucleotide sequence identical to SEQ ID NO: 1. 3 or 4, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical. Alternatively, the nucleic acid may hybridize under stringent hybridization conditions to a nucleic acid comprising SEQ ID NO: 1. 3 or 4 or a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO: 1. 3 or 4, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. In some embodiments, the DNA construct is a plasmid. In some embodiments, the DNA construct comprises a bi-directionally inducible promoter (e.g., pOp 6). The nucleic acid may be operably linked to the bi-directional inducible promoter. In some embodiments, the DNA construct comprises a XVE/OlexA promoter system capable of inducing expression of a nucleic acid upon exposure to β -estradiol. Another reporter nucleic acid (e.g., GUS or tdTomato) may also be operably linked to a bidirectional inducible promoter. The reporter nucleic acid can help select for plants that express the nucleic acid in a leaky manner, for example when no induction of the bi-directional inducible promoter occurs. For example, plants expressing GUS or tdTomato can be selected, which are operably linked to dexamethasone-induced pOp6 when dexamethasone is not administered.

Plant cells

In another aspect, a plant cell is provided comprising one or more of RKD2 or RKD4, a nucleic acid, a recombinant gene, and a DNA construct described herein, preferably a transgene. In some embodiments, RKD2 or RKD4 comprises SEQ ID NO:2 or 5. In some embodiments, RKD2 or RKD4 comprises a nucleotide sequence identical to SEQ ID NO:2 or 5, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. The nucleic acid may comprise a nucleic acid comprising SEQ ID NO: 1. 3 or 4. The nucleic acid may comprise a nucleotide sequence identical to SEQ ID NO: 1. 3 or 4, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical. Under stringent hybridization conditions, the nucleic acid can hybridize to a nucleic acid comprising SEQ ID NO: 1. 3 or 4 or a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO: 1. 3 or 4, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. Also provided are plants, parts of plants, seeds, embryos or callus comprising the plant cells.

The plant cell may be part of or derived from any type of plant material, preferably a shoot, hypocotyl, cotyledon, stem, leaf, petiole, root, embryo, callus, flower, gametophyte or part thereof, or may be a protoplast or be derived from a protoplast. Isolated plant cells can be used as well as plant material, i.e., whole plants or parts of plants comprising plant cells.

A part of a plant, or parts of multiple plants, may be attached to, or separate from, an entire whole plant. These parts of the plant include, but are not limited to, organs, tissues and cells of the plant, preferably seeds.

The plant cell, plant part or plant may be from any plant species, whether monocotyledonous or dicotyledonous. Preferably, the plants which may be suitable for the methods and uses of the present invention are plants selected from the genera: barley (Hordeum), Sorghum (Sorghum), sugarcane (Saccharum), maize (Zea), Setaria (Setaria), rice (Oryza), wheat (Triticum), rye (Secale), Triticale (Triticule), apple (Malus), Brachypodium (Brachypodium), Aegilops (Aegliops), carrot (Daucus), Beta (Beta), Eucalyptus (Eucalyptus), tobacco (Nicotiana), Solanum (Solanum), coffee (Coffea), Vitis (Vitis), Erythrine, Spirulina (Genlisea), cucumber (Cucumis), Arabidopsis (Arabidopsis), Citrus (Cruciferae), Brassica (Brassica), mustard (Brassica), Brassica (Brassica) and (Brassica) including Brassica (Brassica) and (Brassica) including (Brassica) plants (Brassica) including (Brassica) including (Brassica) and (Brassica) including (Brassica) and (Brassica) including (Brassica) including (Brassica) and (Brassica) including (Brassica) and (Brassica) including (L, Brassica (Brassica) including (L (Brassica) including (L, Brassica) including (Brassica (L, Brassica (Brassica) including (L, Brassica (L (Brassica) including (L) including (L (Brassica) including (L) including (L, Cicer (Cicer), Couma (Cajanus), Phaseolus (Phaseolus), Glycine (Glycine), Gossypium (Gossypium), Astragalus (Astragalus), Nelumbo (Lotus), Phalaenopsis (Torenia), Allium (Allium) or Helianthus (Helianthus). More preferably, the plant is selected from the group consisting of barley (Hordeum vulgare), Hordeum bulbusomorium, Mandarin rice biocolor, sugarcane (Saccharum officinarum), Zea spp (Zea spp.), including maize (Zea mays), millet (Setaria italica), Oryza minutissima (Oryza minuta), Oryza sativa (Oryza sativa), Oryza sativa (Oryza australiana), Oryza sativa (Oryza sativa), Triticum aestivum (Triticum aestivum), durum Triticum (Triticum durum), rye (Secale), Triticale, apple (Malus domestica), Brassium brachiatum (Brassimum longissimum), Brassimum nodosum (Brassimum longibrachiatum), Hovenium, mountain sheep weed (Aesius), rye (Malus domestica), tobacco (Carica), Carica sativa), tobacco (Benzyphus grandis), and American rice (maize) Tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), potato (Solanum tuberosum), medium grain coffee (Coffea canephora), grape (Vitis vinifera), Erythrante guttata, Spirulina (Genlisea aureus), cucumber (Cucumis sativus), Morus alba (Marus notubilis), Arabidopsis thaliana (Arabidopsis thaliana), Cardamiana nexosus, Pleurospermum (Lepidium), Brassica campestris (Brassica campestris), Brassica campestris (Capillaria), Brassica oleracea (Brassica oleracea), Brassica campestris (Brassica oleracea), Brassica oleracea (Brassica oleracea), Brassica campechia campechiana (Brassica oleracea), Brassica campechia sativa (Brassica), Brassica campechia (Brassica campechia campestris), Brassica campechia sativa (Brassica) Cicer yamashitae, Cicer bijucum, chickpea (Cicer arietinum), Cicer reticulum, Cicer judaium, Cajanus cajanifolius, cranberry beans (Cajanus scarabaeoides), Phaseolus vulgaris (Phaseolus vulgaris), Glycine max (Glycine max), Cotton gossypii sp.), Astragalus sinicus (Astragalius sinicus), Lotus japonica (Lotus), Pleurotus japonicus (Torrenia fournieri), Allium cepa (Allium cepa), Allium fistulosum (Allium fistulosum), Allium sativum (Allium sativum), Helianthus annuus (Helianthus annuus), Jerusalem artichoke (Helianthus tuberosus) and/or Allium tuberosum (Allium). Particularly preferred are sugar beet (Beta vulgaris), maize (Zea mays), Triticum aestivum (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cereale), sunflower (Helianthus annuus), potato (Solanum tuberosum), Sorghum (Sorghum bicolor), turnip (Brassica rapa), rape (Brassica napus), canola (Brassica juncea), cabbage (Brassica oleracea), radish (Raphanus sativus), rice (Oryza sativa), soybean (Glycine max) and/or cotton (Gossium sp).

The genetically modified plant cell may be part of a whole plant or may be part of such a part. The present invention therefore also relates to plants or plant parts which comprise the above-described genetically modified plant cells.

Cultivating the plant cell into which the genome engineering component has been (co) introduced by integrating the activity of the transgene of interest and the genome engineering component in the presence of RKD2 or RKD4 under conditions that allow genetic modification of the genome of said plant cell.

Genetic modification of plant cells

Methods of making genetic modifications in plant cells are also provided. The method comprises introducing into a plant cell (i) any of the nucleic acids, recombinant genes, or DNA constructs described herein; and (ii) a transgene and/or genome engineering component. Plant cells may be cultured under conditions that allow synthesis of RKD2 or RKD4 polypeptide from the nucleic acid, recombinant gene, or DNA construct. Plant cells may be cultured under conditions that allow genetic modification of the genome of the plant cells by the activity of genome engineering components in the presence of RKD2 or RKD4 polypeptide.

The genome-engineered components may be introduced as proteins and/or nucleic acids encoding the genome-engineered components, in particular as DNA such as plasmid DNA, RNA, mRNA or RNP. Genome engineering can be used to make transgenic, gene-edited or base-edited plant material.

For the plant cell to be modified, biological pathway-based transformation methods, such as Agrobacterium transformation or viral vector-mediated plant transformation, can be used. A common biological method is transformation with Agrobacterium, which has been used in a variety of different plant materials for decades. Viral vector-mediated plant transformation can also be used to introduce genetic material into cells of interest. Agrobacterium-mediated transformation refers to the delivery of exogenous DNA into plant cells using Agrobacterium tumefaciens (an Agrobacterium tumefaciens), a soil bacterium that is used as a natural genetic engineering vector. Agrobacterium tumefaciens is capable of invading plants and transferring foreign DNA in a fairly broad plant range.

Alternatively, transformation methods based on physical delivery methods, such as particle bombardment or microinjection, may be used. Particle bombardment includes genesGun transfection or microparticle-mediated gene transfer, which refers to a physical delivery method for transferring coated microparticles or nanoparticles comprising a nucleic acid or gene construct of interest into a target cell or tissue. The physical introduction means are suitable for introducing nucleic acids, i.e. RNA and/or DNA, and proteins. Particle bombardment and microinjection have become prominent techniques for introducing genetic material into plant cells or tissues of interest. Helenius et al, "Gene delivery in vivo sites using the Helios^TMGene Gun ", Plant Molecular Biology Reporter, 2000, 18(3):287-288 discloses particle bombardment as a physical method for introducing substances into Plant cells. Thus, there are a variety of plant transformation methods for introducing genetic material in the form of a genetic construct into a plant cell of interest, including biological and physical methods known to those skilled in the art of plant biotechnology that can be used to introduce at least one gene encoding at least one wall-associated kinase into at least one cell of at least one of a plant cell, tissue, organ or whole plant.

The term "particle bombardment", also referred to as "biolistic transfection" or "microparticle-mediated gene transfer" as used herein, refers to a physical delivery method for the transfer of coated microparticles or nanoparticles comprising RKD2 or RKD4 genes, genome engineered components, and/or transgenes into a target cell or tissue. The micro-or nanoparticles are propelled and emitted at high pressure towards the target structure of interest using a suitable device (commonly known as a gene gun). Transformation by particle bombardment uses a metal microprojectile coated with the construct of interest, which is then shot at high speed (about 1500km/h) sufficiently fast to penetrate the cell wall of the target tissue, but not sufficiently to cause cell death, using a device called a "gene gun" (Sandford et al 1987). For protoplasts with completely removed cell walls, the conditions are logically different. The at least one microprojectile-precipitated construct is released into the cell after bombardment. Acceleration of the microprojectiles is accomplished by high-voltage electrical discharge or compressed gas (helium). As regards the metal particles used, they must be non-toxic, non-reactive and their diameter is smaller than that of the target cell. Most commonly gold or tungsten. Manufacturers and suppliers of gene guns and related systems provide a wealth of public information regarding their general use.

In a particularly preferred embodiment of microprojectile bombardment, one or more RKD2 or RKD4 genes, genome-engineered components and/or transgenes are co-delivered by a microcarrier comprising gold particles in the size range of 0.4-1.6 micrometer (μm), preferably in the range of 0.4-1.0 μm in an exemplary process, 10-1000 μ g of gold particles, preferably 50-300 μ g, are used per bombardment.

RKD2 or RKD4 genes, genome engineering components, and/or transgenes can be delivered into target cells, for example, using a Bio-Rad PDS-1000/He particle gun or a hand-held Helios gene gun system. When the PDS-1000/He particle gun system is used, the bombardment rupture pressure is 450-. More than one chemical or construct can be co-delivered into the target cell simultaneously with the genome-engineered components.

The delivery methods described above for transformation and transfection may be used simultaneously to introduce the tools of the invention. Also, there are specific transformation or transfection methods for specifically introducing a nucleic acid or amino acid construct of interest into a plant cell, including electroporation, microinjection, nanoparticles, and Cell Penetrating Peptides (CPPs). Furthermore, there are chemical-based transfection methods to introduce gene constructs and/or nucleic acids and/or proteins, including, inter alia, transfection with calcium phosphate, transfection using liposomes (e.g., cationic liposomes), or transfection with cationic polymers (including DEAD-dextran or polyethyleneimine), or combinations thereof. The above delivery techniques may be used alone or in combination in vivo (including in situ) or in vitro methods.

In some embodiments, the genome engineering component comprises:

a) an enzyme that induces a Double Strand Break (DSB) or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, wherein the DSB-inducing enzyme optionally recognizes a predetermined site in the genome of the cell;

b) an enzyme that induces Single Strand Breaks (SSBs), or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, wherein the SSB-inducing enzyme optionally recognizes a predetermined site in the genome of the cell;

c) a base-editing enzyme, optionally fused to a disarmed DSB or SSB inducing enzyme, wherein said base-editing enzyme preferentially recognizes a predetermined site in the genome of said cell; or

In order to be able to break at a predetermined target site, the enzyme preferably comprises a binding/recognition domain and a cleavage domain. Specific enzymes capable of inducing double-or single-strand breaks are nucleases or nickases and variants thereof, including such enzymes that no longer contain a nuclease or nickase function, but rather are used in combination with another enzyme as a recognition molecule. In recent years, many suitable nucleases, especially custom endonucleases, have been developed, including meganucleases, zinc finger nucleases, TALE nucleases, Argonaute nucleases e.g. derived from bacillus griffithii (Natronobacterium gregoryi), and CRISPR nucleases including e.g. Cas9, Cpf1, Csm1, MAD7, casX or CasY nucleases as part of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. In a preferred embodiment of the invention, the genome engineering component comprises a DSB or SSB inducing enzyme or variant thereof selected from the group consisting of: CRISPR/Cas endonuclease, CRISPR/Cas9 endonuclease, CRISPR/Cpf1 endonuclease, CRISPR/Csm1 endonuclease, CRISPR/MAD7 endonuclease, CRISPR/CasX endonuclease, CRISPR/Casy endonuclease, Zinc Finger Nuclease (ZFN), homing endonuclease, meganuclease, and TAL effector nuclease.

Rare-cutting endonucleases are DSBI/SSBI enzymes that have recognition sites of preferably about 14 to 70 contiguous nucleotides and therefore have very low cutting frequencies even in larger genomes (e.g., most plant genomes). Homing endonucleases, also known as meganucleases, constitute a family of such rare-cutting endonucleases. They can be encoded by introns, independent genes or intervening sequences and have surprising structural and functional properties that distinguish them from more classical restriction enzymes (usually derived from bacterial restriction modification type II systems). Their recognition sites have a general asymmetry, as opposed to the characteristic binary symmetry of most restriction endonuclease recognition sites. Several homing endonucleases encoded by introns or endoproteins have been shown to promote homing of their respective genetic elements into intron-free or endoproteinefree sites of the allele. By creating site-specific double-strand breaks in either the intron-free or the intein-free alleles, these nucleases generate recombination ends that participate in the gene conversion process that replicates the coding sequence and results in the insertion of introns or intervening sequences at the DNA level. A further series of rare-cutting meganucleases and their respective recognition sites are provided in table i (table i) of WO 03/004659 (pages 17 to 20) (incorporated herein by reference).

In addition, methods can be used to design customized rare-cutting endonucleases that recognize essentially any nucleotide sequence of interest that is selected. Briefly, chimeric restriction enzymes can be prepared by using hybridization between a zinc finger domain designed to recognize a specific nucleotide sequence and a non-specific DNA cleavage domain from a native restriction enzyme (e.g., fokl). Such methods are described in WO 03/080809, WO 94/18313 or WO 95/09233 and Isalan et al (2001). A rapid and generally applicable zinc finger engineering method is described by taking an HIV-1 promoter as a target. Nature biotechnology, 19 (7): 656; liu et al (1997). The design of multi-finger zinc finger proteins for unique addressing in complex genomes. Proceedings of the National Academy of Sciences, 94 (11): 5525-5530.

Another example of a custom endonuclease includes TALE nucleases (TALENs) based on transcriptional activator-like effector proteins (TALEs) from the genus Xanthomonas (xanthomas) fused to the catalytic domain of a nuclease (e.g., fokl or a variant thereof). The DNA binding specificity of these TALEs is defined by the repeated variable double-stranded Residues (RVDs) of 34/35 amino acid repeat units arranged in tandem such that one RVD specifically recognizes one nucleotide in the target DNA. These repeat units can be assembled to recognize essentially any target sequence and fused to the catalytic domain of a nuclease to generate sequence-specific endonucleases (see, e.g., Boch et al (2009), Breaking the code of DNA binding specificity of TAL-type III effectors. science, 326(5959), 1509-. WO 2012/138927 further describes monomeric (compact) TALENs and TALEs with multiple catalytic domains and combinations thereof.

Recently, a new customizable endonuclease system has been described; referred to as CRISPR/Cas system. The CRISPR System in its natural environment describes a molecular complex comprising at least one small individual non-coding RNA in combination with a Cas nuclease or another CRISPR nuclease (such as Cpf1 nuclease or Csm1 nuclease) (Zetsche et al, "Cpf 1 Is a Single RNA-guidelines of a Class 2 CRISPR-Cas System", Cell, 163, pp.1-13, October 2015.; US 2017/0233756A 1) which can produce specific DNA double strand breaks. Currently, CRISPR systems are divided into class 2, comprising 5 types of CRISPR systems, such as type II systems using Cas9 as effector and type V systems using Cpf1 as effector molecule (Makarova et al, Nature rev. microbiol, 2015). In artificial CRISPR systems, synthetic non-coding RNAs and CRISPR nucleases and/or optionally modified CRISPR nucleases (which are modified to act as nickases or lack any nuclease function) may be used in combination with at least one synthetic or artificial guide RNA or gRNA that binds crRNA and/or tracrRNA functions (Makarova et al, 2015, supra). The immune response mediated by the CRISPR/Cas system in nature should require CRISPR-RNA (crrna), where the maturation of such guide RNA that controls CRISPR nuclease-specific activation varies widely between the various CRISPR systems that have been characterized to date. First, the invading DNA (also called the spacer) integrates between two adjacent repeats proximal to the CRISPR locus. The type II CRISPR system encodes Cas9 nuclease as a key enzyme for the interference step, which system comprises both crRNA and transactivating rna (tracrrna) as guide motifs. These hybridize and form double-stranded (ds) RNA regions that are recognized by RNAseIII and can be cleaved to form mature crRNA. These then in turn bind to the Cas molecule in order to specifically guide the nuclease to the target nucleic acid region. Recombinant gRNA molecules can contain both a variable DNA recognition region and a Cas interaction region and can therefore be specifically designed independently of the particular target nucleic acid and desired Cas nuclease.

As a further safety mechanism, PAM (pro-spacer sequence adjacent motif) must be present in the target nucleic acid region; these are DNA sequences directly following the DNA recognizing the Cas9/RNA complex. The PAM sequence of Cas9 derived from Streptococcus pyogenes (Streptococcus pyogenes) has been described as "NGG" or "NAG" (standard IUPAC nucleotide code) (Jinek et al, "A programmable dual-RNA-protected DNA endonuclease in adaptive bacterial immunity", Science 2012, 337: 816-. The PAM sequence of Cas9 derived from Staphylococcus aureus (Staphylococcus aureus) is "NNGRRT" or "NNGRR (N)". Other variant CRISPR/Cas9 systems are known. Thus, Cas9 of Neisseria meningitidis (Neisseria meningitidis) cleaves at the PAM sequence NNNNGATT. Cas9 of Streptococcus thermophilus (Streptococcus thermophilus) cleaves at the PAM sequence NNAGAAW. Recently, a further PAM motif NNNNRYAC has been described for the CRISPR system of Campylobacter jejuni (Campylobacter) (WO 2016/021973 a 1). For Cpf1 nuclease, it has been described that the Cpf1-crRNA complex without tracrRNA efficiently recognizes and cleaves target DNA preceded by a short T-rich PAM, in contrast to the usual G-rich PAM recognized by Cas9 system (Zetsche et al, supra). Furthermore, by using modified CRISPR polypeptides, specific single-chain breaks can be obtained. The use of Cas nickase in combination with various recombinant grnas can also induce highly specific DNA double strand breaks by generating double DNA nicks. Furthermore, by using two RNAs, the specificity of DNA binding, and thus DNA cleavage, can be optimized. Other CRISPR effectors, such as the CasX and CasY effectors originally described for bacteria, are also available and represent additional effectors that may be used for genomic engineering purposes (Burstein et al, "New CRISPR-Cas systems from uncultivated microorganisms", Nature, 2017, 542, 237-.

The cleavage site of the DSBI/SSBI enzyme is related to the exact position of the induced break on the DNA or RNA. The cleavage site may or may not be included in (overlap with) the recognition site of the DSBI/SSBI enzyme, and thus it is believed that the cleavage site of the DSBI/SSBI enzyme is located at or near its recognition site. The recognition site (sometimes also referred to as binding site) of a DSBI/SSBI enzyme is a nucleotide sequence that is recognized by the DSBI/SSBI enzyme (specificity) and determines its binding specificity. For example, TALEN or ZNF monomers have recognition sites defined by their RVD repeats or ZF repeats, respectively, while their cleavage sites are defined by their nuclease domains (e.g., FokI) and are usually outside the recognition sites. In the case of dimeric TALENs or ZFNs, the cleavage site is located between the two recognition/binding sites of the respective monomers, and this region of the spacer DNA or RNA where cleavage occurs is referred to as the spacer.

The skilled person is able to select or design a DSBI/SSBI enzyme that recognizes a specific recognition site and induces a DSB or SSB at a cleavage site at or near the preselected/predetermined site. Alternatively, the DSBI/SSBI enzyme recognition site may be introduced into the target genome using any conventional transformation method or by hybridization with an organism having a DSBI/SSBI enzyme recognition site in its genome, and then any desired nucleic acid may be introduced at or near the site of cleavage by the DSBI/SSBI enzyme.

In various embodiments, the modification of the genome comprises one or more of: i) replacing at least one nucleotide; ii) deletion of at least one nucleotide; iii) insertion of at least one nucleotide; iv) changes in DNA methylation; and v) changes in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone threonyl, histone ribosylation, or histone citrullination.

In some embodiments, the activity of the genome engineering component induces one or more of a double-strand break in the genome of the plant cell, a single-strand break in the genome of the plant cell, a base editing event in the genome of the plant cell, or DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ubiquitination, histone ribosylation, or histone citrullination in the genome of the plant cell.

In some embodiments, following induction of one or more double-stranded breaks or one or more single-stranded breaks, non-homologous end joining (NHEJ) and/or homologous directed repair of the break by homologous recombination mechanisms (HDR) is performed. NHEJ and HDR are two major and distinct pathways for repairing breaks. Homologous recombination requires the presence of homologous sequences as templates (e.g., repair nucleic acid molecules or "donors") to direct the cellular repair process and the repair results are error-free and predictable. In the absence of a template (or repair nucleic acid molecule or "donor") sequence for homologous recombination, cells typically attempt to repair the break by a non-homologous end joining (NHEJ) process.

In a particularly preferred aspect of this embodiment, the repair nucleic acid molecule is additionally introduced into a plant cell. The repair nucleic acid molecule is a single-or double-stranded DNA molecule or RNA molecule that serves as a template for modifying genomic DNA or RNA at a preselected site that is near or at the cleavage site. In some embodiments, a repair nucleic acid molecule is used as a template for modifying genomic DNA, wherein the repair nucleic acid molecule is replicated or integrated at a preselected site by homologous recombination between the flanking regions and corresponding homologous regions in the genome of interest flanking the preselected site, optionally in combination with a non-homologous end joining (NHEJ) (e.g., in the case of only one flanking region) at one of the two ends of the repair nucleic acid molecule. Integration by homologous recombination allows the repair nucleic acid molecule to be precisely ligated to the target genome up to nucleotide levels, whereas NHEJ may result in minor insertions/deletions at the junction between the repair nucleic acid molecule and the genomic DNA.

In various embodiments of the aspects described herein, a modification of the genome has occurred in which at least one nucleotide change has occurred in the genome. The genome may be modified by insertion of a transgene (preferably an expression cassette comprising the transgene of interest), substitution of at least one nucleotide and/or deletion of at least one nucleotide and/or insertion of at least one nucleotide, as long as the resulting total change of at least one nucleotide compared to the nucleotide sequence of the preselected genomic target site prior to modification is produced, thereby allowing the modification to be recognized, for example, by techniques such as sequencing or PCR analysis, as will be apparent to the skilled person.

The genome can be modified at a preselected, predetermined, or predefined site in the genome (e.g., nuclear or chloroplast genome), i.e., a particular nucleotide sequence, at which one or more nucleotides are desired to be inserted, substituted, and/or deleted. For example, the preselected, predetermined, or predefined site can be an endogenous locus or a particular nucleotide sequence in or linked to a previously introduced exogenous DNA, RNA, or transgene. The preselected site may be a specific nucleotide position at (after) which one or more nucleotides are intended to be inserted. The preselected site may also include a sequence of one or more nucleotides to be exchanged (replaced) or deleted.

In various embodiments, the length and percent sequence identity of the flanking regions are selected to enable homologous recombination between the flanking regions and their corresponding DNA regions upstream or downstream of the preselected site. The regions of DNA flanking the preselected site that have homology to the flanking DNA regions of the repair nucleic acid molecule are also referred to as regions of homology in the genomic DNA.

In order to have sufficient homology for recombination, the flanking DNA regions of the repair nucleic acid molecule may vary in length and should be at least about 10nt, about 15nt, about 20nt, about 25nt, about 30nt, about 40nt, or about 50nt in length. However, the flanking regions may actually be as long as possible (e.g., up to about 100-150kb, such as an intact Bacterial Artificial Chromosome (BAC)). Preferably, the flanking region is about 50nt to about 2000nt, for example about 100nt, 200nt, 500nt or 1000 nt. Furthermore, the regions flanking the DNA of interest need not be identical to the regions of homology (the regions of DNA flanking the preselected site) and may have from about 80% to about 100% sequence identity, preferably from about 95% to about 100% sequence identity with the regions of DNA flanking the preselected site. The longer the flanking region, the less stringent the requirement for homology. Furthermore, in order to achieve an exchange of the target DNA sequence at the preselected site without altering the DNA sequence of the adjacent DNA sequences, the flanking DNA sequences should preferably be identical to the upstream and downstream DNA regions flanking the preselected site.

To effect sequence modification at a preselected site, the flanking regions must be selected such that the 3 'end of the upstream flanking region and/or the 5' end of the downstream flanking region are aligned with the terminus of the predetermined site. Thus, the 3 'end of the upstream flanking region defines the 5' end of the predetermined site, while the 5 'end of the downstream flanking region defines the 3' end of the predetermined site.

The preselected site is located outside or remote from said cleavage (and/or recognition) site such that the site in which the genomic modification is to be performed (the preselected site) does not comprise a cleavage site and/or a recognition site for a DSBI/SSBI enzyme such that the preselected site does not overlap with the cleavage (and/or recognition) site. Thus, in this regard, externally/remotely means upstream or downstream of the cleavage (and/or recognition) site.

In various embodiments, at least one base editor according to the invention is temporarily or permanently linked to at least one site-specific DSBI/SSBI enzyme complex or at least one modified site-specific DSBI/SSBI enzyme complex, or optionally to a component of said at least one site-specific DSBI/SSBI enzyme complex. The linkage may be covalent and/or non-covalent. Any of the base editor or site specific DSBI/SSBI enzyme complexes, or catalytically active fragments thereof, or any component of the base editor complex or site specific DSBI/SSBI enzyme complex disclosed herein may be introduced into a cell as a nucleic acid fragment representing or encoding a DNA, RNA, or protein effector, or it may be introduced as a DNA, RNA, and/or protein, or any combination thereof.

A base editor is a protein or fragment thereof that has the ability to mediate targeted base modifications, i.e., to switch a base of interest to obtain a point mutation of interest. Preferably, in the context of the present invention, the at least one base editor is temporarily or permanently fused to at least one DSBI/SSBI enzyme, or optionally to at least one component of DSBI/SSBI. The fusion may be covalent and/or non-covalent. Various publications show targeted base switching, mainly from cytidine (C) to thymine (T), using CRISPR/Cas9 nickases or non-functional nucleases linked to the cytidine deaminase domain (apolipoprotein B mRNA editing catalytic-like polypeptide (APOBEC1), e.g. rat-derived APOBEC). Deamination of cytosine (C) is catalyzed by cytidine deaminase and produces uracil (U) which has the property of base pairing with thymine (T). Most known cytidine deaminases work on RNA, and a few examples of known DNA acceptance require single-stranded (ss) DNA. Studies of dCas 9-target DNA complexes show that at least 9 nucleotides (nt) in the displaced DNA strands are unpaired when forming the Cas 9-guide RNA-DNA "R-loop" complex (Jore et al, nat. struct. mol. biol., 18, 529. minus 536 (2011)). Indeed, in the structure of Cas 9R loop complex, the first 11 nucleotides of the pre-spacer sequence on the DNA strand that were replaced were disordered, indicating that their motion was not strictly restricted. It is also speculated that Cas9 nickase-induced mutation of cytosines in the non-template strand may be due to the accessibility of cellular cytosine deaminase. It is speculated that this subset of ssDNA in the R loop may serve as an efficient substrate for cytidine deaminase tethered to dCas9 to achieve a direct programmable conversion of C to U in DNA (Komor et al, supra). Recently, an Adenine Base Editor (ABE) mediating the conversion of A.T to G.C in genomic DNA has been described in Goudelli et al, Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage, Nature, 2017, 551(7681), 464.

Enzymes that achieve DNA methylation as well as histone modifying enzymes have been identified in the art. Histone post-translational modifications play an important role in regulating chromatin structure and gene expression. For example, enzymes for histone acetylation are described in Sterner d.e., Berger s.l. (June 2000): "esterification of histones and transformation-related factors", Microbiol. mol. biol. Rev.64 (2): 435-59. Enzymes that achieve protein methylation are described in Zhang y, Reinberg D (2001): "transformation regulation by histone methyl: interplay between differential compatibility modifications of the core histones, Genes Dev.15 (18): 2343-60. Histone ubiquitination is described in shilatiard a (2006): "chromatography modifications by methyl and ubiquitination: injections in the regulation of gene expression ", Annu.Rev.biochem.75: 243-69. Enzymes for histone phosphorylation are described in Nowak s.j., cores V.G (April 2004): "Phosphorylation of histone H3: a balancing act beta chromosome condensation and translational action ", Trends Genet.20 (4): 214-20. Enzymes for histone ubiquitination are described in Nathan d., Ingvarsdottir k., Sterner d.e., et al (April 2006): "Genes Dev.20 (8)" is a novel regulator in Saccharomyces cerevisiae and shows dynamic interactive with positive-acting tissues: 966-76. Enzymes for histone ribosylation are described in Hassa p.o., Haenni s.s., Elser m., hottier M.O (September 2006): "Nuclear ADP-ribosylation interactions in mammalian cells: where are the are wee today and where are going? ", microbiol. mol.biol. rev.70 (3): 789 and 829. Histone citrullination is catalyzed by an enzyme called peptidylarginine deaminase 4(PAD4, also known as PADI4), which converts histone arginine (Arg) and monomethyl arginine residues to citrulline.

Enzymes that effect DNA methylation and histone modifying enzymes can be fused to disarmed DSB or SSB inducing enzymes that preferably recognize a predetermined site in the genome of the cell.

Exemplary transgenes

In various embodiments of the methods of genetically modifying in a plant cell, the transgene may be a gene selected from the group consisting of: encoding a gene having resistance or tolerance to abiotic stress including drought stress, osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, nitrogen deficiency, phosphate deficiency, salt stress or water logging, herbicide resistance including resistance to glyphosate, glufosinate/glufosinate, hygromycin, protoporphyrinogen oxidase (PPO) inhibitor, ALS inhibitor and dicamba; genes encoding resistance or tolerance to biotic stress, including viral resistance genes, fungal resistance genes, bacterial resistance genes, insect resistance genes; or a gene encoding a yield-related trait comprising lodging resistance, flowering time, shattering resistance, seed color, endosperm composition or nutrient content.

In various embodiments of the methods of genetically modifying in a plant cell, the methods are effective to promote cell proliferation or cell regeneration, or to increase the efficiency of regeneration of a transgenic, gene-edited, or base-edited plant. The method is preferably effective after genetic/genomic modification. In various embodiments of the methods of genetically modifying in a plant cell, the methods are effective to induce direct or indirect (somatic) embryogenesis from a single cell (preferably an embryonic cell, a somatic cell, or a protoplast) or from a callus cell. The method is preferably effective after genetic/genomic modification. In various embodiments, the methods are effective to increase the efficiency of stable transformation of a transgene into a plant cell, or to increase the efficiency of production of a genetically modified plant. In various embodiments, the methods are effective to increase the efficiency of genome engineering components to edit the genome of a plant cell, or to increase the efficiency of production of transgenic, gene-edited, or base-edited plants.

In some embodiments, the method is effective to increase the efficiency of regeneration of plants derived from recalcitrant genotypes, to increase the efficiency of regeneration of plants from non-conventional tissue types, or preferably to accelerate the regeneration process after genetic/genomic modification.

Transient expression of RKD2 or RKD4 genes

Also provided are methods for transient expression of RKD2 or RKD4 genes in plant cells. The method comprises introducing into a plant cell (i) a nucleic acid, recombinant gene or DNA construct as described herein; and (ii) a transgene and/or genome engineering component.

In some embodiments, RKD2 and RKD4 are transiently co-expressed. Such co-expression is effective in promoting cell proliferation. Such co-expression is effective in promoting cell regeneration. Such co-expression can efficiently induce embryogenesis from a single cell, thereby providing the ability to regenerate a homogenous plant without selection. Co-expression can increase genome editing efficiency by co-delivery with genome editing components.

Transient co-delivery of RKD2 and RKD4 may be performed as described in U.S. provisional application No. 62/685,626, which is incorporated by reference herein in its entirety.

Transient expression may be performed by transient transformation/transfection of nucleic acid fragments encoding RKD2 or RKD4 proteins/polypeptides, preferably expressed under a chemically inducible promoter. Transient expression of nucleic acids encoding RKD2 polypeptide or nucleic acids encoding RKD4 polypeptide may also be achieved by stable transformation of RKD2 or RKD4 gene under the control of tissue and development specific promoters or inducible promoters. RKD2 or RKD4 genes may be expressed and then have transient activity. RKD2 or RKD4 genes can be turned off and degraded soon after a plant cell development change or induced condition removal. For example, dexamethasone inducible promoter pOp6(SEQ ID NO: 15) can be used to drive transient transformation for RKD2 or RKD4 genes.

Transient transfection, transient transformation, and stable transformation can all result in transient expression. "transient transformation" and "transient transfection" include the transfer of foreign material [ i.e., nucleic acid fragments, proteins, Ribonucleoproteins (RNPs), etc. ] into a host cell, resulting in gene expression and/or activity without integration and stable inheritance of the foreign material. The foreign component is not permanently incorporated into the cell genome, but provides a temporary effect leading to genome modification. Transient conversion events cannot be passed on to the next generation and are therefore unsustainable. "Stable transformation" refers to an event that integrates a transferred nucleic acid fragment into the host cell genome (including both the nucleus and the organelle genome) resulting in stable inheritance of the nucleic acid fragment.

For example, transient expression can be used for transient genome editing. Transient activity and/or transient presence of genome-engineered components in a plant cell can result in one or more of a double-strand break in the genome of the plant cell, a single-strand break or breaks in the genome of the plant cell, a base editing event or events in the genome of the plant cell, or DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ubiquitination, histone ribosylation, or histone citrullination in the genome of the plant cell. The modification produced in the genome of the plant cell may for example be selected from: a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide, a change in DNA methylation, a change in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone benzoylation, histone ribosylation, or histone citrullination, or any combination thereof.

In addition to direct or indirect chemical induction of a heterologous promoter operably linked to a polynucleotide sequence encoding RKD2 polypeptide or RKD4 polypeptide, transient expression may also be achieved by one or more site-directed transcriptional activators. Such site-directed transcriptional activators may be synthetic transcription factors described in U.S. provisional application nos. 62/609,508 and 62/758,068, both of which are incorporated herein by reference. The synthetic transcription factor can include at least one recognition domain and at least one gene expression regulatory domain, particularly an activation domain, wherein the synthetic transcription factor is configured to regulate expression of an endogenous gene in the genome of a plant or plant cell. Such an endogenous gene is preferably a (native) morphogenic gene encoding a polypeptide involved in plant development processes such as root formation or shoot formation. In some embodiments, the endogenous morphogenic gene is selected from endogenous nucleic acids encoding RKD4 polypeptide or endogenous nucleic acids encoding RKD2 polypeptide. In some embodiments, the at least one recognition domain is a molecule or fragment of a molecule selected from the group consisting of: at least one TAL effector, at least one de-armed CRISPR/nuclease system, at least one zinc finger domain, and at least one de-armed homing endonuclease, or any combination thereof.

In some embodiments, the at least one disarmed CRISPR/nuclease system is a CRISPR/dCas9 system, a CRISPR/dCpf1 system, a CRISPR/dCsm1 system, a CRISPR/dMAD7 system, a CRISPR/dCasX system, or a CRISPR/dCasY system, or any combination thereof, and wherein the at least one disarmed CRISPR/nuclease system comprises at least one guide RNA.

In some embodiments, the at least one activation domain is selected from an acidic transcriptional activation domain, preferably wherein the at least one activation domain is selected from a TAL effector gene of fusarium solani (Xanthomonas oryzae), VP16 or tetrameric VP64 from Herpes simplex (Herpes simplex), VPR, SAM, Scaffold, Suntag, P300, VP160, or any combination thereof. In some embodiments, the activation domain is VP 64.

In some embodiments, the synthetic transcription factor is configured to regulate expression, preferably transcription, of the morphogenic gene by binding to a regulatory region located at a distance relative to the initiation codon. In a preferred embodiment, the synthetic transcription factor is configured to increase expression, preferably transcription, of the morphogenic gene by binding to a regulatory region located at a distance relative to the initiation codon.

In some embodiments, the site-directed transcriptional activator/synthetic transcription factor or nucleic acid encoding same comprises at least one recognition domain and at least one activation domain, wherein said site-directed transcriptional activator is configured to increase expression of endogenous RKD4 polypeptide or endogenous RKD2 polypeptide, preferably by binding to a regulatory region located at a distance relative to the initiation codon of said endogenous RKD4 polypeptide or endogenous RKD2 polypeptide.

As used herein, a "regulatory region" refers to the binding site of at least one recognition domain to a target sequence in the genome at or near the morphogenic gene. According to the nature of the at least one activation domain and the at least one recognition domain further disclosed herein, there may be two discrete regulatory regions, or there may be overlapping regulatory regions, and the different domains of the synthetic transcription factor may be assembled in a modular fashion.

In certain embodiments, the at least one recognition domain may target at least one sequence (recognition site) relative to the start codon of the gene of interest that is located at least 1000bp, -700bp to +700bp, -550bp to +500bp, or-550 bp to +425bp upstream (-) or downstream (+) relative to the start codon of the gene of interest. In certain embodiments, it may be preferable to recognize a recognition domain near the promoter, and the high extent of targeting of synthetic transcription factors compared to traditional or naturally occurring transcription factors represents an advantage of specific synthetic transcription factors. As the recognition and/or activation domains can be specifically designed and constructed to specifically recognize and target modulated hot spots.

In certain embodiments, at least one recognition site may be located at-169 bp to-4 bp, -101bp to-48 bp, -104bp to-42 bp, or-175 bp to +450bp (upstream (-) or downstream (+) respectively) relative to the start codon of the gene of interest to provide an optimal spatial binding environment that allows for optimal regulation, preferably transcriptional activation activity. In particular for synthetic CRISPR-based transcription factors that function with guide RNAs as recognition moieties, the binding site may also be located within the coding region of the gene of interest (downstream of the start codon of the gene of interest).

In additional embodiments, the recognition domain of the synthetic transcription factor can be bound to the 5 'and/or 3' untranslated region (UTR) of the gene of interest. In embodiments employing different recognition domains, at least two recognition domains may bind to different target regions of the morphogenic gene, including 5 'and/or 3' UTRs, but they may also bind outside the region of the gene, but still remain at a distance of at most 1 to 1500bp therefrom. A preferred region to which the recognition domain can bind is located about-4 bp to about-300 bp, preferably about-40 bp to about-170 p, upstream of the start codon of the morphogenic gene of interest. Furthermore, in this way, the length of the recognition domain, and thus the length of the corresponding recognition site in the genome of interest, can vary depending on the nature of the synthetic transcription factor and the recognition domain employed. This will also determine the length of the corresponding at least one recognition site based on the molecular characteristics of the at least one recognition domain. For example, where a single zinc finger can be from about 8bp to about 20bp, with an array of 3 to 6 zinc finger motifs being preferred, a single TALE recognition site can be from about 11bp to about 30bp, or more. The recognition site of the gRNA of the CRISPR-based synthetic transcription factor includes the targeting or "spacer" sequence of the gRNA that hybridizes to the genomic region of interest, while the gRNA includes other domains, including domains that interact with disarmed CRISPR effectors. The recognition site for synthetic transcription factors based on the disarmed CRISPR effectors will include the PAM motif, since the PAM sequence is necessary for targeted binding to any CRISPR effector, and the exact sequence depends on the kind of CRISPR effector, i.e. the disarmed CRISPR effector.

Introduction of RKD2 or RKD4 genes

Nucleic acids encoding RKD2 or RKD4 and/or genome engineering components may be introduced as DNA, such as plasmid DNA, RNA, mRNA or RNP.

RKD2 or RKD4 genes can be co-delivered with one or more genome engineering components. As used herein, "co-delivery" or "co-delivery" and "co-introduction" or "co-introduction" are used interchangeably. For the purposes of the present invention, "co-introduction" refers to the process of delivering at least two different components simultaneously into the same plant cell. Thus, the genome engineering components are introduced into the same plant cell together with RKD2 and/or RKD 4. Co-introduction into plant cells can be by particle bombardment, microinjection, agrobacterium-mediated transformation, electroporation, electrofusion, agrobacterium infiltration, or vacuum infiltration.

It is believed that the transformed cells are not as regenerative as the wild-type cells. Transformed cells are susceptible to programmed cell death due to the presence of foreign DNA within the cell. Cell death may also be triggered by stress caused by delivery (e.g., ballistic injury). Thus, promotion of cell division is essential to modify the regeneration of cells. Furthermore, the efficiency of genome engineering is largely governed by the state of the host cell. Cells that undergo rapid cell division, such as those in plant meristems, are the most suitable receptors for genome engineering. Promoting cell division is likely to increase the integration or modification of DNA during DNA replication and division, thereby increasing the efficiency of genome engineering.

When RKD2 or RKD4 polypeptide is expressed in a plant cell with a transgene, the RKD2 or RKD4 polypeptide may increase expression of the transgene and the polypeptide encoded by the transgene. When RKD2 or RKD4 polypeptide is expressed in plant cells with a genome engineered component and a transgene, the activity of the genome engineered component may be increased. This increase may result in more efficient integration of the transgene into the genome of the plant cell.

The RKD4 polypeptide coding sequence may be from any number of plants known in the art. Such plants include, but are not limited to, maize, arabidopsis, and common wheat. In some embodiments, the RKD4 polypeptide coding sequence is from RKD4 of Triticum aestivum. In some embodiments, the RKD4 polypeptide coding sequence is from RKD4 of Arabidopsis thaliana. In some embodiments, the RKD4 polypeptide coding sequence is from RKD4 in maize. In some embodiments, the RKD2 polypeptide coding sequence is from RKD2 of Triticum aestivum. In some embodiments, the RKD2 polypeptide coding sequence is from RKD2 of Arabidopsis thaliana. In some embodiments, the RKD2 polypeptide coding sequence is from RKD2 in maize.

For the purposes of the present invention, "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences having the same residue (× 100) divided by the number of positions compared. Gaps, i.e., positions in an alignment where a residue is present in one sequence but not in another, are considered to be positions having non-identical residues. Alignment of the two sequences was performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970). The above computer-assisted sequence alignment can be conveniently performed using standard Software programs, such as the program NEEDLE implemented in European Molecular Biology Open Software Suite (EMBOSS), e.g., version 6.3.1.2 (Trends in Genetics 16(6), 276(2000)), with default parameters: for example, EBLOSUM62, gapopen 10.0 and gapextend 0.5 are protein matrices.

As used herein, the term "hybridize" refers to the formation of a hybrid between two nucleic acid molecules through base pairing of complementary nucleotides. The term "hybridizes under stringent conditions" refers to hybridization under specific conditions. One example of such conditions includes conditions under which a substantially complementary strand, i.e., a strand consisting of a nucleotide sequence having at least 80% complementarity, hybridizes to a specified strand, while a less complementary strand does not hybridize. Alternatively, such conditions refer to specific hybridization conditions of sodium salt concentration, temperature and wash conditions. As an example, highly stringent conditions include incubation in 42 ℃, 50% formamide, 5 XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate, 5 XDenhardt's solution, 10 XSC dextran sulfate, 20mg/ml sheared salmon sperm DNA, and washing in 0.2 XSSC at about 65 ℃ (SSC stands for 0.15M sodium chloride and 0.015M trisodium citrate buffer). Alternatively, highly stringent conditions may mean hybridization at 68 ℃ in 0.25M sodium phosphate, pH 7.2, 7% SDDS, 1mM EDTA and 1% BSA for 16 hours, and washing 2 times with 2 XSSC and 0.1% SDDS at 68 ℃. In addition, highly stringent hybridization conditions are, for example: hybridization was performed in 4 XSSC at 65 ℃ followed by multiple washes in 0.1 XSSC at 65 ℃ for about 1 hour total, or in 0.25M sodium phosphate, pH 7.2, 7% SDS, 1mM EDTA and 1% BSA at 68 ℃ for 16 hours followed by two washes in 2 XSSC and 0.1% SDS at 68 ℃.

Chemical substances for regulating epigenetics

Epigenetic modulating chemicals, such as protein deacetylase inhibitors (ii.1), can be co-introduced with the genome engineering components. Exemplary epigenetic regulatory chemicals for use in accordance with the present invention include, but are not limited to, histone deacetylase inhibitors (HDACi), such as trichostatin a (TSA) and DNA methyltransferase inhibitors.

It is generally believed that the co-delivered epigenetic modulating chemical (ii.1) (specifically HDACi) relaxes plant chromatin structure, facilitating DNA accessibility to genome engineering components in the bombarded cells, and thus, efficiency of genome engineering (i.e. transformation and genome editing). Without wishing to be bound by theory, the basic structural and functional unit of genetic material is the nucleosome, in which negatively charged DNA is wrapped around positively charged histone octamers and related connective histones. Nucleosome units further fold and assemble into chromatin (Andrews, a.j., and Luger, K. (2011). Nucleosome structures(s) and viability: Variations on a the same. annu. rev. biophysis.40: 99-117.). The accessibility of DNA depends to a large extent on the compactness of nucleosomes and chromatin. Chromatin remodeling enzymes dynamically modify lysine or other amino acids in histones, resulting in changes in their charge and interactions with DNA and other proteins, and thus chromatin folding or unfolding (banister a.j., Kouzarides T. (2011) Regulation of chromosome by tissue modifications. cell Res 21: 381-95.). Acetylation and deacetylation of lysine residues in histones by addition or removal of acetyl groups is often involved in the reversible regulation of eukaryotic chromatin structure and mediates chromatin accessibility and regulation of gene expression. Histone Deacetylases (HDACs) are enzymes that remove acetyl groups from lysine residues on the N-terminal tail of histones to make the histones more positively charged, thereby allowing the histones to more tightly wrap DNA. Inhibiting HDACs may help with chromatin spreading, making DNA more accessible.

Chromatin remodeling and other epigenetic modifications undoubtedly play An important role in the regulation of cell totipotency and regeneration (Zhang, h., and Ogas, J. (2009). Inhibition of Histone Deacetylase (HDAC) activity has been shown to be associated with Plant regeneration and microspore embryogenesis (Miguel, C., and Marum, L., 2011.An epigenetic view of Plant cells in view: somaclonal variation and development. J. Exp. bot.62:3713-3725. Li Hui et al (2014) The tissue Deacetylase Inhibitor A proteins in The spore genome Plant Cell, 26: 195-209.). Inhibition of HDAC activity or downstream HDAC-mediated pathways play a major role in triggering stress-induced haploid embryogenesis. One such HDACi is trichostatin a (tsa). Studies have shown that TSA induces proliferation of large numbers of embryonic cells in male gametophytes of brassica napus (b. TSA treatment results in high frequency of sporozoite cell division in cultured microspores and pollen.

In the presence of one or more epigenetic regulatory chemicals (e.g., protein deacetylase inhibitors, especially HDACi), various approaches can be used to further improve genome engineering efficiency. Such HDACi can be trichostatin a (tsa), N-hydroxy-7- (4-dimethylaminobenzoyl) -aminoheptanoamide (M344), suberoylanilide hydroxamic acid (SAHA), and the like. These HDACis are selected from hydroxamic acid based chemicals directed against zinc dependent HDACs.

Plant hormones

In various embodiments, one or more plant hormones, such as auxins and cytokinins, such as 2,4-D, 6-benzylaminopurine (6-BA) and Zeatin (Zeatin), are co-delivered with one or more of the nucleic acids encoding RKD2 or RKD4, the genome engineered components, and the transgene.

Plant somatic cells are capable of restoring cell division and regenerating to intact plants in ex vivo culture through somatic embryogenesis or organogenesis, which is largely dependent on plant hormones such as auxins and cytokinins.

One of the auxins is 2, 4-dichlorophenoxyacetic acid (2,4-D), which is almost indispensable for somatic embryogenesis and cell regeneration of monocotyledons such as maize and wheat. Also, cytokinins such as 6-benzylaminopurine (6-BA) or Zeatin are essential for plant organogenesis and shoot meristem initiation and development. Methods of increasing the efficiency of genome engineering may include co-delivery of one or more plant hormones (2,4-D, 6-BA, Zeatin, etc.) with genome engineering components.

The genome engineered component and at least one epigenetic regulatory chemical and phytohormone may be co-introduced into a plant cell.

As used herein, "co-delivery" or "co-delivery" and "co-introduction" or "co-introduction" are used interchangeably. For the purposes of the present invention, "co-introduction" refers to the process of delivering at least two different components simultaneously into the same plant cell. Thus, the genome-engineered component and at least one of the epigenetic regulatory chemical and the plant hormone can be introduced together into the same plant cell.

The co-introduction into the plant cell may be by particle bombardment, microinjection, agrobacterium-mediated transformation, electroporation, agrobacterium infiltration, or vacuum infiltration. Methods based on physical delivery, such as particle bombardment, microinjection, electroporation, nanoparticles and Cell Penetrating Peptides (CPP), are particularly preferred for the co-introduction of nucleic acids encoding RKD2 or RKD4, genome engineered components and/or transgenes according to the invention. Particularly preferred is co-introduction by particle bombardment.

Regeneration of plant cells into whole plants

According to another aspect of the invention, the genetically modified plant cell can be regenerated into a whole (fertile) plant. Thus, in a preferred aspect of the invention, the step of regenerating the plant is performed after genetic modification of the plant cell. Accordingly, the present invention provides a method for producing a genetically modified plant comprising the steps of:

a) genetically modifying a plant cell according to any of the methods described above for genetically modifying a plant cell, and

b) regenerating a plant from the modified plant cell of step a).

The single or multiple cells proliferate and develop into tissues, organs, and eventually into whole plants. In some embodiments, the plant produced does not comprise any genome engineering component, nucleic acid encoding RKD2 or RKD4 introduced or co-introduced in step a). Step b) of regenerating a plant may, for example, comprise culturing the genetically modified plant cell from step a) on a regeneration medium.

By introducing into a plant cell any of the nucleic acids, recombinant genes and DNA constructs encoding RKD2 or RKD4 described herein, the efficiency of plant regeneration or the efficiency of the ability of a plant cell to regenerate may be increased.

Production of genetically modified plants

The present invention also provides genetically modified plants obtained or obtainable by the above-described methods for producing genetically modified plants or progeny plants thereof. The genetically modified plant may comprise any of the genetically modified plant cells described herein.

In various embodiments, the plant produced does not comprise any genome engineering components or nucleic acids encoding RKD2 or RKD4 introduced or co-introduced into the plant cell used to produce it.

The invention also provides plants or seeds derived from the above-described genetically modified cells that have not been routinely selected. As used herein, "conventional selection" refers to any process of selecting and purifying transformed cells from wild-type cells by using an integrated selectable marker, such as an antibiotic (e.g., kanamycin, hygromycin) or herbicide (e.g., glufosinate, glyphosate) resistance gene. Without conventional selection, such plants or seeds may not have integrated any genome-engineered components, thus resulting in genetically modified plants that do not contain a transgene.

The genetic modification may be a permanent, heritable change in the genome of the plant cell. Plant tissue culture and genome engineering can be performed using currently available methods, including microprojectile bombardment, agrobacterium transformation, electroporation, and the like. Transformation and transgene expression can be monitored by using a visible reporter gene, for example, the red fluorescent tdomoto gene (tDT), which encodes an exceptionally bright red fluorescent protein with a maximum excitation at 554nm and a maximum emission at 581 nm. Genome editing efficiency can be analyzed by Next Generation Sequencing (NGS), qPCR, labeled capillary electrophoresis analysis, and Droplet Digital PCR, among other methods. Sanger sequencing further confirmed site-specific modifications.

Step of culturing

Plant cells into which nucleic acid encoding RKD2 or RKD4, genome engineering components and/or a transgene are introduced or co-introduced may be cultured under conditions that allow genetic modification of the plant cell genome by activity of the genome engineering components in the presence of one or more nucleic acids encoding RKD2 or RKD4 and one or more transgenes.

As used herein, "genetic modification of a genome" includes any type of manipulation such that an endogenous nucleotide is altered to include a mutation, e.g., a deletion, an insertion, a transition, a transversion, or a combination thereof. For example, the endogenous coding region may be deleted. Such mutations may result in polypeptides having amino acid sequences that differ from the amino acid sequences encoded by the endogenous polynucleotides. Another example of a genetic modification is an alteration in a regulatory sequence, such as a promoter, resulting in increased or decreased expression of an operably linked endogenous coding region.

Conditions that are "suitable" for genetic modification of the plant genome (e.g., cleavage of a polynucleotide), or that do not prevent such an event from occurring. Thus, these conditions allow, enhance, facilitate, and/or favor the event. These conditions may differ depending on the respective genome engineering composition (i).

In the method of the present invention, it is preferred to transiently transform a plant cell with the genome-engineered component (i) and at least one compound (ii). As used herein, "transient transformation" refers to the transfer of a foreign substance [ i.e., a nucleic acid fragment, a protein, a Ribonucleoprotein (RNP), etc. ] into a host cell, resulting in gene expression and/or activity without integration and stabilization of the genetic foreign substance. Thus, the genome-engineered component (i) is transiently active and/or transiently present in the plant cell. The genome-engineered components are not permanently incorporated into the cell genome, but rather provide a temporary effect leading to genome modification. For example, the transient activity and/or the transient presence of a genome-engineered component in a plant cell can result in one or more of a double-strand break in the genome of the plant cell, a single-strand break or breaks in the genome of the plant cell, a base editing event or events in the genome of the plant cell, or DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ubiquitination, histone ribosylation, or histone citrullination in the genome of the plant cell.

After introduction of the one or more double-stranded breaks or the one or more single-stranded breaks, non-homologous end joining (NHEJ) and/or homologous directed repair of the breaks by homologous recombination mechanisms (HDR) is performed.

The modification produced in the genome of the plant cell may for example be selected from: insertion of a transgene, preferably an expression cassette comprising a transgene of interest, substitution of at least one nucleotide, deletion of at least one nucleotide, insertion of at least one nucleotide, change in DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone benzoylation, histone ribosylation, or change in histone citrullination, or any combination thereof. According to a particularly preferred aspect of the invention, no foreign genetic material associated with the gene editing machine/system used is stably integrated into the genome of the plant cell.

The genetic modification may be a permanent, heritable change in the genome of the plant cell.

The subject of the present invention is also the plant cells obtained or obtainable by the above-described method. Thus, one embodiment of the present invention is a genetically modified plant cell obtained or obtainable by the above-described method for performing a genetic modification in a plant cell. Genetic modifications in these plant cells compared to the original plant cells may include, for example: insertion of a transgene, preferably an expression cassette comprising a transgene of interest, substitution of at least one nucleotide, deletion of at least one nucleotide, insertion of at least one nucleotide, change in DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone benzoylation, histone ribosylation, or change in histone citrullination, or any combination thereof. Preferably, the genetically modified plant cell does not comprise any foreign genetic material stably integrated into the genome of the plant cell.

a) genetically modifying a plant cell according to the above-described method for genetically modifying a plant cell, and

b) regenerating a plant from the modified plant cell of step a).

Step b) of regenerating a plant may, for example, comprise culturing the genetically modified plant cell from step a) on a regeneration medium.

Regeneration techniques rely on the manipulation of certain plant hormones in tissue culture growth media, sometimes relying on biocides and/or herbicide markers that can be introduced. Regeneration may be obtained from plant somatic cells, callus cells or embryonic cells, and protoplasts from various explants, such as callus, immature or mature embryos, leaves, shoots, roots, flowers, microspores, embryonic tissue, meristematic tissue, organs, or any part thereof. This regeneration technique is generally described in Klee (1987) Ann. Rev. of Plant Phys.38: 467-486. Plant regeneration from cultured Protoplasts is described in Evans et al, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp.124-176, Macmillan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp.21-73, CRC Press, Boca Raton, 1985. To obtain a complete plant from transformed or genetically edited cells, the cells can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once the whole plant has produced and produced seeds, evaluation of progeny begins.

The present invention also provides genetically modified plants obtained or obtainable by the above-described methods for producing genetically modified plants or progeny plants thereof.

Another subject matter of the invention is plant cells or seeds derived from the above-described genetically modified plants.

Yet another subject of the invention is a plant, plant cell or seed derived from the above-described genetically modified cell without selection based on a marker gene. As used herein, "marker gene-based selection" refers to any process of selecting, identifying and/or purifying modified cells, particularly transformed, gene-edited or base-edited cells, from wild-type cells by using an integrated selection marker (gene), such as an antibiotic resistance gene (e.g., kanamycin resistance gene, hygromycin resistance gene) or a herbicide resistance gene (e.g., glufosinate resistance gene, glyphosate resistance gene). If such selection is not made, the plant, plant cell or seed may not have incorporated any genome-engineered components, and thus may produce (i) a genetically modified plant that does not contain the transgene or (ii) a modified plant that has incorporated only the transgene of interest.

Unless otherwise indicated in the examples, all recombinant DNA techniques were performed according to standard protocols, such as Sambrook et al (1989) Molecular Cloning: a Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and Ausubel et al (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Vol 1 and Vol 2. Standard materials and methods for Plant Molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D.Cray, published in conjunction with BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Other references to standard Molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: a Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; volume I and volume II of Brown (1998) Molecular Biology Labfax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reaction are available in Dieffenbach and Dveksler (1995) PCR Primer: a Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al (2000) PCR-bases: from Background to Bench, First Edition, Springer Verlag, Germany.

Sequence of

All patents, patent applications, and publications or publications (including publications on the internet) mentioned or cited herein are hereby incorporated by reference in their entirety.

Examples

The invention is further illustrated by the following examples. However, it is to be understood that the invention is not limited to such examples. The use of these and other examples anywhere in the specification is illustrative only and does not limit the scope and meaning of the invention or any exemplary terms in any way. Likewise, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention will be apparent to those skilled in the art upon reading the present specification, and such variations may be made without departing from the spirit or scope of the invention. Accordingly, the invention is to be defined only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Example 1 preparation of pZY ZZ-TOP, plasmid containing bidirectional promoter pOp6 operably linked to developmental Gene and GUS reporter Gene and dexamethasone inducible transcription factor activating pOp6

A plasmid (pZY ZZ-TOP) was generated with a Chemical (dexamethasone, Dex) inducible promoter system (Zuo, J., & Chua, N.H. (2000). Chemical-induced systems for regulated expression of plant genes. Current Opinion in Biotechnology, 11(2), 146- & 151.). The map of the plasmid is shown in FIG. 1. The plasmid contained the pOp6 promoter (SEQ ID NO: 15). pOp6 promoter was bidirectional and both developmental genes and GUS genes were expressed following exposure to dexamethasone (Sigma-Aldrich. product # D4902-500MG) (FIG. 1). The plasmid also contains the gene LhGR (DNA (SEQ ID NO: 16 and amino acids (SEQ ID NO: 17)). LhGR is constitutively expressed (e.g., pUbi1 promoter + intron). LhGR is a transcription factor that enters the nucleus only in the presence of dexamethasone.

Expression systems are used for stable transformation. GUS staining was used to identify leaky expression without chemical induction. In all subsequent examples, only plants that do not express the GUS gene without chemical induction were considered. Plants expressing GUS without chemical induction were not used.

Example 2 evaluation of transcription factors for the ability to induce plant regeneration by somatic embryogenesis

In another example, the efficiency of five different transcription factors in inducing plant regeneration via somatic embryogenesis was tested. The coding sequences for AtLEC2(SEQ ID NO: 6), AtWUS (SEQ ID NO: 8), AtBBM (SEQ ID NO: 10), AtAGL15(SEQ ID NO: 12) and AtRKD4(SEQ ID NO: 1) were cloned into the inducible promoter system pZY ZZ-TOP of example 1. Dexamethasone induced the expression of each of AtLEC2, AtWUS, AtBBM, AtAGL15, and AtRKD 4.

These dexamethasone-inducible binary constructs based on the pOp6/LhGR transactivation system were stably transformed into Arabidopsis thaliana Col-0 by the floral method (Craft et al (2005). New pOp/LhG4 vectors for expressing the heterologous glucose-induced expression in Arabidopsis thaliana Plant Journal, 41(6), 899-918.). Arabidopsis thaliana Col-0 was grown in plates containing 50% MS medium supplemented with 1% sucrose under controlled environmental conditions (16 hours light/8 hours dark, 22 ℃). The leaky expression of 40 independent transgenic lines was detected by GUS staining.

Non-leaky transformants were induced 5-7 days after germination. Chemical induction of gene expression was performed by culturing on plates containing 20 μ M dexamethasone for 7 days, and then the plants were transferred to inducer-free medium. Frequency of somatic embryos was determined by microscopic imaging after staining with Sudan Red 7B.

The results are shown in fig. 2. RKD4 has the highest efficiency of embryonic structure formation after exposure to dexamethasone.

Example 3 RKD4 expressed in the pOp6/LHGR transactivation System has the ability to promote dexamethasone-inducible somatic embryogenesis

The function of the inducible promoter system controlling RKD4 was tested in Arabidopsis thaliana. Arabidopsis seeds germinate until primary roots are formed. The plantlets were then transferred to plates containing Dex (as described in example 2). The plantlets were imaged and the data is shown in figure 3. Somatic embryo formation was observed in roots (fig. 3, left panel), leaves (fig. 3, middle panel), or both (fig. 3, right panel). Loss of chlorophyll was also observed. After transfer of the somatic embryo structures to the DEX-free medium, the somatic embryo structures started to turn green again. These data indicate that RKD4 expression is capable of inducing somatic embryogenesis in the absence of hormones.

Example 4 expression of AtRKD4 in the pOp6/LhGR transactivation System induces somatic embryo formation in horticulture-related species

The use of the Agrobacterium tumefaciens GV3101 and dexamethasone induced the transformation of the horticulturally relevant species of Phalaenopsis with the binary construct AtRKD4, as described in example 1. The bases of the leaves of stably transformed plants were incubated for two days in plates containing 50% MS medium with 20 μ M dexamethasone and then transferred to medium without dexamethasone. The presence of somatic embryos was determined at 0, 7 and 14 days post-transplantation and the data is shown in figure 4.

Example 5 expression of AtRKD4 in the pOp6/LhGR transactivation System induces somatic embryo formation in horticulturally relevant species of Triticum aestivum

The effect of induced RKD4 expression in agriculturally relevant species of Triticum aestivum was determined. Wheat immature embryos were transformed with Agrobacterium EHA105 carrying a binary construct of estradiol inducible TarKD2(SEQ ID NO: 3) (Valdivia et al, 2013). Transformed embryos were isolated from seeds 12 days after pollination and cultured in 50% MS medium containing 30. mu.M β -estradiol for 7 days. Induction of embryogenic structures was observed in the medium (callus formation; FIG. 5, left panel). The embryo structures were transferred to inducer-free medium to induce somatic embryos. A green change was observed (fig. 5, middle panel) followed by new leaves (fig. 5, right panel). The examples demonstrate the effect of RKD4 expression in agriculturally relevant species.

Example 6 expression of TaRKD2 in barley immature embryos induces somatic embryo formation

In yet another example, barley immature embryos were transformed with Agrobacterium EHA105 carrying a binary construct of estradiol inducible TarKD2(SEQ ID NO: 3) (Valdivia et al, 2013). Transformed barley immature embryos were isolated from seeds, grown for 7 days in 50% MS medium containing 30 μ M β -estradiol (FIG. 6, top left), and then transferred to inducer-free medium to induce somatic embryos. Shoot and root development was observed after removal of the embryogenic structure from the induction medium (FIG. 6, top right panel). Subsequently, the structures were moved under light and the tissue started to turn green (fig. 6, bottom left). Seedlings could form about 60 days after the initial immature embryo was isolated (FIG. 6, bottom right panel).

Example 7 Co-expression of RKD2 and RKD4 in maize

The benefits of RKD4 (from Arabidopsis) and RKD2 (from wheat (SEQ ID NO: 4)) expression could be demonstrated in maize calli. Both sequences were cloned into the construct described in example 1 and co-bombarded with the red fluorescent tdTomato gene (DNA: SEQ ID NO: 18; amino acid: SEQ ID NO: 19) in the presence of a constitutive promoter, such as the double 35S promoter (SEQ ID NO: 21) and the ubiquitin intron (SEQ ID NO: 20). Following exposure to dexamethasone, induction of embryogenesis was measured based on the formation of massive callus structures and stable tdTomato integration. Callus structures showing red fluorescence were observed after 33 days (FIG. 7, left panel AtRKD4, right panel TaRKD 2). The structures are from a single cell impacted by both constructs, resulting in embryogenesis and expression of a red fluorescent marker.

***

The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. It is also to be understood that all values are approximate and are provided for the purpose of description.

Throughout this application, patents, patent applications, publications, product descriptions, and protocols are referenced, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

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gagaggtatt gcgccattat ggaggagaac ccggctatag agctcaccga gcaaacgaag 960

aagctcaggc aggcttgttt caaagagaat tataagcggc gtagagccgc cgcttctgtt 1020

aatcttctcg atcactgcta taacgatctg gcatctcatg agcagcaaat gcctctccca 1080

caaatgggat tctttggatt ttag 1104

<210> 4

<211> 1104

<212> DNA

<213> Artificial Sequence

<220>

<223> cDNA of TaRKD2 genotype 2 encoding SEQ ID NO: 5

<400> 4

atggagatgc aacagcaata cttcggcggt gatggcgatg ctgactggtt ccaccagctc 60

gccttgctcc cgcctttgcc gatctcttcg tctctgccgc ctctccccat gagcgagggc 120

agctgcttac ccatggccgc cgccgccgcg gcggcgcttc ctcttgggga ttgctcatca 180

gctctcatga ttaggccgga agaacagatg agctgcctgc cgatgaaccc ttcgccagct 240

gttgtcgatg atgtgtacag cagctacgcc cccaacaatg tcgacgtcct cccgccgttt 300

cctgcaggtc tcgacgacgc gctgctcatg gagtccttca gcgatatcga cctggaggag 360

ttcgccgacg ccttcggcca caagattaag accgagcctc tcgacgacgc tatggtgccg 420

gcggatcacg atttcgcggc gcaagcgcaa caggcgtgcc cagtggtgat catgaaccag 480

cagcagctga atgcaccacg cgacgtgcgc ctgctcatag atcccgacga cgatgactca 540

actgtcgtcg ccgggggcta tgaggctgcg gccgttgggt gcgctgagca gaagcaggtg 600

aggccggcgc cacgtcgtgt gcgcaagagc agcggtgggg cacgcccagc cgccggtggg 660

aaaagcctcg atcacatagg gtttgaggag ctacgtacgt atttctacat gcctatcacc 720

aaggcggcgc gggagatgaa cgttggtctc accgtgctca agaagcgctg ccgagagctc 780

ggggtcgccc gctggcctca ccggaagatg aagagcctca ggtcactcat cctcaacatc 840

caggagatgg ggaagggcgc aacgtcgccg gcggctgtgc aaggggaact agaggcgctt 900

gagaggtatt gcgccataat ggaggagaac ccggcgatcg agctgactga gcagaccaag 960

aagctgcggc aggcctgctt taaggagaac tacaagagga ggagagcggc ggcctccgtc 1020

aacttgctcg accattgcta caacgacttg gccagtcatg agcagcagat gccattgcca 1080

cagatgggtt tctttgggtt ctaa 1104

<210> 5

<211> 367

<212> PRT

<213> Triticum aestivum

<400> 5

Met Glu Met Gln Gln Gln Tyr Phe Gly Gly Asp Gly Asp Ala Asp Trp

1 5 10 15

Phe His Gln Leu Ala Leu Leu Pro Pro Leu Pro Ile Ser Ser Ser Leu

20 25 30

Pro Pro Leu Pro Met Ser Glu Gly Ser Cys Leu Pro Met Ala Ala Ala

35 40 45

Ala Ala Ala Ala Leu Pro Leu Gly Asp Cys Ser Ser Ala Leu Met Ile

50 55 60

Arg Pro Glu Glu Gln Met Ser Cys Leu Pro Met Asn Pro Ser Pro Ala

65 70 75 80

Val Val Asp Asp Val Tyr Ser Ser Tyr Ala Pro Asn Asn Val Asp Val

85 90 95

Leu Pro Pro Phe Pro Ala Gly Leu Asp Asp Ala Leu Leu Met Glu Ser

100 105 110

Phe Ser Asp Ile Asp Leu Glu Glu Phe Ala Asp Ala Phe Gly His Lys

115 120 125

Ile Lys Thr Glu Pro Leu Asp Asp Ala Met Val Pro Ala Asp His Asp

130 135 140

Phe Ala Ala Gln Ala Gln Gln Ala Cys Pro Val Val Ile Met Asn Gln

145 150 155 160

Gln Gln Leu Asn Ala Pro Arg Asp Val Arg Leu Leu Ile Asp Pro Asp

165 170 175

Asp Asp Asp Ser Thr Val Val Ala Gly Gly Tyr Glu Ala Ala Ala Val

180 185 190

Gly Cys Ala Glu Gln Lys Gln Val Arg Pro Ala Pro Arg Arg Val Arg

195 200 205

Lys Ser Ser Gly Gly Ala Arg Pro Ala Ala Gly Gly Lys Ser Leu Asp

210 215 220

His Ile Gly Phe Glu Glu Leu Arg Thr Tyr Phe Tyr Met Pro Ile Thr

225 230 235 240

Lys Ala Ala Arg Glu Met Asn Val Gly Leu Thr Val Leu Lys Lys Arg

245 250 255

Cys Arg Glu Leu Gly Val Ala Arg Trp Pro His Arg Lys Met Lys Ser

260 265 270

Leu Arg Ser Leu Ile Leu Asn Ile Gln Glu Met Gly Lys Gly Ala Thr

275 280 285

Ser Pro Ala Ala Val Gln Gly Glu Leu Glu Ala Leu Glu Arg Tyr Cys

290 295 300

Ala Ile Met Glu Glu Asn Pro Ala Ile Glu Leu Thr Glu Gln Thr Lys

305 310 315 320

Lys Leu Arg Gln Ala Cys Phe Lys Glu Asn Tyr Lys Arg Arg Arg Ala

325 330 335

Ala Ala Ser Val Asn Leu Leu Asp His Cys Tyr Asn Asp Leu Ala Ser

340 345 350

His Glu Gln Gln Met Pro Leu Pro Gln Met Gly Phe Phe Gly Phe

355 360 365

<210> 6

<211> 1092

<212> DNA

<213> Artificial Sequence

<220>

<223> cDNA of AtLEC2 encoding SEQ ID NO: 7

<400> 6

atggataact tcttaccctt tccctcttct aacgcaaact ctgtccaaga actctctatg 60

gatcctaaca acaatcgctc gcacttcaca acagtcccta cttatgatca tcatcaggct 120

cagcctcatc acttcttgcc tccgttttca tacccggtgg agcagatggc ggcggtgatg 180

aatcctcagc cggtttactt atcggagtgt tatcctcaga tcccggttac gcaaaccgga 240

agtgaattcg gttctctggt tggtaatcct tgtttgtggc aagagagagg tggttttctt 300

gatccgcgta tgacgaagat ggcaaggatc aacaggaaaa acgccatgat gagatcaaga 360

aacaactcta gccctaattc tagtccaagt gagttggttg attcaaagag acagctgatg 420

atgcttaact tgaaaaataa cgtgcagatc tccgacaaga aagatagcta ccaacagtcc 480

acatttgata acaagaagct tagggttttg tgtgagaagg aattgaagaa cagcgatgtt 540

gggtcactcg ggaggatagt tctaccaaag agagatgcag aagcaaatct tccgaagcta 600

tctgataaag aaggaatcgt tgtacagatg agagatgttt tctctatgca gtcttggtct 660

ttcaaataca agttttggtc caataacaag agcagaatgt atgtcctcga gaacacagga 720

gaatttgtga agcaaaatgg agctgagata ggagactttt taacaatata cgaggacgaa 780

agcaagaatc tctacttcgc catgaatgga aattcgggaa aacaaaatga aggaagagaa 840

aatgagtcga gggaaaggaa ccactacgaa gaggcaatgc ttgattacat accaagagac 900

gaagaggaag cttccattgc aatgctcatc ggaaatctaa acgatcacta tcccatccct 960

aacgatctca tggacctcac cactgacctt cagcaccatc aagccacgtc ctcatcaatg 1020

ccacctgagg atcacgcgta cgtgggttca tccgatgatc aggtgagctt taacgacttt 1080

gagtggtggt ga 1092

<210> 7

<211> 363

<212> PRT

<213> Arabidopsis thaliana

<400> 7

Met Asp Asn Phe Leu Pro Phe Pro Ser Ser Asn Ala Asn Ser Val Gln

1 5 10 15

Glu Leu Ser Met Asp Pro Asn Asn Asn Arg Ser His Phe Thr Thr Val

20 25 30

Pro Thr Tyr Asp His His Gln Ala Gln Pro His His Phe Leu Pro Pro

35 40 45

Phe Ser Tyr Pro Val Glu Gln Met Ala Ala Val Met Asn Pro Gln Pro

50 55 60

Val Tyr Leu Ser Glu Cys Tyr Pro Gln Ile Pro Val Thr Gln Thr Gly

65 70 75 80

Ser Glu Phe Gly Ser Leu Val Gly Asn Pro Cys Leu Trp Gln Glu Arg

85 90 95

Gly Gly Phe Leu Asp Pro Arg Met Thr Lys Met Ala Arg Ile Asn Arg

100 105 110

Lys Asn Ala Met Met Arg Ser Arg Asn Asn Ser Ser Pro Asn Ser Ser

115 120 125

Pro Ser Glu Leu Val Asp Ser Lys Arg Gln Leu Met Met Leu Asn Leu

130 135 140

Lys Asn Asn Val Gln Ile Ser Asp Lys Lys Asp Ser Tyr Gln Gln Ser

145 150 155 160

Thr Phe Asp Asn Lys Lys Leu Arg Val Leu Cys Glu Lys Glu Leu Lys

165 170 175

Asn Ser Asp Val Gly Ser Leu Gly Arg Ile Val Leu Pro Lys Arg Asp

180 185 190

Ala Glu Ala Asn Leu Pro Lys Leu Ser Asp Lys Glu Gly Ile Val Val

195 200 205

Gln Met Arg Asp Val Phe Ser Met Gln Ser Trp Ser Phe Lys Tyr Lys

210 215 220

Phe Trp Ser Asn Asn Lys Ser Arg Met Tyr Val Leu Glu Asn Thr Gly

225 230 235 240

Glu Phe Val Lys Gln Asn Gly Ala Glu Ile Gly Asp Phe Leu Thr Ile

245 250 255

Tyr Glu Asp Glu Ser Lys Asn Leu Tyr Phe Ala Met Asn Gly Asn Ser

260 265 270

Gly Lys Gln Asn Glu Gly Arg Glu Asn Glu Ser Arg Glu Arg Asn His

275 280 285

Tyr Glu Glu Ala Met Leu Asp Tyr Ile Pro Arg Asp Glu Glu Glu Ala

290 295 300

Ser Ile Ala Met Leu Ile Gly Asn Leu Asn Asp His Tyr Pro Ile Pro

305 310 315 320

Asn Asp Leu Met Asp Leu Thr Thr Asp Leu Gln His His Gln Ala Thr

325 330 335

Ser Ser Ser Met Pro Pro Glu Asp His Ala Tyr Val Gly Ser Ser Asp

340 345 350

Asp Gln Val Ser Phe Asn Asp Phe Glu Trp Trp

355 360

<210> 8

<211> 879

<212> DNA

<213> Artificial Sequence

<220>

<223> cDNA of AtWUS encoding SEQ ID NO: 9

<400> 8

atggagccgc cacagcatca gcatcatcat catcaagccg accaagaaag cggcaacaac 60

aacaacaaca agtccggctc tggtggttac acgtgtcgcc agaccagcac gaggtggaca 120

ccgacgacgg agcaaatcaa aatcctcaaa gaactttact acaacaatgc aatccggtca 180

ccaacagccg atcagatcca gaagatcact gcaaggctga gacagttcgg aaagattgag 240

ggcaagaacg tcttttactg gttccagaac cataaggctc gtgagcgtca gaagaagaga 300

ttcaacggaa caaacatgac cacaccatct tcatcaccca actcggttat gatggcggct 360

aacgatcatt atcatcctct acttcaccat catcacggtg ttcccatgca gagacctgct 420

aattccgtca acgttaaact taaccaagac catcatctct atcatcataa caagccatat 480

cccagcttca ataacgggaa tttaaatcat gcaagctcag gtactgaatg tggtgttgtt 540

aatgcttcta atggctacat gagtagccat gtctatggat ctatggaaca agactgttct 600

atgaattaca acaacgtagg tggaggatgg gcaaacatgg atcatcatta ctcatctgca 660

ccttacaact tcttcgatag agcaaagcct ctgtttggtc tagaaggtca tcaagaagaa 720

gaagaatgtg gtggcgatgc ttatctggaa catcgacgta cgcttcctct cttccctatg 780

cacggtgaag atcacatcaa cggtggtagt ggtgccatct ggaagtatgg ccaatcggaa 840

gttcgccctt gcgcttctct tgagctacgt ctgaactag 879

<210> 9

<211> 292

<212> PRT

<213> Arabidopsis thaliana

<400> 9

Met Glu Pro Pro Gln His Gln His His His His Gln Ala Asp Gln Glu

1 5 10 15

Ser Gly Asn Asn Asn Asn Asn Lys Ser Gly Ser Gly Gly Tyr Thr Cys

20 25 30

Arg Gln Thr Ser Thr Arg Trp Thr Pro Thr Thr Glu Gln Ile Lys Ile

35 40 45

Leu Lys Glu Leu Tyr Tyr Asn Asn Ala Ile Arg Ser Pro Thr Ala Asp

50 55 60

Gln Ile Gln Lys Ile Thr Ala Arg Leu Arg Gln Phe Gly Lys Ile Glu

65 70 75 80

Gly Lys Asn Val Phe Tyr Trp Phe Gln Asn His Lys Ala Arg Glu Arg

85 90 95

Gln Lys Lys Arg Phe Asn Gly Thr Asn Met Thr Thr Pro Ser Ser Ser

100 105 110

Pro Asn Ser Val Met Met Ala Ala Asn Asp His Tyr His Pro Leu Leu

115 120 125

His His His His Gly Val Pro Met Gln Arg Pro Ala Asn Ser Val Asn

130 135 140

Val Lys Leu Asn Gln Asp His His Leu Tyr His His Asn Lys Pro Tyr

145 150 155 160

Pro Ser Phe Asn Asn Gly Asn Leu Asn His Ala Ser Ser Gly Thr Glu

165 170 175

Cys Gly Val Val Asn Ala Ser Asn Gly Tyr Met Ser Ser His Val Tyr

180 185 190

Gly Ser Met Glu Gln Asp Cys Ser Met Asn Tyr Asn Asn Val Gly Gly

195 200 205

Gly Trp Ala Asn Met Asp His His Tyr Ser Ser Ala Pro Tyr Asn Phe

210 215 220

Phe Asp Arg Ala Lys Pro Leu Phe Gly Leu Glu Gly His Gln Glu Glu

225 230 235 240

Glu Glu Cys Gly Gly Asp Ala Tyr Leu Glu His Arg Arg Thr Leu Pro

245 250 255

Leu Phe Pro Met His Gly Glu Asp His Ile Asn Gly Gly Ser Gly Ala

260 265 270

Ile Trp Lys Tyr Gly Gln Ser Glu Val Arg Pro Cys Ala Ser Leu Glu

275 280 285

Leu Arg Leu Asn

290

<210> 10

<211> 1755

<212> DNA

<213> Artificial Sequence

<220>

<223> cDNA of AtBBM encoding SEQ ID NO: 11

<400> 10

atgaactcga tgaataactg gttaggcttc tctctctctc ctcatgatca aaatcatcac 60

cgtacggatg ttgactcctc caccaccaga accgccgtag atgttgccgg agggtactgt 120

tttgatctgg ccgctccctc cgatgaatct tctgccgttc aaacatcttt tctttctcct 180

ttcggtgtca ccctcgaagc tttcaccaga gacaataata gtcactcccg agattgggac 240

atcaatggtg gtgcatgcaa taacattaac aataacgaac aaaatggacc aaagcttgag 300

aatttcctcg gccgcaccac cacgatttac aataccaacg agaccgttgt agatggaaat 360

ggcgattgtg gaggaggaga cggtggtggt ggcggctcac taggcctttc gatgataaaa 420

acatggctga gtaatcattc ggttgctaat gctaatcatc aagacaatgg taacggtgca 480

cgaggcttgt ccctctctat gaattcatct actagtgata gcaacaacta caacaacaat 540

gatgatgtcg tccaagagaa gactattgtt gatgtcgtag aaactacacc gaagaaaact 600

attgagagtt ttggacaaag gacgtctata taccgcggtg ttacaaggca tcggtggaca 660

ggtagatacg aggcacattt atgggacaat agttgcaaaa gagaaggcca gactcgcaaa 720

ggaagacaag tttatctggg aggttatgac aaagaagaaa aagcagctag ggcttacgat 780

ttagccgcac taaagtattg gggaaccacc actactacta acttcccctt gagtgaatat 840

gagaaagagg tagaagagat gaagcacatg acgaggcaag agtatgttgc ctctctgcgc 900

aggaaaagta gtggtttctc tcgtggtgca tcgatttatc gaggagtaac aaggcatcac 960

caacatggaa ggtggcaagc taggatcgga agagtcgccg gtaacaaaga cctctacttg 1020

ggaactttcg gcacacagga agaggctgct gaggcttatg acattgcagc cattaaattc 1080

agaggattaa gcgcagtgac taacttcgac atgaacagat acaatgttaa agcaatcctc 1140

gagagcccga gtctacctat tggtagttct gcgaaacgtc tcaaggacgt taataatccg 1200

gttccagcta tgatgattag taataacgtt tcagagagtg caaataatgt tagcggttgg 1260

caaaacactg cgtttcagca tcatcaggga atggatttga gcttattgca gcaacagcag 1320

gagaggtacg ttggttatta caatggagga aacttgtcta ccgagagtac tagggtttgt 1380

ttcaaacaag aggaggaaca acaacacttc ttgagaaact cgccgagtca catgactaat 1440

gttgatcatc atagctcgac ctctgatgat tctgttaccg tttgtggaaa tgttgttagt 1500

tatggtggtt atcaaggatt cgcaatccct gttggaacat cggttaatta cgatcccttt 1560

actgctgctg agattgctta caacgcaaga aatcattatt actatgctca gcatcagcaa 1620

caacagcaga ttcagcagtc gccgggagga gattttccgg tggcgatttc gaataaccat 1680

agctctaaca tgtactttca cggggaaggt ggtggagaag gggctccaac gttttcagtt 1740

tggaacgaca cttag 1755

<210> 11

<211> 584

<212> PRT

<213> Arabidopsis thaliana

<400> 11

Met Asn Ser Met Asn Asn Trp Leu Gly Phe Ser Leu Ser Pro His Asp

1 5 10 15

Gln Asn His His Arg Thr Asp Val Asp Ser Ser Thr Thr Arg Thr Ala

20 25 30

Val Asp Val Ala Gly Gly Tyr Cys Phe Asp Leu Ala Ala Pro Ser Asp

35 40 45

Glu Ser Ser Ala Val Gln Thr Ser Phe Leu Ser Pro Phe Gly Val Thr

50 55 60

Leu Glu Ala Phe Thr Arg Asp Asn Asn Ser His Ser Arg Asp Trp Asp

65 70 75 80

Ile Asn Gly Gly Ala Cys Asn Asn Ile Asn Asn Asn Glu Gln Asn Gly

85 90 95

Pro Lys Leu Glu Asn Phe Leu Gly Arg Thr Thr Thr Ile Tyr Asn Thr

100 105 110

Asn Glu Thr Val Val Asp Gly Asn Gly Asp Cys Gly Gly Gly Asp Gly

115 120 125

Gly Gly Gly Gly Ser Leu Gly Leu Ser Met Ile Lys Thr Trp Leu Ser

130 135 140

Asn His Ser Val Ala Asn Ala Asn His Gln Asp Asn Gly Asn Gly Ala

145 150 155 160

Arg Gly Leu Ser Leu Ser Met Asn Ser Ser Thr Ser Asp Ser Asn Asn

165 170 175

Tyr Asn Asn Asn Asp Asp Val Val Gln Glu Lys Thr Ile Val Asp Val

180 185 190

Val Glu Thr Thr Pro Lys Lys Thr Ile Glu Ser Phe Gly Gln Arg Thr

195 200 205

Ser Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Tyr Glu

210 215 220

Ala His Leu Trp Asp Asn Ser Cys Lys Arg Glu Gly Gln Thr Arg Lys

225 230 235 240

Gly Arg Gln Val Tyr Leu Gly Gly Tyr Asp Lys Glu Glu Lys Ala Ala

245 250 255

Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Thr Thr Thr Thr

260 265 270

Thr Asn Phe Pro Leu Ser Glu Tyr Glu Lys Glu Val Glu Glu Met Lys

275 280 285

His Met Thr Arg Gln Glu Tyr Val Ala Ser Leu Arg Arg Lys Ser Ser

290 295 300

Gly Phe Ser Arg Gly Ala Ser Ile Tyr Arg Gly Val Thr Arg His His

305 310 315 320

Gln His Gly Arg Trp Gln Ala Arg Ile Gly Arg Val Ala Gly Asn Lys

325 330 335

Asp Leu Tyr Leu Gly Thr Phe Gly Thr Gln Glu Glu Ala Ala Glu Ala

340 345 350

Tyr Asp Ile Ala Ala Ile Lys Phe Arg Gly Leu Ser Ala Val Thr Asn

355 360 365

Phe Asp Met Asn Arg Tyr Asn Val Lys Ala Ile Leu Glu Ser Pro Ser

370 375 380

Leu Pro Ile Gly Ser Ser Ala Lys Arg Leu Lys Asp Val Asn Asn Pro

385 390 395 400

Val Pro Ala Met Met Ile Ser Asn Asn Val Ser Glu Ser Ala Asn Asn

405 410 415

Val Ser Gly Trp Gln Asn Thr Ala Phe Gln His His Gln Gly Met Asp

420 425 430

Leu Ser Leu Leu Gln Gln Gln Gln Glu Arg Tyr Val Gly Tyr Tyr Asn

435 440 445

Gly Gly Asn Leu Ser Thr Glu Ser Thr Arg Val Cys Phe Lys Gln Glu

450 455 460

Glu Glu Gln Gln His Phe Leu Arg Asn Ser Pro Ser His Met Thr Asn

465 470 475 480

Val Asp His His Ser Ser Thr Ser Asp Asp Ser Val Thr Val Cys Gly

485 490 495

Asn Val Val Ser Tyr Gly Gly Tyr Gln Gly Phe Ala Ile Pro Val Gly

500 505 510

Thr Ser Val Asn Tyr Asp Pro Phe Thr Ala Ala Glu Ile Ala Tyr Asn

515 520 525

Ala Arg Asn His Tyr Tyr Tyr Ala Gln His Gln Gln Gln Gln Gln Ile

530 535 540

Gln Gln Ser Pro Gly Gly Asp Phe Pro Val Ala Ile Ser Asn Asn His

545 550 555 560

Ser Ser Asn Met Tyr Phe His Gly Glu Gly Gly Gly Glu Gly Ala Pro

565 570 575

Thr Phe Ser Val Trp Asn Asp Thr

580

<210> 12

<211> 807

<212> DNA

<213> Artificial Sequence

<220>

<223> cDNA of AtAGL15 encoding SEQ ID NO: 13

<400> 12

atgggtcgtg gaaaaatcga gataaagagg atcgagaatg cgaatagcag acaagtcact 60

ttttccaaga ggcgttctgg gttacttaag aaagctcgtg agctctctgt tctttgtgat 120

gctgaagttg ctgtcatcgt cttctctaag tctggcaagc tcttcgagta ctccagtact 180

ggaatgaagc aaacactttc cagatacggt aatcaccaga gttcttcagc ttctaaagca 240

gaggaggatt gtgcagaggt ggatatttta aaggatcaac tttcaaagct tcaagagaaa 300

catttacaac tgcagggcaa gggcttgaat cctctgacct ttaaagagct gcaaagcctt 360

gagcagcaac tatatcatgc attgattact gtcagagagc gaaaggaacg attgctgact 420

aaccaacttg aagaatcacg cctcaaggaa caacgagcag agttggaaaa cgagaccttg 480

cgtagacagg ttcaagaact gaggagcttt ctcccgtcgt tcacccacta tgttccatcc 540

tacatcaaat gctttgctat agatccaaag aacgctctca taaaccacga cagtaaatgc 600

agcctccaga acaccgattc agacacaact ttgcaattag ggttgccggg agaggcacat 660

gatagaagga cgaatgaagg agaaagagag agcccgtcaa gcgattcagt gacaacaaac 720

acgagcagcg aaactgcaga aagaggggat cagtctagtt tagcaaattc tccacctgaa 780

gccaaaagac aaaggttctc tgtttag 807

<210> 13

<211> 268

<212> PRT

<213> Arabidopsis thaliana

<400> 13

Met Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Ala Asn Ser

1 5 10 15

Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala

20 25 30

Arg Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Val Ile Val Phe

35 40 45

Ser Lys Ser Gly Lys Leu Phe Glu Tyr Ser Ser Thr Gly Met Lys Gln

50 55 60

Thr Leu Ser Arg Tyr Gly Asn His Gln Ser Ser Ser Ala Ser Lys Ala

65 70 75 80

Glu Glu Asp Cys Ala Glu Val Asp Ile Leu Lys Asp Gln Leu Ser Lys

85 90 95

Leu Gln Glu Lys His Leu Gln Leu Gln Gly Lys Gly Leu Asn Pro Leu

100 105 110

Thr Phe Lys Glu Leu Gln Ser Leu Glu Gln Gln Leu Tyr His Ala Leu

115 120 125

Ile Thr Val Arg Glu Arg Lys Glu Arg Leu Leu Thr Asn Gln Leu Glu

130 135 140

Glu Ser Arg Leu Lys Glu Gln Arg Ala Glu Leu Glu Asn Glu Thr Leu

145 150 155 160

Arg Arg Gln Val Gln Glu Leu Arg Ser Phe Leu Pro Ser Phe Thr His

165 170 175

Tyr Val Pro Ser Tyr Ile Lys Cys Phe Ala Ile Asp Pro Lys Asn Ala

180 185 190

Leu Ile Asn His Asp Ser Lys Cys Ser Leu Gln Asn Thr Asp Ser Asp

195 200 205

Thr Thr Leu Gln Leu Gly Leu Pro Gly Glu Ala His Asp Arg Arg Thr

210 215 220

Asn Glu Gly Glu Arg Glu Ser Pro Ser Ser Asp Ser Val Thr Thr Asn

225 230 235 240

Thr Ser Ser Glu Thr Ala Glu Arg Gly Asp Gln Ser Ser Leu Ala Asn

245 250 255

Ser Pro Pro Glu Ala Lys Arg Gln Arg Phe Ser Val

260 265

<210> 14

<211> 16296

<212> DNA

<213> Artificial Sequence

<220>

<223> plasmid pERV1-hygro carrying TaRKD2 as CDS

<400> 14

ggtttacccg ccaatatatc ctgtcaaaca ctgatagttt aaaccgaagg cgggaaacga 60

caatctgatc gggtaccggg cccaagatct ggcccttaag gccttactag gctgcagtgc 120

agcgtgaccc ggtcgtgccc ctctctagag ataatgagca ttgcatgtct aagttataaa 180

aaattaccac atattttttt tgtcacactt gtttgaagtg cagtttatct atctttatac 240

atatatttaa actttactct acgaataata taatctatag tactacaata atatcagtgt 300

tttagagaat catataaatg aacagttaga catggtctaa aggacaattg agtattttga 360

caacaggact ctacagtttt atctttttag tgtgcatgtg ttctcctttt tttttgcaaa 420

tagcttcacc tatataatac ttcatccatt ttattagtac atccatttag ggtttagggt 480

taatggtttt tatagactaa tttttttagt acatctattt tattctattt tagcctctaa 540

attaagaaaa ctaaaactct attttagttt ttttatttaa taatttagat ataaaataga 600

ataaaataaa gtgactaaaa attaaacaaa taccctttaa gaaattaaaa aaactaagga 660

aacatttttc ttgtttcgag tagataatgc cagcctgtta aacgccgtcg atcgacgagt 720

ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca 780

cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg 840

ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag 900

gcggcctcct cctcctctca cggcaccggc agctacgggg gattcctttc ccaccgctcc 960

ttcgctttcc cttcctcgcc cgccgtaata aatagacacc ccctccacac cctctttccc 1020

caacctcgtg ttgttcggag cgcacacaca cacaaccaga tctcccccaa atccacccgt 1080

cggcacctcc gcttcaaggt acgccgctcg tcctcccccc ccccccctct ctaccttctc 1140

tagatcggcg ttccggtcca tggttagggc ccggtagttc tacttctgtt catgtttgtg 1200

ttagatccgt gtttgtgtta gatccgtgct gctagcgttc gtacacggat gcgacctgta 1260

cgtcagacac gttctgattg ctaacttgcc agtgtttctc tttggggaat cctgggatgg 1320

ctctagccgt tccgcagacg ggatcgatct aggataggta tacatgttga tgtgggtttt 1380

actgatgcat atacatgatg gcatatgcag catctattca tatgctctaa ccttgagtac 1440

ctatctatta taataaacaa gtatgtttta taattatttt gatcttgata tacttggatg 1500

atggcatatg cagcagctat atgtggattt ttttagccct gccttcatac gctatttatt 1560

tgcttggtac tgtttctttt gtcgatgctc accctgttgt ttggtgttac ttctgcaggt 1620

actagtattc cgggcggaat gaaagcgtta acggccaggc aacaagaggt gtttgatctc 1680

atccgtgatc acatcagcca gacaggtatg ccgccgacgc gtgcggaaat cgcgcagcgt 1740

ttggggttcc gttccccaaa cgcggctgaa gaacatctga aggcgctggc acgcaaaggc 1800

gttattgaaa ttgtttccgg cgcatcacgc gggattcgtc tgttgcagga agaggaagaa 1860

gggttgccgc tggtaggtcg tgtggctgcc ggtgaaccgt cgagcgcccc cccgaccgat 1920

gtcagcctgg gggacgagct ccacttagac ggcgaggacg tggcgatggc gcatgccgac 1980

gcgctagacg atttcgatct ggacatgttg ggggacgggg attccccggg tccgggattt 2040

accccccacg actccgcccc ctacggcgct ctggatatgg ccgacttcga gtttgagcag 2100

atgtttaccg atgcccttgg aattgacgag tacggtgggg atccgtctgc tggagacatg 2160

agagctgcca acctttggcc aagcccgctc atgatcaaac gctctaagaa gaacagcctg 2220

gccttgtccc tgacggccga ccagatggtc agtgccttgt tggatgctga gccccccata 2280

ctctattccg agtatgatcc taccagaccc ttcagtgaag cttcgatgat gggcttactg 2340

accaacctgg cagacaggga gctggttcac atgatcaact gggcgaagag ggtgccaggc 2400

tttgtggatt tgaccctcca tgatcaggtc caccttctag aatgtgcctg gctagagatc 2460

ctgatgattg gtctcgtctg gcgctccatg gagcacccag ggaagctact gtttgctcct 2520

aacttgctct tggacaggaa ccagggaaaa tgtgtagagg gcatggtgga gatcttcgac 2580

atgctgctgg ctacatcatc tcggttccgc atgatgaatc tgcagggaga ggagtttgtg 2640

tgcctcaaat ctattatttt gcttaattct ggagtgtaca catttctgtc cagcaccctg 2700

aagtctctgg aagagaagga ccatatccac cgagtcctgg acaagatcac agacactttg 2760

atccacctga tggccaaggc aggcctgacc ctgcagcagc agcaccagcg gctggcccag 2820

ctcctcctca tcctctccca catcaggcac atgagtaaca aaggcatgga gcatctgtac 2880

agcatgaagt gcaagaacgt ggtgcccctc tatgacctgc tgctggagat gctggacgcc 2940

caccgcctac atgcgcccac tagccgtgga ggggcatccg tggaggagac ggaccaaagc 3000

cacttggcca ctgcgggctc tacttcatcg cattccttgc aaaagtatta catcacgggg 3060

gaggcagagg gtttccctgc cacagtctga gagctccctg gcggaattcc cagagatgtt 3120

agctgaaatc atcactaatc agataccaaa atattcaaat ggaaatatca aaaagcttct 3180

gtttcatcaa aaatgactcg acctaactga gtaagctagc ttgttcgagt attatggcat 3240

tgggaaaact gtttttcttg taccatttgt tgtgcttgta atttactgtg ttttttattc 3300

ggttttcgct atcgaactgt gaaatggaaa tggatggaga agagttaatg aatgatatgg 3360

tccttttgtt cattctcaaa ttaatattat ttgttttttc tcttatttgt tgtgtgttga 3420

atttgaaatt ataagagata tgcaaacatt ttgttttgag taaaaatgtg tcaaatcgtg 3480

gcctctaatg accgaagtta atatgaggag taaaacacta gatccccaaa caagcttgga 3540

aactgaaggc gctcgagtta ctagatcggg gaattgatcc cccctcgaca gcttgcatgc 3600

cgcttgggct gcaggtcgag gctaaaaaac taatcgcatt atcatcccct cgacgtactg 3660

tacatataac cactggtttt atatacagca gtactgtaca tataaccact ggttttatat 3720

acagcagtcg acgtactgta catataacca ctggttttat atacagcagt actgtacata 3780

taaccactgg ttttatatac agcagtcgag gtaagattag atatggatat gtatatggat 3840

atgtatatgg tggtaatgcc atgtaatatg ctcgactcta ggatcttcgc aagacccttc 3900

ctctatataa ggaagttcat ttcatttgga gaggacacgc tgaagctagt cgactctagc 3960

ctccctaggc tgccggacga cgagctcctc ccccctcccc ctccgccgcc gccgcgccgg 4020

taaccacccc gcccctctcc tctttctttc tccgtttttt tttccgtctc ggtctcgatc 4080

tttggccttg gtagtttggg tgggcgagag gcggcttcgt gcgcgcccag atcggtgcgc 4140

gggaggggcg ggatctcgcg gctggggctc tcgccggcgt ggatccggcc cggatctcgc 4200

ggggaatggg gctctcggat gtagatctgc gatccgccgt tgttggggga gatgatgggg 4260

ggtttaaaat ttccgccatg ctaaacaaga tcaggaagag gggaaaaggg cactatggtt 4320

tatattttta tatatttctg ctgcttcgtc aggcttagat gtgctagatc ttcttctttc 4380

tttcttcttt ttgtgggtag aatttgaatc cctcagcatt gttcatcggt agtttttctt 4440

ttcatgattt gtgacaaatg cagcctcgtg cggagctttt ttgtaggcgc gggctgcagg 4500

aattcaagct tacgcgtgtc atcacaagtt tgtacaaaaa agcaggctat ggagatgcaa 4560

caacaatact tcggggggga cggcgatgcg gactggttcc atcaactcgc attgcttccc 4620

ccacttccaa tctcatcgtc tctcccccca ctcccgatgt cagagggctc atgtctccct 4680

atggcagcag cagctgcagc tgcactcccc cttggcgatt gctcgagcgc cctcatgata 4740

cgccctgagg aacagatgtc ttgccttcca atgaacccct ctccagcggt cgtcgacgat 4800

gtctactctt cctacgcacc gaacaatgtc gacgtgttgc cgccattccc ggcaggactt 4860

gacgacgctc tgttgatgga gtctttttct gacatcgacc tcgaggagtt tgctgacgca 4920

tttggccaca agatcaagac agaacccctc gacgatgcca tggtccccgc ggaccacgac 4980

ttcgcggctc aagcccaaca ggcctgccct gtggtcatca tgaatcagca acaactcaac 5040

gcacccagag acgtgcgcct gctcattgac ccggatgatg atgacagcac cgtggtggcc 5100

gggggctatg aagctgcagc ggtggggtgc gccgagcaga aacaggtcag gccagcacca 5160

cgtagggtga gaaagagctc aggcggcgca agaccagccg cgggaggaaa gtccctcgat 5220

cacatcggat tcgaggaact caggacctat ttctatatgc caatcaccaa ggcagcgagg 5280

gaaatgaacg tggggctgac agtcctgaag aagagatgcc gggaactggg ggtggcgcgc 5340

tggccacaca gaaagatgaa gtctctgaga agcctgatcc tcaacattca ggagatgggg 5400

aagggcgcaa catctcccgc agccgtgcag ggggaacttg aagcgcttga gaggtattgc 5460

gccattatgg aggagaaccc ggctatagag ctcaccgagc aaacgaagaa gctcaggcag 5520

gcttgtttca aagagaatta taagcggcgt agagccgccg cttctgttaa tcttctcgat 5580

cactgctata acgatctggc atctcatgag cagcaaatgc ctctcccaca aatgggattc 5640

tttggatttt agacccagct ttcttgtaca aagtggtgat gactcgaatt tccccgatcg 5700

ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat 5760

tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac 5820

gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat 5880

agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt 5940

actagatcgc tcgacgcggc cgccatggcc tctagtggat cagcttgcat gcctgcaggt 6000

cactggattt tggttttagg aattagaaat tttattgata gaagtatttt acaaatacaa 6060

atacatacta agggtttctt atatgctcaa cacatgagcg aaaccctata agaaccctaa 6120

ttcccttatc tgggaactac tcacacatta ttctggagaa aaatagagag agatagattt 6180

gtagagagag actggtgatt tttgcggact ccggtcggca tctactctat tcctttgccc 6240

tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg 6300

tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc 6360

ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca 6420

agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagc cgcggcgatc 6480

ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc 6540

acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc 6600

tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat 6660

ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga 6720

gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat 6780

ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg 6840

gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatgg 6900

cctccgcgac cggctgcaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga 6960

caccctgtgc acggcgggag atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa 7020

gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt 7080

agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc 7140

tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact 7200

tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc atatcttatt 7260

gccccccggg gccctcgacc tgcagaagta acaccaaaca acagggtgag catcgacaaa 7320

agaaacagta ccaagcaaat aaatagcgta tgaaggcagg gctaaaaaaa tccacatata 7380

gctgctgcat atgccatcat ccaagtatat caagatcaaa ataattataa aacatacttg 7440

tttattataa tagataggta ctcaaggtta gagcatatga atagatgctg catatgccat 7500

catgtatatg catcagtaaa acccacatca acatgtatac ctatcctaga tcgatcccgt 7560

ctgcggaacg gctagagcca tcccaggatt ccccaaagag aaacactggc aagttagcaa 7620

tcagaacgtg tctgacgtac aggtcgcatc cgtgtacgaa cgctagcagc acggatctaa 7680

cacaaacacg gatctaacac aaacatgaac agaagtagaa ctaccgggcc ctaaccatgg 7740

accggaacgc cgatctagag aaggtagaga gggggggggg gggaggacga gcggcgtacc 7800

ttgaagcgga ggtgccgacg ggtggatttg ggggagatct ggttgtgtgt gtgtgcgctc 7860

cgaacaacac gaggttgggg aaagagggtg tggagggggt gtctatttat tacggcgggc 7920

gaggaaggga aagcgaagga gcggtgggaa aggaatcccc cgtagctgcc ggtgccgtga 7980

gaggaggagg aggccgcctg ccgtgccggc tcacgtctgc cgctccgcca cgcaatttct 8040

ggatgccgac agcggagcaa gtccaacggt ggagcggaac tctcgagagg ggtccagagg 8100

cagcgacaga gatgccgtgc cgtctgcttc gcttggcccg acgcgacgct gctggttcgc 8160

tggttggtgt ccgttagact cgtcgatcga cggcgtttaa caggctggca ttatctactc 8220

gaaacaagaa aaatgtttcc ttagtttttt taatttctta aagggtattt gtttaatttt 8280

tagtcacttt attttattct attttatatc taaattatta aataaaaaaa ctaaaataga 8340

gttttagttt tcttaattta gaggctaaaa tagaataaaa tagatgtact aaaaaaatta 8400

gtctataaaa accattaacc ctaaacccta aatggatgta ctaataaaat ggatgaagta 8460

ttatataggt gaagctattt gcaaaaaaaa aggagaacac atgcacacta aaaagataaa 8520

actgtagagt cctgttgtca aaatactcaa ttgtccttta gaccatgtct aactgttcat 8580

ttatatgatt ctctaaaaca ctgatattat tgtagtacta tagattatat tattcgtaga 8640

gtaaagttta aatatatgta taaagataga taaactgcac ttcaaacaag tgtgacaaaa 8700

aaaatatgtg gtaatttttt ataacttaga catgcaatgc tcattatctc tagagagggg 8760

cacgaccggg tcacgctgca ctgcagacta ctagagccga tcgtgaagtt tctcatctaa 8820

gcccccattt ggacgtgaat gtagacacgt cgaaataaag atttccgaat tagaataatt 8880

tgtttattgc tttcgcctat aaatacgacg gatcgtaatt tgtcgtttta tcaaaatgta 8940

ctttcatttt ataataacgc tgcggacatc tacatttttg aattgaaaaa aaattggtaa 9000

ttactctttc tttttctcca tattgaccat catactcatt gctgatccat gtagatttcc 9060

cggacatgaa gccatttaca attgaatata tcctgccgcc gctgccgctt tgcacccggt 9120

ggagcttgca tgttggtttc tacgcagaac tgagccggtt aggcagataa tttccattga 9180

gaactgagcc atgtgcacct tccccccaac acggtgagcg acggggcaac ggagtgatcc 9240

acatgggact tttaaacatc atccgtcgga tggcgttgcg agagaagcag tcgatccgtg 9300

agatcagccg acgcaccggg caggcgcgca acacgatcgc aaagtatttg aacgcaggta 9360

caatcgagcc gacgttcacg cggaacgacc aagcaagcta tgttgcgatt acttcgccaa 9420

ctattgcgat aacaagaaaa agccagcctt tcatgatata tctcccaatt tgtgtagggc 9480

ttattatgca cgcttaaaaa taataaaagc agacttgacc tgatagtttg gctgtgagca 9540

attatgtgct tagtgcatct aacgcttgag ttaagccgcg ccgcgaagcg gcgtcggctt 9600

gaacgaattg ttagacatta tttgccgact accttggtga tctcgccttt cacgtagtgg 9660

acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa 9720

gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag 9780

tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac 9840

aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt 9900

aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc 9960

gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga 10020

tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct 10080

ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca 10140

atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa 10200

aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc 10260

aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg 10320

gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc 10380

gatacttcgg cgatcaccgc ttccctcatg atgtttaact ttgttttagg gcgactgccc 10440

tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct 10500

tgctgcttgg atgcccgagg catagactgt accccaaaaa aacagtcata acaagccatg 10560

aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt 10620

gagcgcatac gctacttgca ttacagctta cgaaccgaac aggcttatgt ccactgggtt 10680

cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc 10740

gaggcatttc tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag 10800

gcatacccac cggtgccttg atgtgggcgc cggcggtcga gtggcgacgg cgcggcttgt 10860

ccgcgccctg gtagattgcc tggccgtagg ccagccattt ttgagcggcc agcggccgcg 10920

ataggccgac gcgaagcggc ggggcgtagg gagcgcagcg accgaagggt aggcgctttt 10980

tgcagctctt cggctgtgcg ctggccagac agttatgcac aggccaggcg ggttttaaga 11040

gttttaataa gttttaaaga gttttaggcg gaaaaatcgc cttttttctc ttttatatca 11100

gtcacttaca tgtgtgaccg gttcccaatg tacggctttg ggttcccaat gtacgggttc 11160

cggttcccaa tgtacggctt tgggttccca atgtacgtgc tatccacagg aaagagacct 11220

tttcgacctt tttcccctgc tagggcaatt tgccctagca tctgctccgt acattaggaa 11280

ccggcggatg cttcgccctc gatcaggttg cggtagcgca tgactaggat cgggccagcc 11340

tgccccgcct cctccttcaa atcgtactcc ggcaggtcat ttgacccgat cagcttgcgc 11400

acggtgaaac agaacttctt gaactctccg gcgctgccac tgcgttcgta gatcgtcttg 11460

aacaaccatc tggcttctgc cttgcctgcg gcgcggcgtg ccaggcggta gagaaaacgg 11520

ccgatgccgg gatcgatcaa aaagtaatcg gggtgaaccg tcagcacgtc cgggttcttg 11580

ccttctgtga tctcgcggta catccaatca gctagctcga tctcgatgta ctccggccgc 11640

ccggtttcgc tctttacgat cttgtagcgg ctaatcaagg cttcaccctc ggataccgtc 11700

accaggcggc cgttcttggc cttcttcgta cgctgcatgg caacgtgcgt ggtgtttaac 11760

cgaatgcagg tttctaccag gtcgtctttc tgctttccgc catcggctcg ccggcagaac 11820

ttgagtacgt ccgcaacgtg tggacggaac acgcggccgg gcttgtctcc cttcccttcc 11880

cggtatcggt tcatggattc ggttagatgg gaaaccgcca tcagtaccag gtcgtaatcc 11940

cacacactcg ccatgccggc cggccctgcg gaaacctcta cgtgcccgtc tggaagctcg 12000

tagcggatca cctcgccagc tcgtcggtca cgcttcgaca gacggaaaac ggccacgtcc 12060

atgatgctgc gactatcgcg ggtgcccacg tcatagagca tcggaacgaa aaaatctggt 12120

tgctcgtcgc ccttgggcgg cttcctaatc gacggcgcac cggctgccgg cggttgccgg 12180

gattctttgc ggattcgatc agcggccgct tgccacgatt caccggggcg tgcttctgcc 12240

tcgatgcgtt gccgctgggc ggcctgcgcc gccttcaact tctccaccag gtcatcaccc 12300

agcgccgcgc cgatttgtac cgggccggat ggtttgcgac cgctcacgcc gattcctcgg 12360

gcttgggggt tccagtgcca ttgcagggcc ggcagacaac ccagccgctt acgcctggcc 12420

aaccgcccgt tcctccacac atggggcatt ccacggcgtc ggtgcctggt tgttcttgat 12480

tttccatgcc gcctccttta gccgctaaaa ttcatctact catttattca tttgctcatt 12540

tactctggta gctgcgcgat gtattcagat agcagctcgg taatggtctt gccttggcgt 12600

accgcgtaca tcttcagctt ggtgtgatcc tccgccggca actgaaagtt gacccgcttc 12660

atggctggcg tgtctgccag gctggccaac gttgcagcct tgctgctgcg tgcgctcgga 12720

cggccggcac ttagcgtgtt tgtgcttttg ctcattttct ctttacctca ttaactcaaa 12780

tgagttttga tttaatttca gcggccagcg cctggacctc gcgggcagcg tcgccctcgg 12840

gttctgattc aagaacggtt gtgccggcgg cggcagtgcc tgggtagctc acgcgctgcg 12900

tgatacggga ctcaagaatg ggcagctcgt acccggccag cgcctcggca acctcaccgc 12960

cgatgcgcgt gcctttgatc gcccgcgaca cgacaaaggc cgcttgtagc cttccatccg 13020

tgacctcaat gcgctgctta accagctcca ccaggtcggc ggtggcccat atgtcgtaag 13080

ggcttggctg caccggaatc agcacgaagt cggctgcctt gatcgcggac acagccaagt 13140

ccgccgcctg gggcgctccg tcgatcacta cgaagtcgcg ccggccgatg gccttcacgt 13200

cgcggtcaat cgtcgggcgg tcgatgccga caacggttag cggttgatct tcccgcacgg 13260

ccgcccaatc gcgggcactg ccctggggat cggaatcgac taacagaaca tcggccccgg 13320

cgagttgcag ggcgcgggct agatgggttg cgatggtcgt cttgcctgac ccgcctttct 13380

ggttaagtac agcgataacc ttcatgcgtt ccccttgcgt atttgtttat ttactcatcg 13440

catcatatac gcagcgaccg catgacgcaa gctgttttac tcaaatacac atcacctttt 13500

tagacggcgg cgctcggttt cttcagcggc caagctcgcc ggccaggccg cgagcttggc 13560

atcagacaaa ccggccagga tttcatgcag ccgcacggtt gagacgtgcg cgggcggctc 13620

gaacacgtac ccggccgcga tcatctccgc ctcgatctct tcggtaatga aaaacggttc 13680

gtcctggccg tcctggtgcg gtttcatgct tgttcctctt ggcgttcatt ctcggcggcc 13740

gccagggcgt cggcctcggt caatgcgtcc tcacggaagg caccgcgccg cctggcctcg 13800

gtgggcgtca cttcctcgct gcgctcaagt gcgcggtaca gggtcgagcg atgcacgcca 13860

agcagtgcag ccgcctcttt cacggtgcgg ccttcctggt cgatcagctc gcgggcgtgc 13920

gcgatctgtg ccggggtgag ggtagggcgg gggccaaact tcacgcctcg cgccttggcg 13980

gcctcgcgcc cgctccgggt gcggtcgatg attagggaac gctcgaactc ggcaatgccg 14040

gcgaacacgg tcaacaccat gcggccggcc ggcgtggtgg tgtcggccca cggctctgcc 14100

aggctacgca ggcccgcgcc ggcctcctgg atgcgctcgg caatgtccag taggtcgcgg 14160

gtgctgcggg ccaggcggtc tagcctggtc actgtcacaa cgtcgccagg gcgtaggtgg 14220

tcaagcatcc tggccagctc cgggcggtcg cgcctggtgc cggtgatctt ctcggaaaac 14280

agcttggtgc agccggccgc gtgcagttcg gcccgttggt tggtcaagtc ctggtcgtcg 14340

gtgctgacgc gggcatagcc cagcaggcca gcggcggcgc tcttgttcat ggcgtaatgt 14400

ctccggttct agtcgcaagt attctacttt atgcgactaa aacacgcgac aagaaaacgc 14460

caggaaaagg gcagggcggc agcctgtcgc gtaacttagg acttgtgcga catgtcgttt 14520

tcagaagacg gctgcactga acgtcagaag ccgactgcac tatagcagcg gaggggttgg 14580

atcgacctcg acgtacccct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca 14640

catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc 14700

ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg 14760

tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga 14820

gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg 14880

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 14940

gtatcagctc actcaaaggc ggtaatcggt tatccacaga atcaggggat aacgcaggaa 15000

agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 15060

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 15120

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 15180

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 15240

gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 15300

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 15360

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 15420

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 15480

ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 15540

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 15600

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 15660

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 15720

tggtcatgag attatcaaaa aggatcttca cctagatcct tttcggcgtc cacatcaacg 15780

gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg cgatatcttg ctgcgttcgg 15840

atattttcgt ggagttcccg ccacagaccc ggattgaagg cgagatccag caactcgcgc 15900

cagatcatcc tgtgacggaa ctttggcgcg tgatgactgg ccaggacgtc ggccgaaaga 15960

gcgacaagca gatcacgctt ttcgacagcg tcggatttgc gatcgaggat ttttcggcgc 16020

tgcgctacgt ccgcgaccgc gttgagggat caagccacag cagcccactc gaccttctag 16080

ccgacccaga cgagccaagg gatctttttg gaatgctgct ccgtcgtcag gctttccgac 16140

gtttgggtgg ttgaacagaa gtcattatcg cacggaatgc caagcactcc cgaggggaac 16200

cctgtggttg gcatgcacat acaaatggac gaacggataa accttttcac gcccttttaa 16260

atatccgatt attctaataa acgctctttt ctctta 16296

<210> 15

<211> 707

<212> DNA

<213> Artificial Sequence

<220>

<223> W pOp6 W promoter

<400> 15

ctagctgtag ttgtagaatg taaaatgtaa tgttgttgtt gtttgttgtt gttgttggta 60

attgttgtaa aaatacgcgc gtctagcttc agcgtgtcct ctccaaatga aatgaacttc 120

cttatataga ggaagggtct tgcgaagatc gatccactag tctttcaatt gtgagcgctc 180

acaattcttt ctcttccctt tcttctttct agtctagtct ttcaattgtg agcgctcaca 240

attctttctc ttccctttct tctttctagt ctagtctttc aattgtgagc gctcacaatt 300

ctttctcttc cctttcttct ttctagtcta gtctttcaat tgtgagcgct cacaattctt 360

tctcttccct ttcttctttc tagtctagtc tttcaattgt gagcgctcac aattctttct 420

cttccctttc ttctttctag tctagtcttt caattgtgag cgctcacaat tctttctctt 480

ccctttcttc tttctagtct ttcaattgtg agcgctcaca attctttctc ttccctttct 540

tctttctagc tccaccgcgg tggcggccgg ccgctctagt ggatcgatct tcgcaagacc 600

cttcctctat ataaggaagt tcatttcatt tggagaggac acgctgaagc tagacgcgcg 660

tatttttaca acaattacca acaacaacaa caaacaacaa caacatt 707

<210> 16

<211> 2226

<212> DNA

<213> Artificial Sequence

<220>

<223> cDNA of LhGR encoding SEQ ID NO: 17

<400> 16

atggctagtg aagctcgaaa aacaaagaaa aaaatcaaag ggattcagca agccactgca 60

ggagtctcac aagacacttc ggaaaatcct aacaaaacaa tagttcctgc agcattacca 120

cagctcaccc ctaccttggt gtcactgctg gaggtgattg aacccgaggt gttgtatgca 180

ggatatgata gctctgttcc agattcagca tggagaatta tgaccacact caacatgtta 240

ggtgggcgtc aagtgattgc agcagtgaaa tgggcaaagg cgataccagg cttcagaaac 300

ttacacctgg atgaccaaat gaccctgcta cagtactcat ggatgtttct catggcattt 360

gccctgggtt ggagatcata cagacaatca agtggaaacc tgctctgctt tgctcctgat 420

ctgattatta atgagcagag aatgtctcta ccctgcatgt atgaccaatg taaacacatg 480

ctgttcgtct cctctgagct ccagcgattg caggtatcct atgaagagta tctctgtatg 540

aaaaccttac tgcttctctc ctcagttcct aaggaaggtc tgaagagcca agagttattt 600

gatgagattc gaatgactta tatcaaagag ctaggaaaag ccatcgtcaa aagggaaggg 660

aactccagtc agaactggca acggttttac caactgacaa agcttctgga ctccatgcat 720

gaggtggttg agaatctcct tacctactgc ttccagacat ttttggataa gaccatgagt 780

attgaattcc cagagatgtt agctgaaatc atcactaatc agataccaaa gtactcaaac 840

ggtaatatca agaagcttct gtttcatcaa aaatctacta gcaaaccggt aacgttatac 900

gacgtcgctg aatacgccgg cgtttctcat caaaccgttt ctagagtggt taaccaggct 960

tcacatgtta gcgctaaaac ccgggaaaaa gttgaagctg ccatggctga gctcaactac 1020

atcccgaacc gtgttgcgca gcagctggct ggtaaacaaa gcttgctgat cggtgtcgcg 1080

acctcgagct tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc 1140

gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt 1200

aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg 1260

ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt 1320

cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg 1380

cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc 1440

ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc 1500

aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa 1560

caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat 1620

cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat 1680

atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc 1740

accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc 1800

tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa 1860

accactagtg gatcggaatt cgccaatttt aatcaaagtg ggaatattgc tgatagctca 1920

ttgtccttca ctttcactaa cagtagcaac ggtccgaacc tcataacaac tcaaacaaat 1980

tctcaagcgc tttcacaacc aattgcctcc tctaacgttc atgataactt catgaataat 2040

gaaatcacgg ctagtaaaat tgatgatggt aataattcaa aaccactgtc acctggttgg 2100

acggaccaaa ctgcgtataa cgcgtttgga atcactacag ggatgtttaa taccactaca 2160

atggatgatg tatataacta tctattcgat gatgaagata ccccaccaaa cccaaaaaaa 2220

gagtaa 2226

<210> 17

<211> 741

<212> PRT

<213> Artificial Sequence

<220>

<223> LhGR protein

<400> 17

Met Ala Ser Glu Ala Arg Lys Thr Lys Lys Lys Ile Lys Gly Ile Gln

1 5 10 15

Gln Ala Thr Ala Gly Val Ser Gln Asp Thr Ser Glu Asn Pro Asn Lys

20 25 30

Thr Ile Val Pro Ala Ala Leu Pro Gln Leu Thr Pro Thr Leu Val Ser

35 40 45

Leu Leu Glu Val Ile Glu Pro Glu Val Leu Tyr Ala Gly Tyr Asp Ser

50 55 60

Ser Val Pro Asp Ser Ala Trp Arg Ile Met Thr Thr Leu Asn Met Leu

65 70 75 80

Gly Gly Arg Gln Val Ile Ala Ala Val Lys Trp Ala Lys Ala Ile Pro

85 90 95

Gly Phe Arg Asn Leu His Leu Asp Asp Gln Met Thr Leu Leu Gln Tyr

100 105 110

Ser Trp Met Phe Leu Met Ala Phe Ala Leu Gly Trp Arg Ser Tyr Arg

115 120 125

Gln Ser Ser Gly Asn Leu Leu Cys Phe Ala Pro Asp Leu Ile Ile Asn

130 135 140

Glu Gln Arg Met Ser Leu Pro Cys Met Tyr Asp Gln Cys Lys His Met

145 150 155 160

Leu Phe Val Ser Ser Glu Leu Gln Arg Leu Gln Val Ser Tyr Glu Glu

165 170 175

Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu Ser Ser Val Pro Lys Glu

180 185 190

Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu Ile Arg Met Thr Tyr Ile

195 200 205

Lys Glu Leu Gly Lys Ala Ile Val Lys Arg Glu Gly Asn Ser Ser Gln

210 215 220

Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Met His

225 230 235 240

Glu Val Val Glu Asn Leu Leu Thr Tyr Cys Phe Gln Thr Phe Leu Asp

245 250 255

Lys Thr Met Ser Ile Glu Phe Pro Glu Met Leu Ala Glu Ile Ile Thr

260 265 270

Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn Ile Lys Lys Leu Leu Phe

275 280 285

His Gln Lys Ser Thr Ser Lys Pro Val Thr Leu Tyr Asp Val Ala Glu

290 295 300

Tyr Ala Gly Val Ser His Gln Thr Val Ser Arg Val Val Asn Gln Ala

305 310 315 320

Ser His Val Ser Ala Lys Thr Arg Glu Lys Val Glu Ala Ala Met Ala

325 330 335

Glu Leu Asn Tyr Ile Pro Asn Arg Val Ala Gln Gln Leu Ala Gly Lys

340 345 350

Gln Ser Leu Leu Ile Gly Val Ala Thr Ser Ser Leu Ala Leu His Ala

355 360 365

Pro Ser Gln Ile Val Ala Ala Ile Lys Ser Arg Ala Asp Gln Leu Gly

370 375 380

Ala Ser Val Val Val Ser Met Val Glu Arg Ser Gly Val Glu Ala Cys

385 390 395 400

Lys Ala Ala Val His Asn Leu Leu Ala Gln Arg Val Ser Gly Leu Ile

405 410 415

Ile Asn Tyr Pro Leu Asp Asp Gln Asp Ala Ile Ala Val Glu Ala Ala

420 425 430

Cys Thr Asn Val Pro Ala Leu Phe Leu Asp Val Ser Asp Gln Thr Pro

435 440 445

Ile Asn Ser Ile Ile Phe Ser His Glu Asp Gly Thr Arg Leu Gly Val

450 455 460

Glu His Leu Val Ala Leu Gly His Gln Gln Ile Ala Leu Leu Ala Gly

465 470 475 480

Pro Leu Ser Ser Val Ser Ala Arg Leu Arg Leu Ala Gly Trp His Lys

485 490 495

Tyr Leu Thr Arg Asn Gln Ile Gln Pro Ile Ala Glu Arg Glu Gly Asp

500 505 510

Trp Ser Ala Met Ser Gly Phe Gln Gln Thr Met Gln Met Leu Asn Glu

515 520 525

Gly Ile Val Pro Thr Ala Met Leu Val Ala Asn Asp Gln Met Ala Leu

530 535 540

Gly Ala Met Arg Ala Ile Thr Glu Ser Gly Leu Arg Val Gly Ala Asp

545 550 555 560

Ile Ser Val Val Gly Tyr Asp Asp Thr Glu Asp Ser Ser Cys Tyr Ile

565 570 575

Pro Pro Leu Thr Thr Ile Lys Gln Asp Phe Arg Leu Leu Gly Gln Thr

580 585 590

Ser Val Asp Arg Leu Leu Gln Leu Ser Gln Gly Gln Ala Val Lys Gly

595 600 605

Asn Gln Leu Leu Pro Val Ser Leu Val Lys Arg Lys Thr Thr Ser Gly

610 615 620

Ser Glu Phe Ala Asn Phe Asn Gln Ser Gly Asn Ile Ala Asp Ser Ser

625 630 635 640

Leu Ser Phe Thr Phe Thr Asn Ser Ser Asn Gly Pro Asn Leu Ile Thr

645 650 655

Thr Gln Thr Asn Ser Gln Ala Leu Ser Gln Pro Ile Ala Ser Ser Asn

660 665 670

Val His Asp Asn Phe Met Asn Asn Glu Ile Thr Ala Ser Lys Ile Asp

675 680 685

Asp Gly Asn Asn Ser Lys Pro Leu Ser Pro Gly Trp Thr Asp Gln Thr

690 695 700

Ala Tyr Asn Ala Phe Gly Ile Thr Thr Gly Met Phe Asn Thr Thr Thr

705 710 715 720

Met Asp Asp Val Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr Pro Pro

725 730 735

Asn Pro Lys Lys Glu

740

<210> 18

<211> 717

<212> DNA

<213> Artificial Sequence

<220>

<223> cDNA of tdTomato encoding SEQ ID NO: 19

<400> 18

atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag 60

ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120

ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180

atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc 240

cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300

gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc 360

tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag 420

accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480

gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc 540

atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg 600

gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc 660

cgccaccacc tgttcctgta cggcatggac gagctgtaca agtctagagg tacctga 717

<210> 19

<211> 238

<212> PRT

<213> Artificial Sequence

<220>

<223> tdTomato protein

<400> 19

Met Val Ser Lys Gly Glu Glu Val Ile Lys Glu Phe Met Arg Phe Lys

1 5 10 15

Val Arg Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly

20 25 30

Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys

35 40 45

Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

50 55 60

Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile

65 70 75 80

Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg

85 90 95

Val Met Asn Phe Glu Asp Gly Gly Leu Val Thr Val Thr Gln Asp Ser

100 105 110

Ser Leu Gln Asp Gly Thr Leu Ile Tyr Lys Val Lys Met Arg Gly Thr

115 120 125

Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

130 135 140

Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly

145 150 155 160

Glu Ile His Gln Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val

165 170 175

Glu Phe Lys Thr Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly

180 185 190

Tyr Tyr Tyr Val Asp Thr Lys Leu Asp Ile Thr Ser His Asn Glu Asp

195 200 205

Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His His Leu

210 215 220

Phe Leu Tyr Gly Met Asp Glu Leu Tyr Lys Ser Arg Gly Thr

225 230 235

<210> 20

<211> 515

<212> DNA

<213> Artificial Sequence

<220>

<223> Ubiquitin intron

<400> 20

cgccgctcgt cctccccccc cccccctctc taccttctct agatcggcgt tccggtccat 60

ggttagggcc cggtagttct acttctgttc atgtttgtgt tagatccgtg tttgtgttag 120

atccgtgctg ctagcgttcg tacacggatg cgacctgtac gtcagacacg ttctgattgc 180

taacttgcca gtgtttctct ttggggaatc ctgggatggc tctagccgtt ccgcagacgg 240

gatcgatcta ggataggtat acatgttgat gtgggtttta ctgatgcata tacatgatgg 300

catatgcagc atctattcat atgctctaac cttgagtacc tatctattat aataaacaag 360

tatgttttat aattattttg atcttgatat acttggatga tggcatatgc agcagctata 420

tgtggatttt tttagccctg ccttcatacg ctatttattt gcttggtact gtttcttttg 480

tcgatgctca ccctgttgtt tggtgttact tctgc 515

<210> 21

<211> 693

<212> DNA

<213> Artificial Sequence

<220>

<223> double 35S promoter

<400> 21

gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc 60

ctcggattcc attgcccagc tatctgtcac ttgatcaaaa ggacagtaga aaaggaaggt 120

ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc 180

gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 240

ccaaccacgt cttcaaagca agtgaattga tgtgataaca tggtggagca cgacactctc 300

gtctactcca agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt 360

caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat cggtcacttg 420

atcaaaagga cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga 480

aaggctatcg ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg 540

aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt 600

gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca agacccttcc 660

tctatataag gaagttcatt tcatttggag agg 693

<210> 22

<211> 4075

<212> DNA

<213> Artificial Sequence

<220>

<223> XVE/OLexA promoter system

<400> 22

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 60

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 120

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 180

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 240

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 300

tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag 360

cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg 420

ttggacttgc tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg 480

gcacggcagg cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc 540

caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 600

ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 660

tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 720

taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 780

atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 840

cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 900

ctgggatggc tctagccgtt ccgcagacgg gatcgatcta ggataggtat acatgttgat 960

gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1020

cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1080

acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1140

ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1200

tctgcaggta ctagtattcc gggcggaatg aaagcgttaa cggccaggca acaagaggtg 1260

tttgatctca tccgtgatca catcagccag acaggtatgc cgccgacgcg tgcggaaatc 1320

gcgcagcgtt tggggttccg ttccccaaac gcggctgaag aacatctgaa ggcgctggca 1380

cgcaaaggcg ttattgaaat tgtttccggc gcatcacgcg ggattcgtct gttgcaggaa 1440

gaggaagaag ggttgccgct ggtaggtcgt gtggctgccg gtgaaccgtc gagcgccccc 1500

ccgaccgatg tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg 1560

catgccgacg cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt 1620

ccgggattta ccccccacga ctccgccccc tacggcgctc tggatatggc cgacttcgag 1680

tttgagcaga tgtttaccga tgcccttgga attgacgagt acggtgggga tccgtctgct 1740

ggagacatga gagctgccaa cctttggcca agcccgctca tgatcaaacg ctctaagaag 1800

aacagcctgg ccttgtccct gacggccgac cagatggtca gtgccttgtt ggatgctgag 1860

ccccccatac tctattccga gtatgatcct accagaccct tcagtgaagc ttcgatgatg 1920

ggcttactga ccaacctggc agacagggag ctggttcaca tgatcaactg ggcgaagagg 1980

gtgccaggct ttgtggattt gaccctccat gatcaggtcc accttctaga atgtgcctgg 2040

ctagagatcc tgatgattgg tctcgtctgg cgctccatgg agcacccagg gaagctactg 2100

tttgctccta acttgctctt ggacaggaac cagggaaaat gtgtagaggg catggtggag 2160

atcttcgaca tgctgctggc tacatcatct cggttccgca tgatgaatct gcagggagag 2220

gagtttgtgt gcctcaaatc tattattttg cttaattctg gagtgtacac atttctgtcc 2280

agcaccctga agtctctgga agagaaggac catatccacc gagtcctgga caagatcaca 2340

gacactttga tccacctgat ggccaaggca ggcctgaccc tgcagcagca gcaccagcgg 2400

ctggcccagc tcctcctcat cctctcccac atcaggcaca tgagtaacaa aggcatggag 2460

catctgtaca gcatgaagtg caagaacgtg gtgcccctct atgacctgct gctggagatg 2520

ctggacgccc accgcctaca tgcgcccact agccgtggag gggcatccgt ggaggagacg 2580

gaccaaagcc acttggccac tgcgggctct acttcatcgc attccttgca aaagtattac 2640

atcacggggg aggcagaggg tttccctgcc acagtctgag agctccctgg cggaattccc 2700

agagatgtta gctgaaatca tcactaatca gataccaaaa tattcaaatg gaaatatcaa 2760

aaagcttctg tttcatcaaa aatgactcga cctaactgag taagctagct tgttcgagta 2820

ttatggcatt gggaaaactg tttttcttgt accatttgtt gtgcttgtaa tttactgtgt 2880

tttttattcg gttttcgcta tcgaactgtg aaatggaaat ggatggagaa gagttaatga 2940

atgatatggt ccttttgttc attctcaaat taatattatt tgttttttct cttatttgtt 3000

gtgtgttgaa tttgaaatta taagagatat gcaaacattt tgttttgagt aaaaatgtgt 3060

caaatcgtgg cctctaatga ccgaagttaa tatgaggagt aaaacactag atccccaaac 3120

aagcttggaa actgaaggcg ctcgagttac tagatcgggg aattgatccc ccctcgacag 3180

cttgcatgcc gcttgggctg caggtcgagg ctaaaaaact aatcgcatta tcatcccctc 3240

gacgtactgt acatataacc actggtttta tatacagcag tactgtacat ataaccactg 3300

gttttatata cagcagtcga cgtactgtac atataaccac tggttttata tacagcagta 3360

ctgtacatat aaccactggt tttatataca gcagtcgagg taagattaga tatggatatg 3420

tatatggata tgtatatggt ggtaatgcca tgtaatatgc tcgactctag gatcttcgca 3480

agacccttcc tctatataag gaagttcatt tcatttggag aggacacgct gaagctagtc 3540

gactctagcc tccctaggct gccggacgac gagctcctcc cccctccccc tccgccgccg 3600

ccgcgccggt aaccaccccg cccctctcct ctttctttct ccgttttttt ttccgtctcg 3660

gtctcgatct ttggccttgg tagtttgggt gggcgagagg cggcttcgtg cgcgcccaga 3720

tcggtgcgcg ggaggggcgg gatctcgcgg ctggggctct cgccggcgtg gatccggccc 3780

ggatctcgcg gggaatgggg ctctcggatg tagatctgcg atccgccgtt gttgggggag 3840

atgatggggg gtttaaaatt tccgccatgc taaacaagat caggaagagg ggaaaagggc 3900

actatggttt atatttttat atatttctgc tgcttcgtca ggcttagatg tgctagatct 3960

tcttctttct ttcttctttt tgtgggtaga atttgaatcc ctcagcattg ttcatcggta 4020

gtttttcttt tcatgatttg tgacaaatgc agcctcgtgc ggagcttttt tgtag 4075

<210> 23

<211> 687

<212> DNA

<213> Zea mays

<400> 23