阅读说明：本技术 结构特异性识别蛋白1(ssrp1)核酸分子用来控制昆虫害虫 (Use of structure specific recognition protein 1(SSRP1) nucleic acid molecules for controlling insect pests ) 是由 肯尼·E·纳瓦 耿朝现 梅根·弗雷 普莱姆钱德·甘德拉 安德烈亚斯·维尔辛斯卡斯 凯瑟琳· 于 2018-05-18 设计创作，主要内容包括：本公开涉及核酸分子及使用其控制昆虫害虫的方法,该方法通过对包括花粉甲虫的昆虫害虫中的靶编码序列和转录非编码序列进行RNA干扰介导的抑制来实现。本公开还涉及制备转基因植物的方法,所述转基因植物表达可用于控制昆虫害虫的核酸分子；以及由此获得的植物细胞和植物。(The present disclosure relates to nucleic acid molecules and methods of using the same for controlling insect pests by RNA interference-mediated inhibition of target coding and transcribed non-coding sequences in insect pests, including pollen beetles. The present disclosure also relates to methods of making transgenic plants that express nucleic acid molecules useful for controlling insect pests; and plant cells and plants obtained therefrom.)

1. An isolated nucleic acid molecule comprising at least one polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

1, SEQ ID NO; 1 or the reverse complement of SEQ ID NO; a fragment of at least 15 contiguous nucleotides of an endogenously encoded polynucleotide from Brassica napus (Meligethe aeneuus Fabricius) comprising SEQ ID NOs 2-3; a complement or reverse complement of a fragment of at least 15 contiguous nucleotides of an endogenous coding polynucleotide from brassica napus luo ca comprising SEQ ID NOs 2-3; the native coding sequence of a Nitrocellus (Meligethes) organism comprising SEQ ID NO 4; a complementary sequence or reverse complementary sequence comprising the natural coding sequence of the Nitrocera organism of SEQ ID NO 4; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Nitidus organism comprising SEQ ID NO 4; a complement or reverse complement of a fragment comprising at least 15 contiguous nucleotides of the natural coding sequence of the Nitrocera organism of SEQ ID NO. 4.

2. The nucleic acid molecule of claim 1, wherein the nucleotide sequence is selected from the group consisting of: 2-4 of SEQ ID NO; a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs 2-4; as well as the aforementioned complementary sequences and reverse complementary sequences.

3. The nucleic acid molecule of claim 1, wherein the molecule is a vector.

4. An isolated nucleic acid molecule characterized by a polynucleotide operably linked to a heterologous promoter, wherein said polynucleotide is: 4, SEQ ID NO; the complement of SEQ ID NO. 4, or the reverse complement of SEQ ID NO. 4.

5. A ribonucleic acid (RNA) molecule encoded by the nucleic acid molecule of claim 1, wherein the RNA molecule comprises a polyribonucleotide encoded by the nucleotide sequence.

6. The RNA molecule of claim 5, wherein the molecule is a double-stranded ribonucleic acid (dsRNA) molecule.

7. The dsRNA molecule of claim 6, wherein contacting the polyribonucleotide with an insect pest inhibits expression of an endogenous nucleic acid molecule specifically complementary to the polyribonucleotide.

8. The dsRNA molecule of claim 7, wherein contacting the polyribonucleotide with the insect pest kills or inhibits growth and/or feeding of the pest.

9. The dsRNA of claim 6, comprising first, second and third polyribonucleotides, wherein said first polyribonucleotide is transcribed from said polynucleotide, wherein said third polyribonucleotide is linked to said first polyribonucleotide by said second polyribonucleotide, and wherein said third polyribonucleotide is substantially the reverse complement of said first polyribonucleotide, such that said first and said third polyribonucleotides hybridize when transcribed into a ribonucleic acid to form the dsRNA.

10. The dsRNA of claim 6, wherein said molecule comprises first and second polyribonucleotides, wherein said first polyribonucleotide is transcribed from said polynucleotide, wherein said third polyribonucleotide is a separate strand from said second polyribonucleotide, and wherein said first and said second polyribonucleotides hybridize to form said dsRNA.

11. The vector of claim 3, wherein the vector is a plant transformation vector, and wherein the heterologous promoter is functional in a plant cell.

12. A cell comprising the nucleic acid molecule of claim 1.

13. The cell of claim 12, wherein the cell is a prokaryotic cell.

14. The cell of claim 12, wherein the cell is a eukaryotic cell.

15. The cell of claim 14, wherein the cell is a plant cell.

16. A plant comprising the nucleic acid molecule of claim 1.

17. A part of a plant according to claim 16, wherein said plant part comprises said nucleic acid molecule.

18. The plant part of claim 17, wherein said plant part is a seed.

19. A food product or commodity product produced from the plant of claim 16, wherein the product comprises a detectable amount of the polynucleotide.

20. The plant of claim 16, wherein the polynucleotide is expressed in the plant as a double-stranded ribonucleic acid (dsRNA) molecule.

21. The plant cell of claim 15, wherein the cell is a cell from a Brassica (Brassica) plant species.

22. The plant of claim 16, wherein the plant is a brassica plant species.

23. The plant of claim 16, wherein the polynucleotide is expressed in the plant as a double-stranded ribonucleic acid (dsRNA) molecule, and the dsRNA molecule inhibits expression of an endogenous polynucleotide that is specifically complementary to the RNA molecule when an insect pest ingests a portion of the plant.

24. The nucleic acid molecule of claim 1, further comprising at least one additional polynucleotide operably linked to a heterologous promoter, wherein the additional polynucleotide encodes an RNA molecule.

25. The nucleic acid molecule of claim 24, wherein the molecule is a plant transformation vector, and wherein the heterologous promoter is functional in a plant cell.

26. A method of controlling an insect pest population, the method comprising providing an agent comprising a ribonucleic acid (RNA) molecule that functions to inhibit a biological function within the insect pest when contacted with the insect pest, wherein the RNA molecule comprises a polyribonucleotide that is specifically hybridizable to a target polyribonucleotide selected from the group consisting of: 12-15 of SEQ ID NO; the complement of any one of SEQ ID Nos. 12 to 15; the reverse complement of any one of SEQ ID NOs 12-15; a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOS 13-15; the complement of a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOS 13-15; 13-15, the reverse complement of a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs; a transcript of a PB ssrp1 encoding polynucleotide comprising SEQ ID NOS: 2-3; the complement of a transcript of a PB ssrp1 encoding polynucleotide comprising SEQ ID NOS: 2-3; the reverse complement of a transcript of the PB ssrp1 encoding polynucleotide comprising SEQ ID NOS: 2-3; a fragment comprising at least 15 contiguous nucleotides of a transcript of the PB ssrp1 encoding polynucleotide of SEQ ID NOS: 2-3; the complement of a fragment comprising at least 15 contiguous nucleotides of the transcript of PB ssrp1 encoding polynucleotide of SEQ ID NOS: 2-3; and the reverse complement of a fragment comprising at least 15 contiguous nucleotides of the transcript of the PB ssrp1 encoding polynucleotide of SEQ ID Nos. 2-3.

27. The method of claim 26, wherein the RNA molecule is a double-stranded RNA (dsrna) molecule.

28. The method of claim 27, wherein providing the agent comprises contacting the insect pest with a sprayable composition comprising the agent or feeding the insect pest to an RNA bait comprising the agent.

29. The method of claim 27, wherein providing the agent is a transgenic plant cell expressing the dsRNA molecule.

30. A method of controlling an insect pest population, the method comprising:

providing an agent comprising first and second polyribonucleotides that, upon contact with an insect pest, act to inhibit a biological function within the insect pest, wherein the first polyribonucleotide comprises a nucleotide sequence having about 90% to about 100% sequence identity to about 15 to about 30 consecutive nucleotides of a polyribonucleotide selected from the group consisting of SEQ ID NOS 13-15, and wherein the first polyribonucleotide specifically hybridizes to the second polyribonucleotide.

31. A method of controlling an insect pest population, the method comprising:

providing in a host plant of an insect pest a plant cell comprising the nucleic acid molecule of claim 1, wherein the polynucleotide is expressed to produce a double-stranded ribonucleic acid (dsRNA) molecule that, upon contact with an insect pest belonging to the population, acts to inhibit expression of a target sequence within the insect pest and results in reduced growth and/or survival of the insect pest or pest population relative to the development of the same pest species on a plant of the same host plant species that does not comprise the polynucleotide.

32. The method of claim 31, wherein the insect pest population is reduced relative to a population of the same pest species that infests a host plant of the same host plant species lacking the plant cell comprising the nucleic acid molecule.

33. A method of controlling insect pest infestation in a plant, the method comprising providing in the diet of the insect pest a ribonucleic acid (RNA) molecule comprising a polyribonucleotide that can specifically hybridize to a reference polyribonucleotide selected from the group consisting of:

SEQ ID NO:12-15；

the complement or reverse complement of any one of SEQ ID NOs 12-15;

a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOS 13-15;

the complement or reverse complement of a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs 13-15;

a transcript of a PB ssrp1 encoding polynucleotide comprising SEQ ID NOS: 2-3;

the complement or reverse complement of a transcript of a PB ssrp1 encoding polynucleotide comprising SEQ ID NOS: 2-3;

a fragment comprising at least 15 contiguous nucleotides of a transcript of the PB ssrp1 encoding polynucleotide of SEQ ID NOS: 2-3; and

the complement or reverse complement of a fragment comprising at least 15 contiguous nucleotides of the PB ssrp1 encoding polynucleotide of SEQ ID NOS: 2-3.

34. The method of claim 33, wherein the RNA molecule is a double-stranded RNA (dsrna) molecule.

35. The method of claim 34, wherein the diet comprises plant cells comprising a polynucleotide transcribed to express the dsRNA molecule.

36. A method of increasing crop yield, the method comprising:

growing a plant comprising the nucleic acid of claim 1 in the crop plant to allow expression of the polynucleotide.

37. The method of claim 36, wherein the plant is a brassica species.

38. The method of claim 36, wherein expression of the polynucleotide produces a double-stranded rna (dsrna) molecule that inhibits a target gene in an insect pest that has contacted a portion of the plant, thereby inhibiting development or growth of the insect pest and yield loss caused by infection of the insect pest.

39. A method of producing a transgenic plant cell, the method comprising:

transforming a plant cell with the plant transformation vector of claim 11;

culturing the transformed plant cell under conditions sufficient to allow development of a plant cell culture comprising a plurality of transgenic plant cells;

selecting a transgenic plant cell that has integrated the polynucleotide into its genome;

screening the transgenic plant cell for expression of a double-stranded ribonucleic acid (dsRNA) molecule encoded by the polynucleotide; and

selecting a transgenic plant cell expressing said dsRNA.

40. A method of producing a transgenic plant resistant to an insect pest, the method comprising:

regenerating a transgenic plant from a transgenic plant cell comprising the nucleic acid molecule of claim 1, wherein expression of a double-stranded ribonucleic acid (dsRNA) molecule encoded by the polynucleotide is sufficient to modulate expression of a target gene in an insect pest when the insect pest contacts the RNA molecule.

41. A method of producing a transgenic plant cell, the method comprising:

transforming a plant cell with a vector comprising a means for providing ssrp 1-mediated protection of a plant from the louse nori pest;

culturing a plurality of transformed plant cells under conditions sufficient to allow development of a plant cell culture comprising the transformed plant cells;

selecting a transformed plant cell that has integrated into its genome said means for providing ssrp 1-mediated protection of a louse pest to a plant;

screening said transformed plant cells for the expression of a means for inhibiting expression of ssrp1 gene in a louse pest; and

selecting plant cells expressing the means for inhibiting expression of ssrp1 gene in a trichina pest.

42. A method of producing a transgenic plant, the method comprising:

regenerating a transgenic plant from a transgenic plant cell produced by the method of claim 40, wherein the plant cell of said plant comprises a means for inhibiting expression of ssrp1 gene in a Nitida pest.

43. The method of claim 42, wherein expression of the means for inhibiting expression of ssrp1 gene in a Nitraria pest is sufficient to reduce expression of the target ssrp1 gene in a Nitraria pest that infests the transgenic plant.

44. A plant comprising a means for inhibiting expression of ssrp1 gene in a nordlia pest.

45. The nucleic acid of claim 1, further comprising a polynucleotide encoding an insecticidal polypeptide from Bacillus thuringiensis (Bacillus thuringiensis), Alcaligenes spp.

46. The nucleic acid of claim 44, wherein said insecticidal polypeptide is selected from the group consisting of Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C Bacillus thuringiensis insecticidal polypeptides.

47. The plant cell of claim 15, wherein said cell comprises a polynucleotide encoding a pesticidal polypeptide from bacillus thuringiensis, alcaligenes, or pseudomonas.

48. The plant cell of claim 47, wherein said insecticidal polypeptide is selected from the group consisting of Cry1B, Cry1I, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt 2C.

49. The plant of claim 16, wherein the plant comprises a polynucleotide encoding a pesticidal polypeptide from bacillus thuringiensis, alcaligenes, or pseudomonas.

50. The plant of claim 49, wherein said insecticidal polypeptide is selected from the group consisting of Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C, a Bacillus thuringiensis insecticidal polypeptide.

51. The method of claim 31, wherein the plant cell comprises a polynucleotide encoding a pesticidal polypeptide from bacillus thuringiensis, alcaligenes, or pseudomonas.

52. The method according to claim 51, wherein said insecticidal polypeptide is selected from the group consisting of Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C Bacillus thuringiensis insecticidal polypeptides.

Technical Field

The present invention relates generally to genetic control of plant damage caused by insect pests (e.g., pollen beetles). In particular embodiments, the invention relates to the identification of target coding and non-coding polynucleotides, and the use of recombinant DNA techniques to post-transcriptionally repress or inhibit the expression of target coding and non-coding polynucleotides in insect pest cells, thereby providing protection to a plant.

Background

European Pollen Beetles (PB) are a serious pest of rape, and both larvae and adults feed on flowers and pollen. Damage to crops by pollen beetles can cause 20% to 40% yield loss. The major pest species is rape pollen beetle (Meligethe aeneus). Currently, control of pollen beetles in oilseed rape relies primarily on pyrethroids, which are expected to be rapidly eliminated in view of the environmental and regulatory conditions of such substances. In addition, resistance of pollen beetles to existing chemical pesticides has been reported. Therefore, there is an urgent need for an environmentally friendly pollen beetle control scheme with a novel mechanism of action.

In nature, pollen beetles live through the winter as adults in the soil or under the deciduous layer. In the spring, adults emerge from hibernation and begin to feed on the flowers of the weeds and migrate to the flowering rape plants. Lay eggs in rape flower buds. The larvae develop in and feed on flower buds and flowers. The later stage larvae find pupation sites in the soil. Second generation adults emerged in seventy-eight months and feed on a variety of flowering plants before finding a place to live through winter.

RNA interference (RNAi) is a method that utilizes an endogenous cellular pathway whereby Interfering RNA (iRNA) molecules (e.g., dsRNA molecules) specific for the entire target gene or any portion of the target gene that is sufficiently large in size cause degradation of the mRNA encoded by the target gene. In recent years, RNAi has been used to perform gene "knockdown" in many species and experimental systems, such as cells in Caenorhabditis elegans (Caenorhabditis elegans), plants, insect embryos, and tissue cultures. See, e.g., Fire et al, (1998) Nature 391: 806-11; martinez et al, (2002) Cell 110: 563-74; McManus and Sharp, (2002) Nature Rev. genetics 3: 737-47.

RNAi effects mRNA degradation via an endogenous pathway involving the DICER protein complex. DICER cleaves long dsRNA molecules into short fragments of approximately 20 nucleotides, called small interfering RNAs (sirnas). siRNA unwinds into two single-stranded RNAs: passenger strand (passenger strand) and guide strand (guide strand). The passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC).

The authors of us patent 7,612,194 and us patent publication No. 2007/0050860 demonstrated that in situ (in planta) RNAi of plants can serve as a potential pest management tool in the context of providing plant protection against western corn rootworm (d.v. virgifera LeConte), while demonstrating that even for a relatively small set of candidate genes, no effective RNAi target can be identified accurately a priori. Baum et al, (2007) nat. Biotechnol.25(11) 1322-6. By using the high throughput in vivo dietary RNAi system to screen potential target genes to develop transgenic RNAi maize, researchers found that only 14 of the first 290 target gene pools showed larval control potential.

Disclosure of Invention

Disclosed herein are nucleic acid molecules (e.g., target genes, DNA, dsRNA, siRNA, miRNA, shRNA, and hpRNAs) and methods of use thereof for controlling insect pests including, for example, canola beetle (meligethe aeneus Fabricius) (pollen beetle, "PB"). In particular examples, exemplary nucleic acid molecules are disclosed that can be homologous to at least a portion of one or more native nucleic acids in the PB.

In these and other examples, the native nucleic acid sequence may be a target gene, the product of which may be, for example, but not limited to: participate in metabolic processes; or participate in the development of larvae. In some examples, post-transcriptional inhibition of target gene expression by a nucleic acid molecule comprising a polynucleotide homologous to the target gene may be lethal to PB or result in reduced growth and/or development of PB. In a particular example, a structure specific recognition protein 1 (structural specific recognitionprotein 1), referred to herein as ssrp1) or ssrp1 homologue can be selected as the target gene for post-transcriptional silencing. In particular examples, the target gene that can be used for post-transcriptional inhibition is PB ssrp 1; SEQ ID NO:1 (i.e., a PBssrp1 polynucleotide characterized by the inclusion of SEQ ID NOS: 2-3). Thus, disclosed herein are isolated nucleic acid molecules comprising a polynucleotide of SEQ ID NO. 1; a PB ssrp1 polynucleotide comprising SEQ ID NOS: 2-3; fragments of PB ssrp1 (i.e., SEQ ID NOS: 2-4); and/or a complement or reverse complement of any of the foregoing.

Also disclosed are nucleic acid molecules comprising a polynucleotide encoding a polypeptide that is at least about 85% identical to an amino acid sequence within a target gene product (e.g., the PB ssrp1 product). For example, a nucleic acid molecule may comprise a polynucleotide encoding: a polypeptide having at least 85% identity to PB SSRP 1; 5 (i.e., the SSRP1 polypeptide characterized by comprising SEQ ID NOS: 6-7); and/or the amino acid sequence within the ssrp1 gene product (i.e., SEQ ID NOS: 6-7). Also disclosed are nucleic acid molecules comprising a polynucleotide that is the complement or reverse complement of a polynucleotide encoding a polypeptide having at least 85% identity to an amino acid sequence within a target gene product.

Also disclosed are cDNA polynucleotides useful for producing iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecules that are complementary to all or a portion of an insect pest target gene, such as the ssrp1 gene. In particular embodiments, the dsRNA, siRNA, shRNA, miRNA, and/or hpRNA may be produced in vitro or in vivo by a genetically modified organism, such as a plant or bacterium. In particular examples, cDNA molecules are disclosed that can be used to produce iRNA molecules that are complementary or reverse complementary to all or part of ssrp1 (e.g., SEQ ID NO:1, PB ssrp1 polynucleotide characterized as comprising SEQ ID NOS: 2-3), or fragments thereof.

Further disclosed are means (means) for inhibiting the expression of ssrp1 gene in a lounge (meligethe) pest, and means for providing ssrp 1-mediated protection of the lounge pest to a plant. The means for inhibiting the expression of ssrp1 gene in a Nitraria pest is a double stranded RNA molecule wherein one strand of the molecule consists of the polyribonucleotide of SEQ ID NO. 15. Functional equivalents of the means for inhibiting the expression of ssrp1 gene in a Nitidus pest include double stranded RNA molecules comprising polyribonucleotides substantially homologous to all or part of the Nitidus brassicae ssrp1 gene having SEQ ID NOS: 2-3. The means for providing ssrp 1-mediated protection of a plant from a nitida pest is a DNA molecule comprising a polynucleotide encoding a ssrp1 gene for inhibiting expression of a ctrp 1 gene in a nitida pest operably linked to a functional promoter in a plant cell (e.g., a canola (canola) cell).

Additionally, methods for controlling insect pest (e.g., pollen beetle) populations are disclosed that include providing to an insect pest an iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecule that functions when ingested by the pest to inhibit a biological function within the pest. In some embodiments, an iRNA molecule that acts to inhibit a biological function within a pest when ingested by the pest comprises all or part of a polynucleotide selected from the group consisting of: 12 is SEQ ID NO; the complement or reverse complement of SEQ ID NO. 12; 13 in SEQ ID NO; the complement or reverse complement of SEQ ID NO 13; 14, SEQ ID NO; the complement or reverse complement of SEQ ID NO. 14; (ii) native polyribonucleotides from PB comprising SEQ ID NO 13-14; 13-14, the complement or reverse complement of a natural polyribonucleotide from PB; 15, SEQ ID NO; the complement or reverse complement of SEQ ID NO. 15; a polyribonucleotide that hybridizes to a transcript of a naturally-encoded polynucleotide of a clearweed organism (e.g., PB) comprising all or part of any one of SEQ ID NOs 2-4; and the complement or reverse complement of a polyribonucleotide that hybridizes to a transcript of a naturally-encoded polynucleotide of a Nitidus organism comprising all or part of any one of SEQ ID NOs 2-4.

In particular embodiments, the iRNA that acts to inhibit a biological function within the pest when ingested by the insect pest is transcribed from a DNA comprising all or part of a polynucleotide selected from the group consisting of: 1, SEQ ID NO; 1 or the reverse complement of SEQ ID NO; 2-3, a naturally encoded polynucleotide from PB; 2-3, the complement of the naturally encoded polynucleotide from PB; 4, SEQ ID NO; the complement or reverse complement of SEQ ID NO. 4; a naturally-encoded polynucleotide of a Nitidus organism comprising all or a portion of any of SEQ ID NOs 2-4; and a complement or reverse complement of a naturally encoded polynucleotide of a Nitidus organism comprising all or part of any of SEQ ID NOs 2-4.

Also disclosed herein are methods wherein the dsRNA, siRNA, shRNA, miRNA, and/or hpRNA can be provided to an insect pest in a diet-based assay or in a genetically modified plant cell expressing the dsRNA, siRNA, shRNA, miRNA, and/or hpRNA. In these and other examples, the dsRNA, siRNA, shRNA, miRNA, and/or hpRNA may be ingested by the pest. Ingestion of the dsRNA, siRNA, shRNA, miRNA, and/or hpRNA of the invention can then produce RNAi in the pest, which in turn can result in silencing of genes essential for pest viability and ultimately death. In a particular example, the insect pest controlled by use of the nucleic acid molecule of the invention may be pollen beetles (rape pollen beetles).

The foregoing and other features will become more apparent upon consideration of the following detailed description of several embodiments taken in conjunction with the accompanying fig. 1-2.

Drawings

Figure 1 includes a description of the strategy employed to provide dsRNA from a single transcription template using a single pair of primers.

Figure 2 includes a description of the strategy employed to provide dsRNA from two transcription templates.

Sequence listing

The nucleic acid sequences listed in the accompanying sequence listing are represented using standard letter abbreviations for nucleotide bases as defined in 37c.f.r. § 1.822. The listed nucleotide and amino acid sequences define molecules (i.e., polynucleotides and polyribonucleotides, respectively, and polypeptides) having nucleotide and amino acid monomers arranged in the described manner. The listed nucleotide and amino acid sequences also each define a class of polynucleotide/polyribonucleotide or polypeptides comprising nucleotide and amino acid monomers arranged in the described manner. In view of the redundancy of the genetic code, it is understood by those skilled in the art that a nucleotide sequence comprising a coding sequence also describes a class of polynucleotides that encode the same polypeptide as the polynucleotide consisting of the reference sequence. It will also be understood that the amino acid sequence describes a class of polynucleotide ORFs that encode the polypeptide.

Only one strand of each nucleotide sequence is shown, but any reference to the shown strand includes the complementary strand. Since the complement and reverse complement of a primary nucleic acid sequence are also necessarily disclosed by the primary sequence, any reference to a nucleotide sequence includes the complement and reverse complement of the nucleotide sequence unless specifically indicated otherwise (or otherwise evident from the context in which the sequence appears). Furthermore, as is understood in the art, the ribonucleotide sequence of an RNA strand is determined by the sequence of the DNA it transcribes (except for the replacement of thymine (T) with a uracil (U) nucleobase), so any reference to a DNA sequence encoding an RNA sequence includes the RNA sequence. In the accompanying sequence listing:

1 shows an exemplary pollen beetle (coleoptera) ssrp1 DNA, referred to herein in some places as PBssrp 1:

SEQ ID NO:2 shows a characteristic fragment of an exemplary pollen beetle ssrp1 DNA:

FIG. 3 shows another characteristic fragment of the exemplary pollen beetle ssrp1 DNA:

SEQ ID No. 4 shows another exemplary genus meligetes (Meligethes) ssrp1 DNA, referred to herein in some places as PB ssrp1 reg1 (region 1), which in some instances is used to produce dsRNA:

SEQ ID NO:5 shows the amino acid sequence of the Nitraria SSRP1 polypeptide encoded by exemplary PB SSRP1 DNA:

SEQ ID NO 6 shows the characteristic amino acid sequence of the Nitidus SSRP1 polypeptide:

SEQ ID NO 7 shows another characteristic amino acid sequence of the Nitraria SSRP1 polypeptide:

the nucleotide sequence of the T7 phage promoter is shown in SEQ ID NO 8.

9-10 show primers for PCR amplification of the ssrp1 sequence, including PB ssrp1 reg1 used in some examples to generate dsRNA, the ssrp1 sequence.

Exemplary DNA encoding PB ssrp1 reg1 hairpin-forming RNA is shown in SEQ ID NO:11, comprising a sense nucleotide sequence, a loop sequence comprising an intron (underlined) and an antisense nucleotide sequence (bold): .

12-16 show exemplary RNAs transcribed from and processed from exemplary ssrp1 polynucleotide and fragments thereof, for example, by DICER activity.

Detailed Description

I. Brief summary of several embodiments

We have developed RNA interference (RNAi) as a pest management tool using the possible target pest species, european pollen beetle, to transgenic plants expressing dsRNA. Herein, we describe RNAi-mediated knockdown of structure-specific recognition protein 1(ssrp1) in the exemplary insect pest european pollen beetle, e.g., shown to have a lethal phenotype for european pollen beetles when iRNA molecules are delivered via ingestion or injection of ssrp1 dsRNA. In embodiments herein, the ability to deliver ssrp1dsRNA by feeding an insect confers RNAi effects, which are very useful for the management of insect pests. By combining ssrp 1-mediated RNAi with other useful RNAi targets, the possibility of influencing (e.g., by inhibiting the target sequence with multiple modes of action to achieve synergistic control) multiple target sequences increases the opportunity to develop sustainable insect pest management approaches involving RNAi technology.

Disclosed herein are methods and compositions for genetically controlling insect (e.g., PB) pest infestation. Also provided are methods of identifying one or more genes essential to the life cycle of an insect pest for use as target genes for RNAi-mediated control of insect pest populations. DNA plasmid vectors encoding RNA molecules can be designed to repress (represses) one or more target genes necessary for growth, survival and/or development. In some embodiments, methods of post-transcriptionally repressing (post-transcriptional repression) a target gene expression or inhibiting a target gene via a nucleic acid molecule complementary to a coding or non-coding sequence of the target gene in an insect pest are provided. In these and other embodiments, the pest may ingest one or more dsRNA, siRNA, shRNA, miRNA, and/or hpRNA molecules transcribed from all or a portion of a nucleic acid molecule that is complementary to a coding or non-coding sequence of a target gene, thereby providing a plant protective effect. Thus, some embodiments relate to sequence-specific inhibition of expression of a target gene product using dsRNA, siRNA, shRNA, miRNA, and/or hpRNA complementary to coding and/or non-coding sequences of one or more target genes to achieve at least partial control of an insect (e.g., Coleoptera) pest. In some embodiments, the dsRNA molecule (e.g., SEQ ID NO:16) may be capable of forming a miRNA or siRNA molecule of 21-23 ribonucleotides in length, for example, by processing the dsRNA with DICER enzymes.

Disclosed are isolated and purified nucleic acid molecules characterized by a polynucleotide comprising at least one nucleotide sequence (e.g., the sequences shown in SEQ ID NO:1 and SEQ ID NOS: 2-3, fragments thereof, and the complementary sequences and reverse complementary sequences described above). In some embodiments, stabilized dsRNA molecules can be expressed from these polynucleotides, fragments thereof, or genes comprising one or more of these polynucleotides for post-transcriptional silencing or suppression of target genes. In certain embodiments, the isolated and purified nucleic acid molecule comprises all or a portion of the PB ssrp1 polynucleotide comprising SEQ ID NO:1, SEQ ID NOS: 2-3 (e.g., SEQ ID NO:4), and/or a complement or reverse complement thereof.

Some embodiments relate to a recombinant host cell (e.g., a plant cell) having in its genome at least one recombinant DNA encoding at least one iRNA (e.g., dsRNA) molecule. In particular embodiments, the iRNA molecule can be provided upon ingestion by an insect pest, thereby post-transcriptionally silencing or inhibiting expression of a target gene in the pest. The recombinant DNA may comprise, for example: 1, SEQ ID NO; all or a portion of the PB ssrp1 polynucleotide comprising SEQ ID NOS: 2-3; a fragment of the PB ssrp1 polynucleotide comprising SEQ ID NOS: 2-3; 4, SEQ ID NO; a polynucleotide consisting of a partial sequence of a gene comprising one of SEQ ID NOs 2 to 4; the complementary sequences described above; and/or the reverse complement of the above.

Some embodiments relate to a recombinant host cell having in its genome recombinant DNA encoding at least one iRNA (e.g., dsRNA) molecule comprising a ribonucleotide sequence selected from the group consisting of: 12 is SEQ ID NO; all or part of a PB polyribonucleotide comprising SEQ ID NO. 13-14; as well as the aforementioned complementary sequences and reverse complementary sequences. When ingested by an insect pest (e.g., PB), the iRNA molecule can silence or inhibit expression of a target ssrp1 DNA (e.g., a DNA comprising all or a portion of the PB ssrp1 polynucleotide comprising SEQ ID NOs: 2-3 (SEQ ID NO: 4)) in the pest, thereby causing the pest to stop growth, development, and/or feeding.

In some embodiments, a recombinant host cell having in its genome at least one recombinant DNA encoding at least one RNA molecule capable of forming a dsRNA molecule may be a transformed plant cell. Some embodiments relate to transgenic plants comprising such transformed plant cells. In addition to such transgenic plants, progeny plants of any transgenic plant generation, transgenic seeds, and transgenic plant products are also provided, each of which comprises one or more recombinant DNA. In particular embodiments, RNA molecules capable of forming dsRNA molecules can be expressed in transgenic plant cells. Thus, in these and other embodiments, dsRNA molecules can be isolated from transgenic plant cells. In a particular embodiment, the transgenic plant is a plant selected from the group comprising brassicaceae (Brassica) plants, such as Brassica napus (Brassica napus).

Some embodiments relate to methods of modulating the expression of a target gene in an insect pest cell. In these and other embodiments, a nucleic acid molecule may be provided, wherein the nucleic acid molecule comprises a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule. In particular embodiments, a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule can be operably linked to a promoter, and can also be operably linked to a transcription termination sequence. In particular embodiments, a method for modulating expression of a target gene in an insect pest cell can comprise: (a) transforming a plant cell with a vector comprising a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule; (b) culturing the transformed plant cell under conditions sufficient to allow development of a plant cell culture comprising a plurality of transformed plant cells; (c) selecting a transformed plant cell that has integrated the polynucleotide into its genome; and (d) determining that the selected transformed plant cell comprises an RNA molecule capable of forming a dsRNA molecule encoded by the polynucleotide. A plant can be regenerated from a plant cell that has the polynucleotide integrated in its genome and that comprises a dsRNA molecule encoded by the polynucleotide.

Accordingly, also disclosed is a transgenic plant having integrated in its genome a polynucleotide encoding a dsRNA molecule, wherein the transgenic plant comprises the dsRNA molecule encoded by the polynucleotide. In particular embodiments, expression of the dsRNA molecule in a plant is sufficient to modulate expression of a target gene in an insect pest cell that is in contact with the transformed plant or plant cell (e.g., by feeding on the transformed plant, a portion of the plant (e.g., a leaf), or a plant cell) such that growth and/or survival of the pest is inhibited. The transgenic plants disclosed herein can exhibit resistance to and/or enhanced tolerance to attack by an insect pest. Particular transgenic plants may exhibit resistance and/or enhanced protection against brassica napus pollen beetles.

Also disclosed herein are methods of delivering control agents (such as iRNA molecules) to insect pests. Such control agents may directly or indirectly impair the ability of an insect pest population to feed on, grow, or otherwise cause damage to a host. In some embodiments, a method is provided comprising delivering a stabilized dsRNA molecule to an insect pest to repress at least one target gene in the pest, thereby causing RNAi and reducing or eliminating plant damage in the pest host. In some embodiments, the method of inhibiting expression of a target gene in an insect pest can cause growth, survival, and/or cessation of development of the pest.

In some embodiments, compositions (e.g., topical compositions) comprising iRNA (e.g., dsRNA) molecules for use by plants, insects, and/or in the environment of plants or insects are provided to achieve elimination or mitigation of insect pest infestation. In particular embodiments, the composition may be a nutritional composition or food source to be fed to the insect pest. Some embodiments include making the nutritional composition or food source available to pests. Ingestion of a composition comprising an iRNA molecule can result in uptake of the molecule by one or more cells of the pest, which in turn can result in inhibition of expression of at least one target gene in the one or more cells of the pest. By providing one or more compositions comprising iRNA molecules in a host of a pest, uptake or damage to a plant or plant cell caused by infestation by the insect pest can be limited or eliminated in or on any host tissue or environment in which the pest is present.

The compositions and methods disclosed herein can be used in combination with other methods and compositions for controlling damage caused by insect pests. For example, iRNA molecules as described herein for protecting plants from insect pests can be used in such methods: the methods include the additional use of one or more chemical agents effective against insect pests, biopesticides effective against such pests, crop rotation, recombinant genetic techniques that exhibit characteristics different from those of RNAi-mediated methods and RNAi compositions (e.g., recombinant production of proteins (e.g., Bt toxins and PIP-1 polypeptides (see U.S. patent publication No. US2014/0007292a1)) that are harmful to insect pests in plants, and/or recombinant expression of other iRNA molecules.

Abbreviation of II

dsRNA double-stranded ribonucleic acid

EST expression sequence tag

NCBI national center for Biotechnology information

gDNA genomic DNA

iRNA inhibitory ribonucleic acid

ORF open reading frame

RNAi ribonucleic acid interference

miRNA micro ribonucleic acid

shRNA short hairpin ribonucleic acid

Small inhibitory ribonucleic acid (siRNA)

hpRNA hairpin ribonucleic acid

UTR untranslated region

PB pollen beetle (rape outcrop beetle)

PCR polymerase chain reaction

qPCR quantitative polymerase chain reaction

RISC RNA-induced silencing complexes

Standard error of SEM mean

Term of

In the following description and tables, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims (including the scope to be accorded such terms), the following definitions are provided:

coleopteran pests: as used herein, the term "coleopteran pest" refers to a coleopteran (Coleoptera) pest insect, and specifically includes a louse pest insect that feeds on crops and crop products, including canola (canola). In a particular example, the coleopteran pest is a coleopteran beetle.

(with the organism): as used herein, terms such as "in contact with" or "taken up by" an organism (e.g., an insect pest) include, with respect to a nucleic acid molecule, internalization of the nucleic acid molecule into the organism, including, for example and without limitation: the organism ingests the molecule (e.g., by eating); contacting an organism with a composition comprising a nucleic acid molecule; and soaking the organism with a solution comprising the nucleic acid molecule.

Contig (contig): as used herein, the term "contig" refers to a DNA sequence that is reconstructed from a set of overlapping DNA segments derived from a single genetic source.

Expressing: as used herein, "expression" of an encoding polynucleotide (e.g., a gene or transgene) refers to the process by which the encoded information of a nucleic acid transcription unit (including, for example, gDNA or cDNA) is converted into a manipulated, non-manipulated or structural portion of a cell, typically including the synthesis of a protein. Gene expression may be affected by external signals, such as exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Gene expression can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, by controlling transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or by activation, inactivation, compartmentalization, or degradation after a specific protein molecule has been produced, or a combination of these. Gene expression can be measured at the RNA or protein level by any method known in the art, including but not limited to northern blotting, RT-PCR, western blotting, or in vitro, in situ, or in vivo protein activity assays.

Genetic material: as used herein, the term "genetic material" includes all genes and nucleic acid molecules, such as DNA and RNA.

Inhibition: as used herein, the term "inhibit" when used to describe the action on an encoding polynucleotide (e.g., a gene) means that mRNA transcribed from the encoding polynucleotide and/or the peptide, polypeptide, or protein product of the encoding polynucleotide is measurably reduced at the cellular level. In some examples, inhibiting expression of the encoding polynucleotide can result in an approximate disappearance of expression. By "specifically inhibits" is meant inhibiting the target-encoding polynucleotide in the cell in which specific inhibition is being achieved, without ultimately affecting the expression of other encoding polynucleotides (e.g., genes).

Insects: as used herein, the term "insect pest" specifically includes pollen beetles, in relation to pests.

After separation: an "isolated" biological component (such as a nucleic acid molecule or protein) has been substantially separated, produced separately, or purified away from other biological components (i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins) in the cell of the organism in which the component naturally occurs, while effecting a chemical or functional change in the component (e.g., a polynucleotide can be isolated from a chromosome by breaking a chemical bond that links the polynucleotide to the remaining DNA in the chromosome). Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also includes RNA molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules, proteins, and peptides.

Nucleic acid molecule (A): as used herein, the term "nucleic acid molecule" may refer to a polymeric form of nucleotides, which may include both sense and antisense strands of RNA, cDNA, gDNA, as well as synthetic forms and mixed polymers of the foregoing. A nucleotide or nucleobase may refer to a ribonucleotide, a deoxyribonucleotide, or a modified form of either type of nucleotide. As used herein, a "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise indicated, nucleic acid molecules are typically at least 10 bases in length. Conventionally, the nucleotide sequence of a nucleic acid molecule is read from the 5 'end to the 3' end of the molecule. "complementary sequence" of a nucleic acid molecule refers to a polynucleotide having nucleobases that can form base pairs (i.e., A-T/U and G-C) with the nucleobases of the nucleic acid molecule.

Some embodiments include nucleic acids that: which comprises a template DNA transcribed into an RNA molecule comprising a polyribonucleotide hybridized to an mRNA molecule. In some examples, the template DNA is the complement of the polynucleotide that is transcribed into the mRNA molecule, which is present in the 5 'to 3' direction, such that RNA polymerase (which transcribes the DNA in the 5 'to 3' direction) will transcribe polyribonucleotides from this complement that can hybridize to the mRNA molecule. Thus, unless otherwise specified or clear from context to be so, the term "complementary sequence" refers to a polynucleotide whose nucleobases 5 'to 3' can form base pairs with nucleobases of a reference nucleic acid. In some examples, the template DNA is the reverse complement of the polynucleotide transcribed into an mRNA molecule. Thus, unless otherwise specifically indicated (or clear from context to the template DNA), the "reverse complement" of a polynucleotide refers to a complementary sequence of opposite orientation. The foregoing is demonstrated in the following diagram:

ATGATGATG Polynucleotide

TACTACTAC Polynucleotide "complementary sequence"

CATCATCAT polynucleotide.

Some embodiments of the invention include RNAi molecules that form hairpin RNAs. In these RNAi molecules, both the nucleotide sequence of the polynucleotide targeted for RNA interference and its reverse complement may be present in the same molecule, such that a single-stranded RNA molecule can "fold over" and hybridize to itself over a region comprising the nucleotide sequence and the reverse complement of the nucleotide sequence.

"nucleic acid molecule" includes all polynucleotides, such as: DNA in single-and double-stranded forms; RNA in single-stranded form; and double-stranded forms of rna (dsrna). The term "nucleotide sequence" or "nucleic acid sequence" refers to both the sense and antisense strands of a nucleic acid as single strands alone or in a duplex. The term "ribonucleic acid" (RNA) includes iRNA (inhibitory RNA), dsRNA (double-stranded RNA), siRNA (small interfering RNA), shRNA (small hairpin RNA), mRNA (messenger RNA), miRNA (microrna), hpRNA (hairpin RNA), tRNA (transfer RNA, whether loaded with or not with a corresponding acylated amino acid), and cRNA (complementary RNA). The term "deoxyribonucleic acid" (DNA) includes cDNA, gDNA and DNA-RNA hybrids. The term "polynucleotide" and "nucleic acid" and "fragments thereof" will be understood by those skilled in the art as such terms: including two gdnas, ribosomal RNAs, transfer RNAs, messenger RNAs, operons, and smaller engineered polynucleotides (that encode or may be adapted to encode a peptide, polypeptide, or protein).

Oligonucleotide: oligonucleotides are short polymers of nucleic acids. Oligonucleotides can be formed by cleaving a longer nucleic acid segment, or by polymerizing individual nucleotide precursors. Automated synthesizers allow synthesis of oligonucleotides up to several hundred bases in length. Since oligonucleotides bind to complementary nucleic acids, they can be used as probes for detecting DNA or RNA. Oligonucleotides (oligodeoxyribonucleotides) consisting of DNA can be used in PCR, a technique for amplifying DNA. In PCR, oligonucleotides are often referred to as "primers" which allow a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

Nucleic acid molecules may include either or both naturally occurring nucleotides and/or modified nucleotides that are linked together by naturally occurring nucleotide linkages and/or non-naturally occurring nucleotide linkages. As will be readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., no electrical linkage: e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.; charged linkages: e.g., phosphorothioate, phosphorodithioate, etc.; pendent moieties: e.g., peptides; intercalators: e.g., acridine, psoralen, etc.; chelators; alkylators; and modified linkages: e.g., alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially-duplexed, triplex, hairpin, circular, and padlock (padlocked) conformations.

As used herein, with respect to DNA, the term "encoding polynucleotide", "structural polynucleotide" or "structural nucleic acid molecule" refers to a polynucleotide that: when placed under the control of appropriate regulatory elements, translation into a polypeptide is ultimately via transcription and mRNA. With respect to RNA, the term "encoding polynucleotide" refers to a polynucleotide that is translated into a peptide, polypeptide, or protein. The boundaries of the encoding polynucleotide are determined by a translation start codon at the 5 'end and a translation stop codon at the 3' end. Encoding polynucleotides include, but are not limited to: gDNA, cDNA, ESTs and recombinant polynucleotides.

As used herein, "transcribed non-coding polynucleotide" refers to a segment of an mRNA molecule that is not translated into a peptide, polypeptide, or protein, such as 5'UTR, 3' UTR, and intron segments. Further, "transcribed non-coding polynucleotide" refers to a nucleic acid that is transcribed into RNA that functions in a cell, such as structural RNA (e.g., ribosomal RNA (rRNA), e.g., 5S rRNA, 5.8S rRNA, 16S rRNA, 18S rRNA, 23S rRNA, and 28S rRNA, etc.), transfer RNA (trna), and snRNA such as U4, U5, U6, and the like. Transcribed non-coding polynucleotides also include, for example and without limitation, small RNAs (srnas), which term is commonly used to describe small bacterial non-coding RNAs, small nucleolar RNAs (snornas), micrornas, small interfering RNAs (sirnas), Piwi-interacting RNAs (Piwi-interacting RNAs), and long non-coding RNAs. Still further, a "transcribed non-coding polynucleotide" is a polynucleotide that may naturally occur in a nucleic acid as a "spacer sequence" within a gene and is transcribed into an RNA molecule.

Lethal RNA interference: as used herein, the term "lethal RNA interference" refers to RNA interference that causes death or reduced viability in a subject individual when, for example, dsRNA, miRNA, siRNA, shRNA, and/or hpRNA is delivered to the subject individual.

Genome: as used herein, the term "genome" refers to chromosomal DNA present within the nucleus of a cell, and also refers to organelle DNA present within subcellular components of a cell. In some embodiments of the invention, the DNA molecule may be introduced into a plant cell such that the DNA molecule is integrated into the genome of the plant cell. In these and other embodiments, the DNA molecule may be integrated into the nuclear DNA of the plant cell, or into the DNA of the chloroplast or mitochondria of the plant cell. The term "genome" as applied to a bacterium refers to both chromosomes and plasmids within a bacterial cell. In some embodiments of the invention, a DNA molecule may be introduced into a bacterium such that the DNA molecule is integrated into the genome of the bacterium. In these and other embodiments, the DNA molecule may be integrated into the chromosome, or located as or in a stable plasmid.

Sequence identity: as used herein, the term "sequence identity" or "identity," in the context of two polynucleotides or polypeptides, refers to the residues in the sequences of the two molecules that are the same when aligned for maximum correspondence over a specified comparison window.

As used herein, the term "percent sequence identity" can refer to a value determined by comparing two optimally aligned sequences (e.g., nucleic acid or polypeptide sequences) of a molecule over a comparison window, wherein the portion of the sequences in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) in order to achieve optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at each position compared to a reference sequence is considered 100% identical to the reference sequence, and vice versa.

Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described, for example, in the following documents: smith and Waterman (1981) adv.Appl.Math.2: 482; needleman and Wunsch (1970) J.mol.biol.48: 443; pearson and Lipman (1988) Proc.Natl.Acad.Sci.U.S.A.85: 2444; higgins and Sharp (1988) Gene 73: 237-44; higgins and Sharp (1989) CABIOS 5: 151-3; corpet et al, (1988) Nucleic Acids Res.16: 10881-90; huang et al, (1992) Comp.appl.biosci.8: 155-65; pearson et al (1994) Methods mol. biol.24: 307-31; tatiana et al (1999) FEMSMICrobiol. Lett.174: 247-50. Detailed considerations for sequence alignment methods and homology calculations can be found, for example, in Altschul et al, (1990) J.mol.biol.215: 403-10.

National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST)^TM(ii) a Altschul et al (1990)) are available from a variety of sources, including the national center for biotechnology information (Bethesda, MD) and are available on the internet and can be used in conjunction with several sequence analysis programs. An explanation of how to use this program to determine sequence identity is BLAST on the Internet^TMThe "help" portion of (1). For comparison of nucleic acid sequences, BLAST may be used with the default BLOSUM62 matrix set to default parameters^TM(Blastn) the "Blast 2 sequence" function of the program. Nucleic acids having even greater sequence similarity to the sequence of the reference polynucleotide will show an increase in percent identity when evaluated in this way.

Specific hybridizable/specific complementary: as used herein, the terms "specifically hybridizable" and "specifically complementary" are terms which indicate a sufficient degree of complementarity such that stable and specific binding occurs between the nucleic acid molecule and the target nucleic acid molecule. Hybridization between two nucleic acid molecules involves the formation of antiparallel alignments between the nucleobases of the two nucleic acid molecules. These two molecules are then able to form hydrogen bonds with corresponding bases on the opposite strand to form a duplex molecule which, if sufficiently stable, can be detected using methods well known in the art. A polynucleotide is not necessarily 100% complementary to a target nucleic acid to which it can specifically hybridize. However, the amount of complementarity that must be present for specific hybridization varies with the hybridization conditions used.

Hybridization conditions that result in a particular degree of stringency will vary depending upon the nature of the hybridization method chosen, as well as the composition and length of the hybridizing nucleic acids. In general, the temperature of hybridization and the ionic strength of the hybridization buffer (in particular Na)⁺And/orMg⁺⁺Concentration) will determine the stringency of hybridization, but the number of washes will also affect stringency. Calculations concerning the hybridization conditions required to achieve a particular degree of stringency are known to those of ordinary skill in the art and are discussed, for example, in the following documents: sambrook et al (eds.),Molecular Cloning:A Laboratory Manual2 nd edition, volumes 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11; and Hames and Higgins (editors),Nucleic Acid HybridizationIRL Press, Oxford, 1985. Further details and guidance regarding nucleic acid hybridization can be found in the following references: for example, Tijssen, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", described inLaboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid ProbesPart I, chapter 2, Elsevier, NY, 1993; and Ausubel et al (eds.),Current Protocols in Molecular Biologychapter 2, Greene Publishing and Wiley-Interscience, NY, 1995.

As used herein, "stringent conditions" encompass conditions under which hybridization occurs only when there is less than 20% mismatch between the sequence of the hybridizing molecule and the homologous polynucleotide within the target nucleic acid molecule. "stringent conditions" include additional levels of specific stringency. Thus, as used herein, a "medium stringency" condition is one in which molecules with sequence mismatches of more than 20% do not hybridize; "high stringency" conditions are conditions under which sequences that are mismatched by more than 10% do not hybridize; whereas "very high stringency" conditions are those in which sequences that are mismatched by more than 5% do not hybridize.

Representative, non-limiting hybridization conditions are as follows.

High stringency conditions (polynucleotides detected that share at least 90% sequence identity): hybridization in 5x SSC buffer at 65 ℃ for 16 hours; wash twice in 2x SSC buffer at room temperature for 15 minutes each; and washed twice in 0.5x SSC buffer at 65 ℃ for 20 minutes each.

Medium stringency conditions (polynucleotides detected that share at least 80% sequence identity): hybridization in 5x-6x SSC buffer at 65-70 ℃ for 16-20 hours; wash twice in 2x SSC buffer at room temperature for 5-20 minutes each; and washed twice in 1 XSSC buffer at 55-70 ℃ for 30 minutes each.

Non-stringent control conditions (polynucleotides sharing at least 50% sequence identity will hybridize): hybridization in 6 XSSC buffer at room temperature to 55 ℃ for 16-20 hours; wash at least twice each for 20-30 minutes in 2x-3x SSC buffer at room temperature to 55 ℃.

As used herein, the terms "substantially/substantially homologous", "substantially/essentially identical" or "substantially/substantially homologous" with respect to a reference polynucleotide or polyribonucleotide refer to a polynucleotide or polyribonucleotide having consecutive nucleobases that hybridize under stringent conditions to an oligonucleotide consisting of the nucleotide sequence of the reference polynucleotide or polyribonucleotide. For example, polynucleotides that are substantially homologous to a reference polynucleotide of any of SEQ ID NOS: 2-4 are those that hybridize under stringent conditions (e.g., the moderately stringent conditions described above) to an oligonucleotide consisting of the nucleotide sequence of the reference polynucleotide. Substantially homologous or substantially identical polynucleotides may have at least 80% sequence identity. For example, substantially identical polynucleotides may have about 80% to 100% sequence identity, such as 79%, 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 99%, about 99.5%, and about 100%. The property of substantial identity is closely related to specific hybridization. For example, a nucleic acid molecule can specifically hybridize when there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to the non-target polynucleotide under conditions in which specific binding is desired (e.g., under stringent hybridization conditions).

As used herein, the term "ortholog" refers to a gene in two or more species that has evolved from a common ancestral nucleic acid, and that can retain the same function in the two or more species.

As used herein, two polynucleotides are considered to exhibit "complete complementarity" when each nucleotide of the polynucleotide read in the 5 'to 3' direction is complementary to each nucleotide of the other polynucleotide read in the 5 'to 3' direction. Similarly, a polynucleotide that is fully reverse complementary to a reference polynucleotide will display a nucleotide sequence that: wherein each nucleotide of the polynucleotide read in the 5 'to 3' direction is complementary to each nucleotide of the reference polynucleotide read in the 3 'to 5' direction. These terms and descriptions are well defined in the art and are readily understood by one of ordinary skill in the art.

Operatively connected to: when a first polynucleotide is in a functional relationship with a second polynucleotide, the first polynucleotide is said to be operably linked to the second polynucleotide. When produced recombinantly, operably linked polynucleotides are typically contiguous and, if necessary, the two protein coding regions may be joined in frame (e.g., in a translationally fused ORF). However, operably linked nucleic acids need not be contiguous.

The term "operably linked" when used in reference to a regulatory genetic element and an encoding polynucleotide means that the regulatory element affects the expression of the linked encoding polynucleotide. "regulatory element" or "control element" refers to a polynucleotide that affects the timing and level/amount of transcription, RNA processing or stability, or translation of an associated encoding polynucleotide. Regulatory elements may include promoters, translation leader sequences, introns, enhancers, stem-loop structures, repressor binding polynucleotides, polynucleotides with termination sequences, polynucleotides with polyadenylation recognition sequences, and the like. Specific regulatory elements may be located upstream and/or downstream of the encoding polynucleotide to which they are operably linked. Furthermore, the particular regulatory elements operably linked to the encoding polynucleotide may be located on the relevant complementary strand of the double-stranded nucleic acid molecule.

A promoter: as used herein, the term "promoter" refers to a region of DNA that can be upstream from the initiation of transcription and that can participate in the recognition and binding of RNA polymerase and other proteins to initiate transcription. The promoter may be operably linked to a coding polynucleotide for expression in a cell, or the promoter may be operably linked to a polynucleotide encoding a signal peptide, which may be operably linked to a coding polynucleotide for expression in a cell. A "plant promoter" may be a promoter capable of initiating transcription in a plant cell. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue-preferred" promoters. Promoters that initiate transcription only in certain tissues are referred to as "tissue-specific" promoters. A "cell-type specific" promoter primarily drives expression in certain cell types (e.g., vascular cells in roots or leaves) in one or more organs. An "inducible" promoter may be a promoter that may be under environmental control. Examples of environmental conditions under which transcription can be initiated by an inducible promoter include anaerobic conditions and the presence of light. Tissue-specific promoters, tissue-preferred promoters, cell-type specific promoters and inducible promoters constitute the category of "non-constitutive" promoters. A "constitutive" promoter is a promoter that can be active under most environmental conditions or in most tissues or cell types.

Any inducible promoter may be used in some embodiments of the invention. See Ward et al, (1993) Plant mol.biol.22: 361-366. With inducible promoters, the rate of transcription increases in response to an inducing agent. Exemplary inducible promoters include, but are not limited to: copper-responsive promoters from the ACEI system; an In2 gene from maize that responds to a benzenesulfonamide herbicide safener; the Tet repressor from Tn 10; and inducible promoters from steroid hormone genes, the transcriptional activity of which can be induced by glucocorticosteroids (Schena et al, 1991, Proc. Natl. Acad. Sci. USA 88: 0421).

Exemplary constitutive promoters include, but are not limited to: promoters from plant viruses, such as the 35S promoter from cauliflower mosaic virus (CaMV), the promoter from the rice actin gene, the ubiquitin promoter, pEMU, MAS, maize H3 histone promoter and ALS promoter, the Xba1/NcoI fragment (or a polynucleotide similar to the Xba1/NcoI fragment) of brassica napus ALS3 structural gene 5' (international PCT publication No. WO 96/30530).

Furthermore, any tissue-specific or tissue-preferential promoter may be utilized in some embodiments of the invention. A plant transformed with a nucleic acid molecule comprising an encoding polynucleotide operably linked to a tissue-specific promoter can produce the product of the encoding polynucleotide exclusively or preferentially in specific tissues. Exemplary tissue-specific or tissue-preferential promoters include, but are not limited to: seed-preferred promoters, such as the promoter from the phaseolin gene; leaf-specific and light-inducible promoters, such as those from cab or rubisco; an anther-specific promoter, such as the promoter from LAT 52; pollen-specific promoters, such as the promoter from Zm 13; and microspore-preferred promoters, such as the promoter from apg.

Rapeseed (Rapeseed)/canola (oiled seed) plants: as used herein, the term "rapeseed" or "canola" refers to brassica plants; such as canola plants of the brassica napus species.

And (3) transformation: as used herein, the term "transformation" or "transduction" refers to the transfer of one or more nucleic acid molecules into a cell. In the case of a nucleic acid molecule that is stably replicated by a cell, either by incorporation of the nucleic acid molecule into the genome of the cell, or by episomal replication, the cell is "transformed" by the nucleic acid molecule transduced into the cell. As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to: transfection with viral vectors; transforming with a plasmid vector; electroporation (Fromm et al, 1986, Nature 319: 791-3); lipofectation (Felgner et al, 1987, Proc. Natl. Acad. Sci. USA 84: 7413-7); microinjection (Mueller et al, 1978, Cell 15: 579-85); agrobacterium (Agrobacterium) mediated transfer (Fraley et al, 1983, Proc. Natl. Acad. Sci. USA80: 4803-7); direct DNA uptake; and particle bombardment (Klein et al, 1987, Nature 327: 70).

And (3) transgenosis: an exogenous polynucleotide. In some examples, the transgene may be DNA encoding one or both strands of RNA capable of forming a dsRNA molecule comprising a nucleotide sequence complementary to a nucleic acid molecule present in pollen beetles. In other examples, the transgene may be a gene (e.g., a herbicide tolerance gene, a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desired agricultural trait). In these and other examples, the transgene may contain regulatory elements (e.g., a promoter) operably linked to the encoding polynucleotide of the transgene.

Carrier: a nucleic acid molecule introduced into a cell, for example, to produce a transformed cell. A vector may comprise genetic elements that allow it to replicate in a host cell, such as an origin of replication. Examples of carriers include, but are not limited to: a plasmid, cosmid, phage, or virus that carries exogenous DNA into a cell. The vector may also include one or more genes (including genes that produce antisense molecules and/or selectable marker genes) as well as other genetic elements known in the art. The vector may transduce, transform, or infect a cell, thereby causing the cell to express the nucleic acid molecule and/or protein encoded by the vector. The carrier optionally includes a substance (e.g., liposome, protein coating, etc.) that aids in the entry of the nucleic acid molecule into the cell.

Yield: stabilized yield of about 100% or greater relative to yield of a test variety grown at the same time and under the same conditions at the same growth location. In particular embodiments, "increased yield" or "increased yield" means a cultivar having a stabilized yield of 105% or greater relative to the yield of a cultivar grown at the same growth location containing a substantial density of insect pests detrimental to a crop (which are the pests targeted by the compositions and methods herein) at the same time and under the same conditions.

As used herein, a noun modified by an indefinite article means "at least one" unless specifically stated or implied.

Unless specified otherwiseUnless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in the following publications: for exampleLewin's Genes X，Jones&Bartlett Publishers, 2009(ISBN 100763766321); krebs et al (ed.),The Encyclopediaof Molecular Biologyblackwell Science Ltd, 1994(ISBN 0-632-02182-9); and Meyers R.A (editors),Molecular Biology and Biotechnology:A Comprehensive Desk ReferenceVCH Publishers, Inc., 1995(ISBN 1-56081-. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise indicated. All temperatures are in degrees celsius.

Nucleic acid molecules comprising insect pest sequences

A. Overview

Nucleic acid molecules useful for controlling insect pests are described herein. In some examples, the insect pest is brassica napus ludiflora. Nucleic acid molecules described include target polynucleotides (e.g., native genes and non-coding polynucleotides), dsRNA, siRNA, shRNA, hpRNA, and miRNA. For example, dsRNA, siRNA, miRNA, shRNA, and/or hpRNA molecules are described in some embodiments, which molecules may be specifically complementary to all or part of one or more native nucleic acids in an insect pest. In these and other embodiments, the one or more native nucleic acids may be one or more target genes, the products of which may be, for example, but not limited to: involved in metabolic processes or in the development of larvae. The nucleic acid molecules described herein, when introduced into a cell comprising at least one native nucleic acid that is specifically complementary to the nucleic acid molecule, can initiate RNAi in the cell, thereby reducing or eliminating expression of the native nucleic acid. In some examples, reducing or eliminating expression of a target gene by a nucleic acid molecule specifically complementary to the target gene can result in slowing or stopping the growth, development, and/or feeding of the insect pest.

In some embodiments, at least one target gene in an insect pest may be selected, wherein the target gene comprises a ssrp1 polynucleotide. In particular examples, a target gene comprising a ssrp1 polynucleotide is selected, wherein the target gene is the PB ssrp1 gene comprising SEQ ID NOs 2-3 or the condominium gene comprising SEQ ID No. 4.

In some embodiments, the target gene may be a nucleic acid molecule comprising a polynucleotide that: the polynucleotide can be back-translated in silico (in silico) into a polypeptide comprising a contiguous amino acid sequence that is at least about 85% identical (e.g., at least 84%, 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or 100% identical) to the amino acid sequence of the protein product of the ssrp1 polynucleotide. In particular examples, the target gene is a nucleic acid molecule comprising a polynucleotide that can be reverse translated in silico into a polypeptide comprising a contiguous amino acid sequence that is at least about 85% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 100% identical, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:5, PBSSRP1 comprising SEQ ID NOS: 6-7, and peptide fragments of the foregoing.

According to the invention there is provided DNA, the expression of which produces an RNA molecule comprising a polynucleotide which is specifically complementary to all or part of a native RNA molecule encoded by a coding polynucleotide in pollen beetles. In some embodiments, down-regulation of an encoding polynucleotide in a pest cell can be obtained following ingestion of an expressed RNA molecule by an insect pest. In particular embodiments, down-regulation of a coding sequence in an insect pest cell has a deleterious effect on the growth development and/or survival of the pest.

In some embodiments, the target polynucleotide includes transcribed non-coding RNAs such as 5' UTRs, 3' UTRs, splice leader sequences, introns, terminal introns (outron) (e.g., 5' UTR RNA that is subsequently modified in trans-splicing), donitrons (e.g., non-coding RNAs required to provide a trans-spliced donor sequence), and other non-coding transcribed RNAs of target insect pest genes. Such polynucleotides may be derived from both monocistronic and polycistronic genes.

Thus, also described herein in connection with some embodiments are iRNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) comprising at least one nucleotide sequence that is specifically complementary to all or part of a target polynucleotide in a pollen beetle. In some embodiments, an iRNA molecule can comprise one or more nucleotide sequences that are complementary to all or part of a plurality of target polynucleotides (e.g., 2, 3,4, 5,6, 7,8, 9, 10, or more target polynucleotides). In particular embodiments, the iRNA molecule can be produced in vitro, or in vivo by genetically modifying an organism, such as a plant or bacterium. Also disclosed are cdnas that can be used to generate dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules that are specifically complementary to all or a portion of a target polynucleotide in an insect pest. Further described are recombinant DNA constructs for use in achieving stable transformation of a particular host target. The transformed host target can express effective levels of dsRNA, siRNA, miRNA, shRNA, and/or hpRNA molecules from the recombinant DNA construct. Thus, also described is a plant transformation vector comprising at least one polynucleotide operably linked to a heterologous promoter functional in a plant cell, wherein expression of the one or more polynucleotides results in an RNA molecule comprising at least one contiguous nucleotide sequence that is specifically complementary to all or part of a target polynucleotide in an insect pest.

In particular examples, nucleic acid molecules useful for controlling insect pests include: 1, SEQ ID NO; a naturally encoded polynucleotide isolated from pollen beetles comprising SEQ ID NOs 2-3; all or a portion of a native ssrp1 polynucleotide isolated from genus nitida comprising any one of SEQ ID NOs 2-4; DNA that when expressed produces an RNA molecule comprising a polyribonucleotide that is specifically complementary or reverse-complementary to all or part of a native RNA molecule encoded by the genus melissa ssrp 1; an iRNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) comprising at least one polyribonucleotide that is specifically complementary or reverse-complementary to all or part of the genus melissa ssrp 1; can be used to generate cDNA for dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules that are specifically complementary or reverse complementary to all or part of the genus melissa ssrp 1; and/or a recombinant DNA construct for effecting stable transformation of a particular host target, wherein the transformed host target comprises one or more of the foregoing polynucleotides.

B. Nucleic acid molecules

The present invention provides, among other things, iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules that inhibit expression of a target gene in a cell, tissue, or organ of an insect pest; and DNA molecules capable of being expressed as iRNA molecules in cells or microorganisms to inhibit expression of target genes in cells, tissues or organs of insect pests.

Some embodiments of the invention provide an isolated or recombinant nucleic acid molecule characterized by a polynucleotide comprising at least one (e.g., one, two, three, or more) nucleotide sequence selected from the group consisting of: 1, SEQ ID NO; the complementary sequence or reverse complementary sequence of SEQ ID NO. 1; a PB ssrp1 polynucleotide comprising SEQ ID NOS: 2-3; the complement or reverse complement of the PB ssrp1 polynucleotide comprising SEQ ID NOS: 2-3; a fragment comprising at least 15 (e.g., at least 19) contiguous nucleotides of the PBssrp1 polynucleotide of SEQ ID NOS: 2-3 (e.g., SEQ ID NO: 4); a complement or reverse complement of a fragment comprising at least 15 contiguous nucleotides of the PB ssrp1 polynucleotide of SEQ ID NOS: 2-3; a naturally-encoding polynucleotide of a Nitidus organism (e.g., PB) comprising SEQ ID NO. 4; a complementary sequence or reverse complementary sequence of a naturally encoded polynucleotide of a Nitidus organism comprising SEQ ID NO 4; a fragment of at least 15 contiguous nucleotides of a naturally encoded polynucleotide of a clearweed organism comprising SEQ ID NO 4; and a complement or reverse complement of a fragment of at least 15 contiguous nucleotides of a naturally encoded polynucleotide of a Nitidus organism comprising SEQ ID NO 4.

In particular embodiments, the iRNA transcribed from the aforementioned polynucleotide is contacted with or taken up by an insect pest to inhibit growth, development and/or feeding of the pest. In some embodiments, the contacting with or uptake by the insect occurs via feeding plant material comprising iRNA. In some embodiments, the contacting with or uptake by the insect occurs by spraying the plant comprising the insect with a component comprising the iRNA.

In some embodiments, the nucleic acid molecule of the invention is an iRNA molecule having the following characteristics: a polyribonucleotide comprising a nucleotide sequence of at least one (e.g., one, two, three, or more) selected from the group consisting of: 12 is SEQ ID NO; the complement or reverse complement of SEQ ID NO 12; 13 in SEQ ID NO; the complement or reverse complement of SEQ ID NO 13; 14, SEQ ID NO; the complement or reverse complement of SEQ ID NO. 14; 15, SEQ ID NO; the complement or reverse complement of SEQ ID NO. 15; a fragment of at least 15 (e.g., at least 19) contiguous nucleotides of any one of SEQ ID NOs 13-15; the complement or reverse complement of a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs 13-15; a natural polyribonucleotide transcribed in pollen beetles comprising SEQ ID NO. 13-14; (ii) a complementary sequence or reverse complementary sequence comprising a natural polyribonucleotide transcribed from the pollen beetle of SEQ ID NOs 13-14; a fragment comprising at least 15 contiguous nucleotides of a natural polyribonucleotide transcribed in pollen beetles of SEQ ID NO. 13-14; (ii) a complement or reverse complement of a fragment comprising at least 15 contiguous nucleotides of a natural polyribonucleotide transcribed from a pollen beetle of SEQ ID NOs 13-14; a natural polyribonucleotide transcribed in a clearweed organism comprising SEQ ID NO. 15; a complementary sequence or reverse complementary sequence of a natural polyribonucleotide transcribed in a clearweed organism comprising SEQ ID NO 15; a fragment of at least 15 contiguous nucleotides of a natural polyribonucleotide transcribed in a clearweed organism of SEQ ID NO. 15; 15 or a complementary sequence or reverse complementary sequence of a fragment of at least 15 contiguous nucleotides of a natural polyribonucleotide transcribed in an organism of the genus nitidula comprising SEQ ID NO.

In particular embodiments, contact with or uptake by an insect pest inhibits growth, development, and/or feeding of the pest. In some embodiments, contact with or contact by an insect pest or uptake by an insect occurs by feeding a plant material or bait comprising an iRNA.

In certain embodiments, the invention provides dsRNA molecules comprising a polyribonucleotide comprising at least one nucleotide sequence complementary (or reverse complementary) to a transcript from a target gene comprising any one of SEQ ID NOs 1-4, and fragments thereof, inhibition of which in an insect pest results in the reduction or elimination of a polypeptide or polynucleotide agent essential to the growth, development, or other biological function of the pest. The selected target polynucleotide may exhibit from about 80% to about 100% sequence identity to a reference polynucleotide selected from the group consisting of: any one of SEQ ID NOs 1-4; a continuous fragment comprising the PB ssrp1 gene of SEQ ID NO. 2-3; 2-4; and the aforementioned complementary sequence and reverse complementary sequence; . For example, the selected polynucleotide may exhibit a 79% identity to any of the aforementioned reference polynucleotides; 80 percent; about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94%; about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; or about 100% sequence identity.

In some examples, the dsRNA molecule is transcribed from a polynucleotide comprising: a sense nucleotide sequence substantially identical or identical to a contiguous fragment comprising the PB ssrp1 gene of SEQ ID NOS: 2-3 (SEQ ID NO: 4); an antisense nucleotide sequence that is at least substantially the reverse complement of the sense nucleotide sequence; and an intervening nucleotide sequence (intervening sequence) located between the sense and antisense sequences, whereby sense and antisense polyribonucleotides transcribed from the respective sense and antisense nucleotide sequences hybridize to form a "stem" structure in the dsRNA, and polyribonucleotides transcribed from the intervening sequence form a "loop". Such dsRNA molecules may be referred to as hairpin rna (hprna) molecules. An example of such an hpRNA molecule is SEQ ID NO. 16 encoded by the polynucleotide of SEQ ID NO. 11, which contains the sense nucleotide sequence of SEQ ID NO. 4.

In some embodiments, the polynucleotide capable of being expressed as an iRNA molecule in a cell or microorganism to inhibit expression of a target gene may comprise a single nucleotide sequence that is specifically complementary or reverse complementary to all or part of a native polynucleotide present in pollen beetles, or the polynucleotide may be constructed as a chimera comprising a plurality of such specifically complementary or reverse complementary nucleotide sequences.

In some embodiments, a polynucleotide may comprise a first nucleotide sequence and a second nucleotide sequence separated by a "spacer sequence". The spacer sequence may be a region comprising any nucleotide sequence that promotes (where desired) the formation of secondary structure between the first and second polynucleotides or transcripts thereof. In one embodiment, the spacer sequence is a portion of a sense or antisense encoding polyribonucleotide of an mRNA. Alternatively, the spacer sequence may comprise any combination of nucleotides or homologues thereof capable of being covalently linked to the nucleic acid molecule. In some examples, the spacer sequence may be an intron.

For example, in some embodiments, a DNA molecule can comprise one or more polynucleotides encoding one or more different iRNA molecules, wherein each of the different iRNA molecules comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are complementary to each other. The first nucleotide sequence and the second nucleotide sequence may be linked within the iRNA molecule by a spacer sequence. The spacer sequence may form part of the first or second nucleotide sequence. Expression of an iRNA molecule comprising first and second nucleotide sequences can result in the formation of an hpRNA molecule by specific intramolecular base pairing of the first and second nucleotide sequences. The first nucleotide sequence or the second nucleotide sequence can be substantially identical to a polyribonucleotide encoded by a polynucleotide native to the insect pest (e.g., a target gene fragment, or a transcribed non-coding polynucleotide), or a complement or reverse complement thereof.

dsRNA nucleic acid molecules comprise a double strand of polymeric ribonucleotides and may comprise modifications to either the phosphate-sugar backbone or the nucleosides. Modifications in the RNA structure can be suitably adjusted to enable specific inhibition to occur. In one embodiment, the dsRNA molecule can be modified by ubiquitous enzymatic processes so that siRNA molecules can be generated. The enzymatic process may utilize an RNase III enzyme in vitro or in vivo, such as DICER in eukaryotes. See Elbashir et al, (2001) Nature411: 494-8; and Hamilton and Baulcombe, (1999) Science 286(5441) 950-2. DICER or a functionally equivalent RNase III enzyme cleaves larger dsRNA strands and/or hpRNA molecules into smaller oligonucleotides (e.g., sirnas), each of which is about 19-25 nucleotides in length. siRNA molecules generated by these enzymes have a 3' overhang of 2 to 3 nucleotides, and a 5' phosphate end and a 3' hydroxyl end. The siRNA molecules generated by the RNase III enzyme unwind and separate into single-stranded RNA in the cell. The siRNA molecule then specifically hybridizes to RNA transcribed from the target gene, which is subsequently degraded by the inherent cellular RNA degradation mechanism. This process can result in efficient degradation or removal of the RNA encoded by the target gene in the target organism. The result is post-transcriptional silencing of the targeted gene. In some embodiments, siRNA molecules produced from heterologous nucleic acid molecules by endogenous RNase III enzymes can be effective to mediate down-regulation of target genes in insect pests.

In some embodiments, a nucleic acid molecule can include at least one non-naturally occurring polynucleotide that can be transcribed into a single-stranded RNA molecule capable of forming a dsRNA molecule in vivo by intermolecular hybridization. Such dsrnas typically self-assemble and can be provided in a nutritional source for insect pests to achieve post-transcriptional inhibition of target genes. In these and other embodiments, the nucleic acid molecule can comprise two different non-naturally occurring polynucleotides, wherein each polynucleotide comprises at least one nucleotide sequence that is specifically complementary or reverse complementary to a different target gene in the insect pest. When such a nucleic acid molecule is provided in the form of a dsRNA molecule to, for example, pollen beetles, the dsRNA molecule inhibits the expression of at least two different target genes in the pest.

C. Obtaining nucleic acid molecules

Nucleic acid molecules, such as irnas and DNA molecules encoding irnas, can be designed using a variety of polynucleotides in insect pests as targets. However, the selection of the native polynucleotide is not a straightforward process. For example, only a few of the natural polynucleotides in insect pests will be effective targets. It cannot be predicted with certainty whether a particular native polynucleotide can be effectively down-regulated by a nucleic acid molecule of the invention, or whether down-regulation of a particular native polynucleotide will have an adverse effect on the growth, development and/or survival of an insect pest. Most natural insect pest polynucleotides, such as ESTs isolated therefrom (e.g., the western corn rootworm polynucleotides listed in U.S. Pat. No. 7,612,194), have no adverse effect on the growth, development, and/or survival of the pest. It is also unpredictable which of the natural polynucleotides that may have an adverse effect on insect pests can be used in recombinant techniques to express nucleic acid molecules complementary to such natural polynucleotides in a host plant and adversely affect the pests after feeding without harming the host plant.

In some embodiments, a nucleic acid molecule (e.g., a dsRNA molecule to be provided in a host plant of an insect pest) targets a cDNA encoding a protein or protein portion (such as a polypeptide involved in anabolic or catabolic biochemical pathways, cell division, energy metabolism, digestion, host plant recognition, etc.) necessary for development and/or survival of the pest. As described herein, ingestion of a composition containing one or more dsRNA (at least one segment of which is specifically complementary to at least one substantially identical segment of RNA produced in a cell of a target pest organism) by a target pest organism can result in the death or other inhibition of the target. Polynucleotides derived from natural insect pest genes can be used to construct plant cells that are resistant to pest infestation. For example, a host plant of an insect pest (e.g., brassica napus) can be transformed to contain one or more polynucleotides derived from pollen beetles as provided herein. The polynucleotide transformed into the host may encode one or more RNAs that form a dsRNA structure in a cell or biological fluid within the transformed host, thus making the dsRNA available if or when the pest forms a nutritional relationship with the transgenic host. This can cause repression of the expression of one or more genes in the pest cell and ultimately result in death or inhibition of pest growth or development.

In particular embodiments, targeted are genes that are substantially involved in the growth and development of insect pests. Other target genes for use in the present invention may include, for example, those that play an important role in pest viability, motility, translocation, growth, development, infectivity, and establishment of feeding sites. Thus, the target gene may be a housekeeping gene or a transcription factor.

In some embodiments, the invention provides methods for obtaining a nucleic acid molecule comprising a polynucleotide for producing an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule. One such embodiment comprises: (a) analyzing the expression, function and phenotype of one or more target genes in an insect pest (e.g., pollen beetle) following dsRNA-mediated gene suppression; (b) probing a cDNA or gDNA library with a probe comprising all or part of a polynucleotide from a targeted pest, or a homolog thereof, that exhibits an altered (e.g., reduced) growth or development phenotype in a dsRNA-mediated suppression assay; (c) identifying DNA clones that specifically hybridize to the probe; (d) isolating the DNA clone identified in step (b); (e) sequencing the cDNA or gDNA fragments comprising the clones isolated in step (d), wherein the sequenced nucleic acid molecule comprises all or a substantial portion of said RNA or a homologue thereof; and (f) a chemically synthesized gene or all or a substantial portion of a siRNA, miRNA, hpRNA, mRNA, shRNA, or dsRNA.

In other embodiments, methods for obtaining a nucleic acid fragment comprising a polynucleotide for producing a substantial portion of an iRNA molecule comprise: (a) synthesizing a first oligonucleotide primer and a second oligonucleotide primer that are specifically complementary to a portion of a natural polynucleotide from the targeted insect pest; and (b) amplifying the cDNA or gDNA insert present in the cloning vector using the first and second oligonucleotide primers of step (a), wherein the amplified nucleic acid molecule comprises a substantial portion of an iRNA molecule.

Polynucleotides may be isolated, amplified, or produced by a variety of methods. For example, iRNA molecules can be obtained by PCR amplification of target polynucleotides (e.g., target genes, target gene fragments, and target transcribed non-coding polynucleotides) derived from gDNA or cDNA libraries, or portions thereof. DNA or RNA can be extracted from the target organism and a nucleic acid library can be prepared from the DNA or RNA using methods known to those of ordinary skill in the art. The target gene may be PCR amplified and sequenced using a gDNA library or cDNA library generated from the target organism. The confirmed PCR products can be used as templates for in vitro transcription to generate sense and antisense RNA with minimal promoter. Alternatively, Nucleic acid molecules can be synthesized by any of a number of techniques (see, e.g., Ozaki et al, (1992) Nucleic Acids Research, 20: 5205-. See, e.g., Beaucage et al, (1992) Tetrahedron, 48: 2223-2311; U.S. patent nos. 4,980,460, 4,725,677, 4,415,732, 4,458,066 and 4,973,679. Alternative chemicals that produce non-natural backbone groups, such as phosphorothioates, phosphoramidates, and the like, may also be employed.

The RNA, dsRNA, siRNA, miRNA, shRNA or hpRNA molecules of the invention can be produced chemically or enzymatically by a person skilled in the art by manual or automated reactions or in vivo in a cell comprising a nucleic acid molecule comprising a polynucleotide encoding the RNA, dsRNA, siRNA, miRNA, shRNA or hpRNA molecule. RNA can also be produced by partial or complete organic synthesis, and any modified polyribonucleotide can be introduced by in vitro enzymatic or organic synthesis. RNA molecules can be synthesized by cellular RNA polymerases or bacteriophage RNA polymerases (e.g., T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA polymerase). Expression constructs useful for cloning and expressing polynucleotides are known in the art. See, for example, international PCT publication No. WO97/32016, and U.S. patent nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693. RNA molecules that are chemically synthesized or synthesized by in vitro enzymatic synthesis can be purified prior to introduction into the cell. For example, RNA molecules can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination of these means. Alternatively, RNA molecules that are chemically synthesized or synthesized by in vitro enzymatic synthesis can be used without purification or minimal purification, e.g., to avoid losses due to sample processing. The RNA molecules may be dried for storage or dissolved in an aqueous solution. The solution may contain a buffer or salt to facilitate annealing and/or stabilization of the dsRNA molecule duplex strands.

In some embodiments, the dsRNA molecule can be formed by a single self-complementary RNA strand or by two complementary RNA strands. dsRNA molecules can be synthesized in vivo or in vitro. The cell's endogenous RNA polymerase may mediate transcription of one or both RNA strands in vivo, or cloned RNA polymerase may be used to mediate transcription in vivo or in vitro. The dsRNA may be post-transcriptionally processed into, for example, miRNA and/or siRNA molecules by an enzyme endogenous to the cell. Post-transcriptional inhibition of a target gene in an insect pest may be host-targeted by: specific transcription in an organ, tissue, or cell type of the host (e.g., by using a tissue-specific promoter); stimulating environmental conditions in the host (e.g., by using inducible promoters responsive to infection, stress, temperature, and/or chemical inducers); and/or engineering transcription of a certain developmental stage or developmental age of the host (e.g., by using a developmental stage specific promoter). The RNA strand (whether transcribed in vitro or in vivo) that forms the dsRNA molecule may or may not be polyadenylated and may or may not be capable of being translated into a polypeptide by the translation machinery of the cell.

D. Recombinant vectors and host cell transformation

In some embodiments, the invention also provides a DNA molecule for introduction into a cell (e.g., a bacterial cell, a yeast cell, or a plant cell), wherein the DNA molecule comprises a polynucleotide that, upon expression as an RNA and uptake by an insect pest, effects repression of a target gene in a cell, tissue, or organ of the pest. Accordingly, some embodiments provide a recombinant nucleic acid molecule comprising a polynucleotide capable of being expressed as an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule in a plant cell to inhibit expression of a target gene in an insect pest. To initiate or enhance expression, such recombinant nucleic acid molecules can comprise one or more regulatory elements that can be operably linked to a polynucleotide capable of being expressed as an iRNA. Methods for expressing gene repressor molecules in plants are known and can be used to express the polynucleotides of the invention. See, e.g., International PCT publication Nos. WO 06/073727; and U.S. patent publication No. 2006/0200878 Al).

In particular embodiments, the recombinant DNA molecules of the invention may comprise polynucleotides encoding RNAs that can form dsRNA molecules. Such recombinant DNA molecules can encode RNA that can form dsRNA molecules capable of inhibiting the expression of one or more endogenous target genes in insect pest cells upon ingestion. In many embodiments, the transcribed RNA can form a dsRNA molecule that can be provided in a stabilized form; for example, in hairpin form and stem-loop structures.

In some embodiments, one strand of the dsRNA molecule can be formed by transcription from a polynucleotide comprising a nucleotide sequence substantially identical to any one of: 1, SEQ ID NO; 1 or the reverse complement of SEQ ID NO; a PB ssrp1 polynucleotide comprising SEQ ID NOS: 2-3; the complement or reverse complement of the PB ssrp1 polynucleotide comprising SEQ ID NOS: 2-3; a fragment comprising at least 15 (e.g., at least 19) contiguous nucleotides of the PB ssrp1 polynucleotide of SEQ ID NOS: 2-3 (e.g., SEQ ID NO: 4); a complement or reverse complement of a fragment comprising at least 15 contiguous nucleotides of the PB ssrp1 polynucleotide of SEQ ID NOS: 2-3; a naturally encoded polynucleotide of a Nitidus organism comprising any one of SEQ ID NOs 2-4; a complementary sequence or reverse complementary sequence of a naturally encoded polynucleotide of a Nitidus organism comprising any one of SEQ ID NOs 2-4; a fragment of at least 15 contiguous nucleotides of a naturally encoded polynucleotide of a Nitidus organism comprising any one of SEQ ID NOs 2-4; a complementary sequence or reverse complementary sequence of a fragment of at least 15 contiguous nucleotides of a naturally encoded polynucleotide of a leiocassis organism comprising any one of SEQ ID NOs 2-4.

In some embodiments, one strand of the dsRNA molecule can be formed by transcription from a polynucleotide that is substantially identical to a polynucleotide selected from the group consisting of: 4, SEQ ID NO; the complement of SEQ ID NO. 4; the reverse complement of SEQ ID NO. 4; a fragment of at least 15 (e.g., at least 19) contiguous nucleotides of SEQ ID NO. 4; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO. 4; the reverse complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO. 4.

In particular embodiments, a recombinant DNA molecule encoding an RNA that can form a dsRNA molecule can comprise an encoding polynucleotide that: wherein the at least two nucleotide sequences are arranged such that one nucleotide sequence is in a sense orientation and the other nucleotide sequence is in an antisense orientation relative to the at least one promoter, wherein the sense nucleotide sequence and the antisense nucleotide sequence are linked or connected by a spacer sequence of, for example, about 100 to about 1000 nucleotides. The spacer sequence may form a loop between the sense nucleotide sequence and the antisense nucleotide sequence. The sense nucleotide sequence can be substantially identical to the target gene (e.g., the ssrp1 gene comprising SEQ ID NOS: 2-3) or a fragment thereof. However, in some embodiments, the recombinant DNA molecule may encode RNA that can form a dsRNA molecule that does not contain a spacer sequence. In some embodiments, the sense nucleotide sequence and the antisense nucleotide sequence of the polynucleotide encoding the dsRNA molecule may have different lengths.

Polynucleotides identified as having a deleterious effect on an insect pest or as having plant protection against a pest can be readily incorporated into expressed dsRNA molecules by creating appropriate expression cassettes in the recombinant nucleic acid molecules of the invention. For example, such polynucleotides may be expressed as hairpins having a stem-loop structure by: obtaining a first nucleotide sequence corresponding to a polynucleotide of a target gene (e.g., ssrp1 gene comprising SEQ ID NOS: 2-3 and fragments thereof); linking the nucleotide sequence to a second spacer nucleotide sequence that is not homologous or complementary to the first nucleotide sequence; the linker is then ligated to a third nucleotide sequence, wherein at least a portion of the third nucleotide sequence is substantially the reverse complement of the first nucleotide sequence. The transcript of such a polynucleotide forms a stem loop structure by intramolecular base pairing of the first nucleotide sequence with the third nucleotide sequence, wherein the loop structure is formed by the transcript of the second nucleotide sequence. See, e.g., U.S. patent publication nos. 2002/0048814 and 2003/0018993; and International PCT publication Nos. WO94/01550 and WO 98/05770. dsRNA molecules can be produced, for example, in the form of double-stranded structures such as stem-loop structures (e.g., hairpins), whereby production of miRNA or siRNA targeted to a native insect pest polynucleotide is enhanced by, for example, co-expressing a fragment of the target gene on an additional cassette expressible in a plant, which results in enhanced siRNA production, or mitigating methylation to prevent transcriptional gene silencing of a promoter operably linked to a polynucleotide encoding a dsRNA molecule.

Certain embodiments of the invention comprise introducing (i.e., transforming) a recombinant nucleic acid molecule of the invention into a plant to achieve a level of inhibitory insect pest expression of one or more iRNA molecules. The recombinant DNA molecule may, for example, be a vector, such as a linear or closed circular plasmid. The vector system may be a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the host genome. In addition, the vector may be an expression vector. The polynucleotides of the invention may be suitably inserted into a vector, for example under the control of a suitable promoter which functions in one or more hosts to drive expression of the linked coding polynucleotide or other DNA element. Many vectors can be used for this purpose, and the selection of an appropriate vector will depend primarily on the size of the polynucleotide to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains a variety of components, depending on the function of the vector (e.g., amplifying or expressing DNA) and the particular host cell with which it is compatible.

To confer protection of the transgenic plant from insect pests, the recombinant DNA may be transcribed into iRNA molecules (e.g., RNA molecules forming dsRNA molecules), for example, within the tissues or fluids of the recombinant plant. An iRNA molecule can comprise a polyribonucleotide that is substantially identical to and specifically hybridizable with a corresponding transcribed polyribonucleotide in an insect pest (e.g., pollen beetle) that can cause damage to a host plant species. The pest may contact the iRNA molecule transcribed in the cells of the transgenic host plant, for example, by ingesting cells or fluids of the transgenic host plant comprising the iRNA molecule. Thus, in a particular example, expression of a target gene is repressed by an iRNA molecule within an insect pest that infests a transgenic host plant. In some embodiments, suppression of expression of a target gene in an insect pest protects a plant from attack by the pest.

In order for an iRNA molecule to be delivered to an insect pest in trophic relationship to a plant cell comprising a recombinant polynucleotide of the invention, it is generally desirable to express (i.e., transcribe) the iRNA molecule in the plant cell, although delivery can also be achieved by, for example, treating or coating the cell with a preparation comprising the iRNA molecule. Thus, a recombinant nucleic acid molecule can comprise a polynucleotide of the invention operably linked to one or more regulatory elements (such as a heterologous promoter element that functions in a host cell), such as a bacterial cell in which the nucleic acid molecule is to be amplified or expressed, or a plant cell in which the nucleic acid molecule is to be expressed.

Promoters suitable for use in the nucleic acid molecules of the present invention include inducible promoters, viral promoters, synthetic promoters or constitutive promoters, all of which are well known in the art. Non-limiting examples describing such promoters include U.S. Pat. No. 6,437,217 (maize RS81 promoter), 5,641,876 (rice actin promoter), 6,426,446 (maize RS324 promoter), 6,429,362 (maize PR-1 promoter), 6,232,526 (maize A3 promoter), 6,177,611 (maize constitutive promoter), 5,322,938, 5,352,605, 5,359,142 and 5,530,196(CaMV 35S promoter), 6,433,252 (maize L3 oleosin)Promoter), 6,429,357 (rice actin 2 promoter and rice actin 2 intron), 6,294,714 (light inducible promoter), 6,140,078 (salt inducible promoter), 6,252,138 (pathogen inducible promoter), 6,175,060 (phosphorus deficiency inducible promoter), 6,388,170 (bidirectional promoter), 6,635,806 (gamma-coixin (coixin) promoter), and U.S. patent publication No. 2009/757,089 (maize chloroplast aldolase promoter). Additional promoters include the nopaline synthase (NOS) promoter (Ebert et al, (1987) Proc. Natl. Acad. Sci. USA84(16):5745-9) and the octopine synthase (OCS) promoter (both carried on a tumor-inducible plasmid of Agrobacterium tumefaciens); cauliflower mosaic virus promoters, such as the cauliflower mosaic virus (CaMV)19S promoter (Lawton et al, (1987) Plant mol.biol.9: 315-24); the CaMV 35S promoter (Odell et al, (1985) Nature 313: 810-2); the figwort mosaic virus 35S-promoter (Walker et al, (1987) Proc. Natl. Acad. Sci. USA84(19): 6624-8); the sucrose synthase promoter (Yang and Russell, (1990) Proc. Natl. Acad. Sci. USA 87: 4144-8); the R gene complex promoter (Chandler et al, (1989) Plant Cell 1: 1175-83); a chlorophyll a/b binding protein gene promoter; CaMV 35S (U.S. Pat. nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196); FMV35S (U.S. Pat. nos. 6,051,753 and 5,378,619); the PC1SV promoter (U.S. patent No. 5,850,019); the SCP1 promoter (U.S. patent No. 6,677,503); nos promoter (GenBank)^TMAccession number V00087; depicker et al, (1982) J.mol.appl.Genet.1: 561-73; bevan et al, (1983) Nature 304: 184-7).

In a particular embodiment, the nucleic acid molecule of the invention comprises a tissue-specific promoter, such as a root-specific promoter or a leaf-specific promoter. In some embodiments, a polynucleotide for coleopteran pest control according to the present invention may be cloned between two leaf-specific promoters that are oriented in opposite transcriptional directions relative to the polynucleotide or fragment, operable in a transgenic plant cell, and expressed in the transgenic plant cell to produce therein RNA molecules that may subsequently form dsRNA molecules, as described previously. Insect pests can ingest iRNA molecules expressed in plant tissues, thereby effecting repression of target gene expression.

Additional regulatory elements that may optionally be operably linked to the nucleic acid include a 5'UTR, the 5' UTR serving as a translation leader sequence element between the promoter element and the encoding polynucleotide. The translation leader sequence elements are present in fully processed mRNA and may affect processing of the primary transcript and/or stability of the RNA. Examples of translation leader elements include maize and petunia (petunia) heat shock protein leaders (U.S. Pat. No. 5,362,865), plant viral coat protein leaders, plant ribulose diphosphate carboxylase (rubisco) leaders, and the like. See, e.g., Turner and Foster, (1995) Molecular Biotech.3(3): 225-36. Non-limiting examples of 5' UTRs include GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and free, (1990) J.Virol.64:1590-7), and AGRsunos (GenBank)^TMAccession number V00087; bevan et al, (1983) Nature 304: 184-7).

Additional regulatory elements that may optionally be operably linked to the nucleic acid also include 3 'untranslated elements, 3' transcription termination regions, or polyadenylation regions. These are genetic elements located downstream of the polynucleotide, including polynucleotides that provide polyadenylation signals and/or other regulatory signals capable of affecting transcription or mRNA processing. Polyadenylation signals act in plants, causing polyadenylic acid nucleotides to be added to the 3' end of the mRNA precursor. Polyadenylation elements may be derived from a variety of plant genes or T-DNA genes. A non-limiting example of a 3' transcription termination region is the nopaline synthase 3' region (nos 3 '; Fraley et al, (1983) Proc. Natl. Acad. Sci.USA80: 4803-7). An example of the use of different 3' untranslated regions is provided in Ingelbrecht et al, (1989) Plant Cell 1: 671-80. Non-limiting examples of polyadenylation signals include signals from the pea (Pisum sativum) RbcS2 gene (Ps. RbcS 2-E9; Coruzzi et al, (1984) EMBO J.3:1671-9) and AGRtu. nos (GenBank)^TMAccession number E01312).

Some embodiments may include a plant transformation vector comprising at least one of the aforementioned regulatory elements operably linked to one or more polynucleotides of the present invention. The one or more polynucleotides, when expressed, produce one or more iRNA molecules comprising a polyribonucleotide that is specifically complementary or reverse-complementary to all or part of a native RNA molecule in the insect pest. Thus, the one or more polynucleotides may comprise a segment encoding all or a portion of the polyribonucleotide present within the targeted insect pest RNA transcript, and may comprise an inverted repeat of all or a portion of the targeted transcript. The plant transformation vector may contain a nucleotide sequence encoding a polyribonucleotide that is specifically complementary to more than one target polynucleotide, thereby allowing the production of more than one dsRNA to inhibit expression of two or more genes in cells of one or more populations or species of target insect pests. Polynucleotides comprising nucleotide sequences encoding polyribonucleotides specifically complementary or reverse complementary to fragments of different target genes can be combined into a single composite nucleic acid molecule for expression in transgenic plants. Such segments may be contiguous or separated by a spacer sequence.

In some embodiments, a plasmid already containing at least one polynucleotide of the present invention may be modified by the sequential insertion of additional polynucleotide(s) into the same plasmid, wherein the additional polynucleotide(s) are operably linked to the same regulatory element as the original polynucleotide(s). In some embodiments, the constructs may be designed to inhibit multiple target genes. In some particular embodiments, the multiple genes to be suppressed are obtained from the same insect pest species (e.g., PB), which can enhance the effectiveness of the construct. In other embodiments, the genes may be derived from different insect pests, which may broaden the range of pests for which the construct is effective. Polycistronic DNA elements can be engineered when multiple genes are targeted to achieve repression or a combination of expression and repression.

The recombinant nucleic acid molecules or vectors of the invention may comprise a selectable marker that confers a selectable phenotype on a transformed cell, such as a plant cell. Selectable markers can also be used to select plants or plant cells comprising the recombinant nucleic acid molecules of the invention. The marker may encode for biocide resistance, antibiotic resistance (e.g., kanamycin, geneticin (G418), bleomycin, hygromycin, etc.), or herbicide tolerance (e.g., glyphosate, etc.). Examples of selectable markers include, but are not limited to: a neo gene which encodes kanamycin resistance and can be selected using kanamycin, G418, or the like; a bar gene encoding bialaphos resistance; a mutant EPSP synthase gene encoding glyphosate tolerance; a nitrilase gene conferring resistance to bromoxynil; a mutant acetolactate synthase (ALS) gene that confers imidazolinone or sulfonylurea tolerance; and a methotrexate resistant DHFR gene. There are a variety of selectable markers available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, glufosinate, puromycin, spectinomycin, rifampin, streptomycin, tetracycline and the like. Examples of such selectable markers are illustrated in, for example, U.S. Pat. nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047.

The recombinant nucleic acid molecule or vector of the invention may further comprise a screenable marker. Expression can be monitored using screenable markers. Exemplary screenable markers include: β -glucuronidase or uidA Gene (GUS) encoding enzymes for which a variety of chromogenic substrates are known (Jefferson et al (1987) Plant mol. biol. Rep.5: 387-405); r-locus gene encoding a product that regulates the production of anthocyanin pigment (red) in plant tissues (Dellaporta et al, (1988)' molecular cloning of the mail R-nj allel by transposon tagging with Ac18 th item^WhereuponStudler seminar of genetics(Stadler Genetics Symposium), edited by P.Gustafson and R.appliances, New York: Plenum, pages 263-82); beta-lactamase gene (Sutcliffe et al, (1978) Proc. Natl. Acad. Sci. USA75: 3737-41); genes encoding enzymes known to produce various chromogenic substrates (e.g., PADAC, a chromogenic cephalosporin); luciferase gene (Ow et al, (1986) Science 234: 856-9); xylE gene encoding a product capable of converting a protocatechuic acidCatechol dioxygenase (Zukowski et al, (1983) Gene46(2-3): 247-55); the amylase gene (Ikatu et al, (1990) Bio/technol.8: 241-2); tyrosinase gene, which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to melanin (Katz et al, (1983) j.gen.microbiol.129: 2703-14); and an alpha-galactosidase.

In some embodiments, in methods for creating transgenic plants and expressing heterologous nucleic acids in plants, transgenic plants exhibiting reduced susceptibility to insect pests can be prepared using recombinant nucleic acid molecules as described previously. Plant transformation vectors can be prepared, for example, by inserting a polynucleotide encoding an iRNA molecule into a plant transformation vector, which is then introduced into a plant.

Suitable methods for transforming host cells include any method by which DNA can be introduced into the cells, such as by transforming protoplasts (see, e.g., U.S. patent No. 5,508,184), by drying/inhibiting mediated DNA uptake (see, e.g., Potrykus et al, (1985) mol.gen. gene.199: 183-8), by electroporation (see, e.g., U.S. patent No. 5,384,253), by agitation with silicon carbide fibers (see, e.g., U.S. patent nos. 5,302,523 and 5,464,765), by agrobacterium-mediated transformation (see, e.g., U.S. patent nos. 5,563,055, 5,591,616, 5,693,512, 5,824,877, 5,981,840, and 6,384,301), and by accelerating DNA-coated particles (see, e.g., U.S. patent nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865), among others. By applying techniques such as these, cells of almost any species can be stably transformed. In some embodiments, transformation results in the integration of the heterologous polynucleotide into the genome of the host cell. In the case of multicellular species, the transgenic cell can be regenerated into a transgenic organism. Any of these techniques can be used to produce transgenic plants, e.g., transgenic plants that comprise in their genome one or more polynucleotides encoding iRNA molecules.

The most widely used method for introducing expression vectors into plants is based on the natural transformation system of Agrobacterium. Agrobacterium tumefaciens and agrobacterium rhizogenes (a. rhizogenes) are phytopathogenic soil bacteria that genetically transform plant cells. The Ti and Ri plasmids of Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively, carry genes responsible for genetic transformation of plants. The Ti (tumor-inducing) plasmid contains a large segment of DNA, called T-DNA, which is transferred to the transformed plant. The Vir region of the other segment of the Ti plasmid is responsible for T-DNA transfer. The T-DNA region is bounded by terminal repeats. In the modified binary vector, the tumor-inducing gene has been deleted and the function of the Vir region is used to transfer foreign DNA bordered by T-DNA border elements. The T region may also contain selectable markers for efficient recovery of transgenic cells and plants, and a multiple cloning site for insertion of transfer polynucleotides such as dsRNA encoding nucleic acids.

Thus, in some embodiments, the plant transformation vector is derived from a Ti plasmid of agrobacterium tumefaciens (see, e.g., U.S. Pat. nos. 4,536,475, 4,693,977, 4,886,937 and 5,501,967, and european patent No. EP 0122791) or a Ri plasmid of agrobacterium rhizogenes. Additional plant transformation vectors include, for example and without limitation, those described by Herrera-Estralla et al, (1983) Nature 303: 209-13; bevan et al, (1983) Nature 304: 184-7; klee et al, (1985) Bio/technol.3: 637-42; and those described in european patent No. EP 0120516, as well as those derived from any of the aforementioned vectors. Other bacteria that naturally interact with plants can be modified to mediate gene transfer to many different plants, such as Sinorhizobium (Sinorhizobium), Rhizobium (Rhizobium), and Mesorhizobium (Mesorhizobium). These plant-associated symbiotic bacteria can be made competent for gene transfer by obtaining both disarmed (disarmed) Ti plasmids and suitable binary vectors.

After transformation of recipient cells with a heterologous polynucleotide, the transformed cells are typically identified for further culture and plant regeneration. To improve the ability to identify transformed cells, the skilled artisan may desire to employ selectable or screenable marker genes as set forth previously, wherein the transformation vector is used to generate transformants. In the case of the use of selectable markers, transformed cells are identified within a population of potentially transformed cells by exposing the cells to one or more selection agents. Where a screenable marker is used, the cells may be screened for the desired marker gene trait.

Cells that survive exposure to the selection agent, or cells that have been scored positive in the screening assay, can be placed in culture in a medium that supports plant regeneration. In some embodiments, any suitable plant tissue culture medium (e.g., MS medium and N6 medium) may be modified by inclusion of additional substances, such as growth regulators. The tissue can be maintained on a basal medium with growth regulators until sufficient tissue is available to initiate plant regeneration work, or after repeated rounds of manual selection until the tissue morphology is suitable for regeneration (e.g., for at least 2 weeks), and then transferred to a medium that induces shoot formation. Cultures were transferred periodically until sufficient bud formation had occurred. Once shoots are formed, they are transferred to a medium that induces root formation. Once sufficient roots are formed, the plants can be transferred to soil for further growth and maturation.

To confirm the presence of a polynucleotide of interest (e.g., a polynucleotide encoding one or more iRNA molecules that inhibit expression of a target gene in an insect pest) in a regenerated plant, a variety of assays can be performed. Such assays include, for example: molecular biological assays such as Southern and northern blots, PCR and nucleic acid sequencing; biochemical assays, such as detecting the presence or absence of a protein product, for example by immunological means (ELISA and/or western blot) or by enzymatic function; plant part assays, such as leaf or root assays; and analysis of the phenotype of the whole regenerated plant.

Integration events can be analyzed, for example, by PCR amplification using, for example, oligonucleotide primers specific for the polynucleotide of interest. PCR genotyping should be understood to include, but is not limited to: polymerase Chain Reaction (PCR) amplification of gDNA from callus of the isolated host plant is predicted to contain the polynucleotide of interest integrated into the genome, followed by standard cloning and sequence analysis of the PCR amplification product. Methods of PCR genotyping are well described (e.g.Rios, G., et al, (2002) Plant J.32:243-53) and can be applied to gDNA derived from any Plant species (e.g.Brassica napus) or tissue type (including cell cultures).

Transgenic plants formed using agrobacterium-dependent transformation methods typically contain a single recombinant DNA inserted into one chromosome. The polynucleotide of this single recombinant DNA is referred to as a "transgenic event" or an "integration event". Such transgenic plants are heterozygous for the inserted heterologous polynucleotide. In some embodiments, the transgenic plant is obtained by segregating a separate isolated transgenic plant containing a single exogenous gene from itself (e.g., T₀Plants) sexual mating (selfing) to produce T₁Seeds, and obtaining transgenic plants homozygous for the transgenes. Generated T₁One quarter of the seeds will be homozygous for the transgene. Plants produced from germinating T1 seeds can be used to test for heterozygosity, typically using SNP assays or thermal amplification assays, so as to allow for discrimination between heterozygotes and homozygotes (i.e., zygosity assay).

In particular embodiments, at least 2, 3,4, 5,6, 7,8, 9, or 10, or more different iRNA molecules are produced in a plant cell that have an insect pest inhibitory effect. iRNA molecules (e.g., dsRNA molecules) can be expressed from multiple polynucleotides introduced in different transformation events, or from a single polynucleotide introduced in a single transformation event. In some embodiments, multiple iRNA molecules are expressed under the control of a single promoter. In other embodiments, multiple iRNA molecules are expressed under the control of multiple promoters. A single iRNA molecule can be expressed that comprises a plurality of polyribonucleotides, each of which is at least substantially complementary or reverse-complementary, respectively, to a different locus (e.g., the locus defined by SEQ ID NOS: 2-3) in a different population of the same insect pest species or within one more insect pests of different insect pest species.

In addition to direct transformation of plants with a recombinant nucleic acid molecule, transgenic plants can be prepared by crossing a first plant having at least one transgenic event with a second plant lacking such an event. For example, a recombinant nucleic acid molecule comprising a polynucleotide encoding an iRNA molecule can be introduced into a first plant line that is susceptible to transformation to produce a transgenic plant comprising the polynucleotide, which transgenic plant can be crossed with a second plant line to introgress (introgresses) the polynucleotide encoding the iRNA molecule into the second plant line.

In some aspects, seed and commodity products produced from transgenic plants derived from the transgenic plant cells are included, wherein the seed or commodity product comprises a detectable amount of a polynucleotide or polyribonucleotide of the invention. In some embodiments, such commercial products can be produced, for example, by obtaining transgenic plants and preparing food or feed therefrom. Commercial products comprising one or more of the polynucleotides or polyribonucleotides of the invention include (for example, but are not limited to): meal, oil, crushed or whole grain or seed of a plant, and any food product comprising any meal, oil, or crushed or whole grain of a transgenic plant or seed comprising one or more of the polynucleotides or polyribonucleotides of the invention. The detection of one or more of the polynucleotides or polyribonucleotides of the invention in one or more commercial or commodity products actually demonstrates that the commercial or commodity product is produced by a transgenic plant designed to express one or more of the iRNA molecules of the invention for the purpose of controlling insect pests.

In some embodiments, a transgenic plant or seed comprising a polynucleotide of the invention may also comprise in its genome at least one other transgenic event, including but not limited to: transgenic events by which iRNA molecules are transcriptionally targeted to other coleopteran pest loci than the locus defined by SEQ ID NOs 2-3, such as, for example, one or more loci selected from: caf1-180 (U.S. patent application publication No. 2012/0174258), VatpaseC (U.S. patent application publication No. 2012/0174259), Rhol (U.S. patent application publication No. 2012/0174260), vatpash (U.S. patent application publication No. 2012/0198586), PPI-87B (U.S. patent application publication No. 2013/0091600), RPA70 (U.S. patent application publication No. 2013/0091601), RPS6 (U.S. patent application publication No. 2013/0097730), ROP (U.S. patent application publication No. 14/577,811), RNA polymerase II (U.S. patent application publication No. 62/133,214), RNA polymerase III 40 (U.S. patent application publication No. 14/577,854), RNA polymerase II215 (U.S. patent application publication No. 62/133,202), RNA polymerase II33 (U.S. patent application publication No. 62/133,210), transcription elongation factor spt5 (U.S. patent application publication No. 62/168,613), Transcriptional elongation factors spt6 (U.S. patent application No. 62/168,606), ncm (U.S. patent application No. 62/095487), dre4 (U.S. patent application No. 14/705,807), COPI α (U.S. patent application No. 62/063,199), COPI β (U.S. patent application No. 62/063,203), COPI γ (U.S. patent application No. 62/063,192), and COPI δ (U.S. patent application No. 62/063,216); a transgenic event from which an iRNA molecule is transcribed that targets: organisms other than coleopteran pests (e.g., genes in plant parasitic nematodes); genes encoding insecticidal proteins (e.g., Bacillus thuringiensis insecticidal proteins and PIP-1 polypeptides); herbicide tolerance genes (e.g., genes that are tolerant to glyphosate); and genes that cause a desired phenotype in the transgenic plant, such as increased yield, altered fatty acid metabolism, or restoration of cytoplasmic male sterility. In particular embodiments, polynucleotides encoding iRNA molecules of the present invention can be combined with other insect control and disease traits in plants to obtain desired traits to enhance control of plant disease and insect damage. In some examples, combining insect control traits having different modes of action provides better and synergistic persistence of the protected transgenic plants than plants having a single control trait, for example, because the likelihood of developing resistance to one or more traits in the field is reduced.

Target gene suppression in insect pests

A. Overview

In some embodiments of the invention, an insect pest (e.g., pollen beetle) can be provided with at least one nucleic acid molecule useful for controlling an insect pest, wherein the nucleic acid molecule causes RNAi-mediated gene silencing in the pest. In particular embodiments, iRNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) can be provided to a pest. In some embodiments, a nucleic acid molecule useful for controlling an insect pest may be provided to the pest by contacting the pest with the nucleic acid molecule. In particular embodiments, nucleic acid molecules useful for controlling insect pests may be provided in feeding substrates (e.g., nutritional compositions) for the pests. In particular embodiments, the nucleic acid molecule can be provided by ingestion of plant material ingested by an insect pest that comprises a nucleic acid molecule useful for controlling the pest. In certain embodiments, the nucleic acid molecule is present in plant material by expression of a heterologous polynucleotide introduced into the plant material, for example by transforming a plant cell with a vector comprising the heterologous polynucleotide, and then regenerating plant material or a whole plant from the transformed plant cell.

RNAi mediated repression of target genes

In some embodiments, the invention provides iRNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) that can be designed to target an essential native polynucleotide (e.g., ssrp1 mRNA) in the transcriptome of an insect pest (e.g., pollen beetle), e.g., by designing an iRNA molecule with at least one strand comprising a polynucleotide nucleotide that is specifically complementary or reverse-complementary to a target polynucleotide. The sequence of an iRNA molecule so designed may be identical to that of the target polynucleotide, or may incorporate mismatches that do not prevent specific hybridization between the iRNA molecule and its target polynucleotide.

The iRNA molecules of the invention can be used in methods for gene suppression in insect pests, thereby reducing the level or incidence of damage caused by the pest to a plant (e.g., a protected transgenic plant comprising an iRNA molecule). As used herein, the term "gene suppression" refers to any one of the well-known methods for reducing the level of protein produced as a result of transcription of a gene into mRNA and subsequent translation of the mRNA, including reducing the expression of the protein by the gene or encoding polynucleotide, including post-transcriptional inhibition of expression and transcriptional repression. Post-transcriptional inhibition is mediated by specific homology between all or part of the mRNA transcribed from the gene targeted to be inhibited and the corresponding iRNA molecule used for repression. Furthermore, post-transcriptional inhibition refers to a substantial and measurable decrease in the amount of available mRNA in a cell for binding by ribosomes.

In embodiments where the iRNA molecules of the invention are dsRNA molecules, the DICER enzyme can cleave the dsRNA molecules into short miRNA or siRNA molecules (approximately 20 nucleotides in length, e.g., 19 to 23 nucleotides in length). The double-stranded siRNA molecules generated by DICER activity on dsRNA molecules can be divided into two single-stranded sirnas: the "passenger chain" and the "leader chain". The passenger chains can be degraded and the guide chains can be incorporated into the RISC. Post-transcriptional inhibition occurs by specific hybridization of the guide strand to the mRNA molecule, followed by cleavage by the enzyme Argonaute (the catalytic component of the RISC complex).

In embodiments of the invention, any form of iRNA molecule may be used. It will be appreciated by those skilled in the art that dsRNA molecules are generally more stable than single stranded RNA molecules, and are also generally more stable in cells, both during the preparation process and during the step of providing the iRNA molecules to the cells. Thus, for example, while siRNA molecules and miRNA molecules may be equally effective in some embodiments, dsRNA molecules may be selected for their stability. Certain embodiments include, for example, polynucleotides encoding only one strand of a dsRNA molecule such that they can bind in a transgenic cell to polynucleotides encoding the other strand of the dsRNA molecule, wherein the dsRNA molecule is formed in the cell by hybridization of the two strands encoded by separate polynucleotides.

In particular embodiments, nucleic acid molecules are provided that comprise a polynucleotide that can be expressed in vitro to produce an iRNA molecule comprising polyribonucleotides that are substantially homologous to polyribonucleotides of an RNA molecule encoded by the polynucleotide within the genome of an insect pest. In certain embodiments, the in vitro transcribed iRNA molecule can be a stabilized dsRNA molecule comprising a stem-loop structure. Post-transcriptional inhibition of a target gene in an insect pest may occur after the pest contacts an iRNA molecule that is transcribed in vitro.

In some embodiments of the invention, expression of a polynucleotide comprising at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of a target gene or its complement or reverse complement is used in a method of post-transcriptional suppression of a target gene in an insect pest, wherein the polynucleotide is selected from the group consisting of SEQ ID NO: 1; 1 or the reverse complement of SEQ ID NO; comprises the PB ssrp1 coding sequence of SEQ ID NOS: 2-3; the complement or reverse complement of the PB ssrp1 coding sequence comprising SEQ ID NOS: 2-3; a fragment comprising at least 15 contiguous nucleotides of the PB ssrp1 coding sequence of SEQ ID NOS: 2-3 (e.g., SEQ ID NO: 4); the complement of a fragment comprising at least 15 contiguous nucleotides of the PB ssrp1 coding sequence of SEQ ID NOs 2-3; the reverse complement of a fragment comprising at least 15 contiguous nucleotides of the PB ssrp1 coding sequence of SEQ ID NOS: 2-3; a naturally-encoding polynucleotide of a Nitidus organism (e.g., PB) comprising SEQ ID NO. 4; a complementary sequence of a naturally encoded polynucleotide of a Nitidus organism comprising SEQ ID NO 4; a reverse complement of a naturally encoded polynucleotide of a Nitidus organism comprising SEQ ID NO. 4; a fragment of at least 15 contiguous nucleotides of a naturally encoded polynucleotide of a clearweed organism comprising SEQ ID NO 4; a complementary sequence comprising a fragment of at least 15 contiguous nucleotides of a naturally encoded polynucleotide of a Nitidus organism of SEQ ID NO. 4; the reverse complement of a fragment of at least 15 contiguous nucleotides of a naturally encoded polynucleotide of a Nitidus organism comprising SEQ ID NO. 4. In certain embodiments, expression of a nucleic acid molecule having at least about 80% identity (e.g., 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, and 100%) to any of the foregoing may be used. In these and other embodiments, a nucleic acid molecule that specifically hybridizes to an RNA molecule present in at least one cell of an insect pest may be expressed.

An important feature of some embodiments herein is that the RNAi post-transcriptional repression system is able to tolerate sequence variations in the target gene that are expected to occur due to genetic mutations, strain polymorphisms, or evolutionary divergence. The iRNA molecule may not necessarily be absolutely identical to the primary transcript of the target gene (or its complement and reverse complement) or to the fully processed mRNA, as long as the iRNA molecule specifically hybridizes to the primary transcript of the target gene or the fully processed mRNA. In addition, the iRNA molecule need not be full length relative to the primary transcript of the target gene or the fully processed mRNA.

Inhibition of target genes using the iRNA technique of the invention is sequence specific; that is, genetic suppression is targeted to a polynucleotide that is substantially identical to one or more iRNA molecules or their complement or reverse complement. In some embodiments, inhibition can be performed using RNA molecules that: which comprises a polyribonucleotide having a nucleotide sequence identical to that of a portion of an mRNA transcribed from a target gene or a complementary sequence or a reverse complementary sequence thereof. In these and other embodiments, RNA molecules comprising polyribonucleotides having one or more insertions, deletions, and/or point mutations relative to a target polynucleotide can be used. In particular embodiments, the iRNA molecule and the target gene or a portion of its complement or reverse complement can share, for example, at least from about 80%, at least from about 81%, at least from about 82%, at least from about 83%, at least from about 84%, at least from about 85%, at least from about 86%, at least from about 87%, at least from about 88%, at least from about 89%, at least from about 90%, at least from about 91%, at least from about 92%, at least from about 93%, at least from about 94%, at least from about 95%, at least from about 96%, at least from about 97%, at least from about 98%, at least from about 99%, at least from about 100%, and 100% sequence identity. In some examples, the duplex region of the dsRNA molecule can be specifically hybridizable to a portion of a target gene transcript. In a specifically hybridizable molecule, a less than full-length polyribonucleotide exhibiting a greater degree of sequence identity compensates for a longer, less identical polyribonucleotide. The polyribonucleotide in the duplex region of the dsRNA molecule that is identical or substantially identical to a portion of the target gene transcript or its complement or reverse complement sequence may be at least about 25, 50, 100, 200, 300, 400, 500, or at least about 1000 bases in length. In some examples, polyribonucleotides of greater than 20 to 100 nucleotides may be used. In particular examples, polyribonucleotides greater than about 200 to 300 nucleotides may be used. In these and other specific examples, polyribonucleotides greater than about 500 to 1000 nucleotides may be used, depending on the size of the target gene.

In certain embodiments, expression of a target gene in an insect pest can be inhibited within cells of the pest by at least 10%, at least 33%, at least 50%, or at least 80%, such that significant inhibition occurs. Significant inhibition refers to inhibition above a threshold that results in a detectable phenotype (e.g., growth arrest, feeding arrest, development arrest, induced death, etc.), or a detectable reduction in the appearance of RNA and/or gene products corresponding to the target gene being inhibited. While in certain embodiments of the invention inhibition occurs in substantially all cells of the pest, in other embodiments inhibition occurs only in a subset of cells expressing the target gene.

In some embodiments, transcriptional repression in a cell is mediated by the presence of dsRNA molecules exhibiting substantial sequence identity to the promoter DNA or its complement, thereby effecting so-called "promoter transrepression". Gene suppression may be effective against a target gene in an insect pest that may ingest or contact such dsRNA molecules (e.g., by ingesting or contacting plant material containing the dsRNA molecules). dsRNA molecules used in promoter transrepression can be specifically designed to inhibit or repress expression of one or more homologous or complementary polynucleotides in insect pest cells. U.S. Pat. nos. 5,107,065, 5,759,829, 5,283,184 and 5,231,020 disclose the regulation of gene expression in plant cells by post-transcriptional gene suppression with RNA in either the antisense or sense orientation.

C. Expression of iRNA molecules to provide insect pests

iRNA molecules for RNAi-mediated gene suppression in insect pests can be expressed in any of a number of in vitro or in vivo formats. The iRNA molecule can then be provided to the insect pest, for example, by contacting the iRNA molecule with the pest, or by causing the pest to ingest or otherwise internalize the iRNA molecule. Some embodiments include transformed host plants, transformed plant cells, and progeny of transformed plants of the insect pest. The transformed plant cells and transformed plants can be engineered to express one or more of the iRNA molecules, e.g., under the control of a heterologous promoter, to provide a pest protection effect. Thus, when an insect pest eats a transgenic plant or plant cell during feeding, the pest can ingest iRNA molecules expressed in the transgenic plant or cell. The polynucleotides of the invention can also be introduced into a wide variety of prokaryotic and eukaryotic microbial hosts to produce iRNA molecules. The term "microorganism" includes prokaryotic and eukaryotic species, such as bacteria and fungi.

Regulation of gene expression may include partial or complete suppression of such expression. In other embodiments, a method for suppressing gene expression in an insect pest comprises providing in a tissue of the pest host a gene suppressing amount of at least one dsRNA molecule formed upon transcription from a polynucleotide as described herein, at least one segment of which is complementary to an mRNA within an insect pest cell. dsRNA molecules ingested by insect pests, including modified forms thereof such as siRNA, miRNA, shRNA or hpRNA molecules, can be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identical to RNA molecules transcribed from a gene, e.g., PB ssrp1 comprising SEQ ID NOs 2-3. Thus, isolated and substantially purified nucleic acid molecules, including but not limited to non-naturally occurring polynucleotides and recombinant DNA constructs, are provided for providing dsRNA molecules that repress or inhibit expression of a target endogenous encoding polynucleotide therein upon introduction into an insect pest.

Particular embodiments provide a population delivery system for delivering iRNA molecules for post-transcriptional suppression of one or more target genes in insect plant pests and control of the plant pests. In some embodiments, the delivery system comprises uptake of host transgenic plant cells or host cell contents comprising an RNA molecule transcribed in the host cell. In these and other embodiments, a transgenic plant cell or transgenic plant is created that contains a recombinant DNA construct encoding the stabilized dsRNA molecule of the invention. Transgenic plant cells and transgenic plants comprising a nucleic acid encoding a particular iRNA molecule can be produced by: plant transformation vectors comprising polynucleotides encoding iRNA molecules of the invention (e.g., stabilized dsRNA molecules) are constructed using recombinant DNA techniques, the underlying techniques of which are well known in the art, plant cells or plants are transformed, and transgenic plant cells or plants containing the transcribed iRNA molecules are generated.

To confer protection of transgenic plants against insect pests, recombinant DNA molecules may be transcribed into iRNA molecules, such as dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules or hpRNA molecules, for example. In some embodiments, the RNA molecule transcribed from the recombinant DNA may form a dsRNA molecule within the tissue or fluid of the recombinant plant. Such dsRNA molecules may be composed in part of the same polyribonucleotide as a corresponding target polyribonucleotide transcribed from DNA within the type of insect pest that may attack the host plant. Expression of the target gene within the pest is suppressed by the dsRNA molecule, and suppression of expression of the target gene in the pest results in the transgenic plant being resistant to the pest. The regulatory role of dsRNA molecules has been shown to apply to a variety of genes expressed in pests, including, for example, endogenous genes responsible for cellular metabolism or cellular transformation, including housekeeping genes; a transcription factor; an molting-associated gene; and other genes encoding polypeptides involved in cellular metabolism or normal growth and development.

For transcription from a transgene or expression construct in vivo, regulatory regions (e.g., promoters, enhancers, silencers, and polyadenylation signals) can be used in some embodiments to transcribe one or more RNA strands. Thus, in some embodiments, as indicated previously, the polynucleotide used to produce the iRNA molecule can be operably linked to one or more promoter elements that are functional in a plant host cell. The promoter may be an endogenous promoter that normally resides in the host genome. The polynucleotides of the invention may be further flanked by additional elements that advantageously affect their transcription and/or the stability of the resulting transcript, under the control of the operably linked promoter element. Such elements may be located upstream of an operably linked promoter, downstream of the 3 'end of an expression construct, and may be present both upstream of the promoter and downstream of the 3' end of an expression construct.

Some embodiments provide methods for reducing damage to a host plant (e.g., canola) caused by an insect pest feeding on the plant, wherein the method comprises providing in the host plant a transgenic plant cell expressing at least one nucleic acid molecule of the invention, wherein the nucleic acid molecule, upon ingestion by one or more pests, acts to inhibit expression of the target polynucleotide within the pest, which inhibition of expression causes death and/or reduced growth of the pest, thereby reducing damage to the host plant caused by the pest. In some embodiments, the nucleic acid molecule is a dsRNA molecule. In particular embodiments, the dsRNA molecule comprises more than one polyribonucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a cell of the insect pest. In some embodiments, the nucleic acid molecule comprises a polyribonucleotide that specifically hybridizes to a nucleic acid molecule expressed in an insect pest cell.

In some embodiments, methods are provided for increasing yield of a crop plant (e.g., a brassica plant, such as canola), wherein the method comprises introducing into the crop plant at least one nucleic acid molecule comprising a polynucleotide of the invention; cultivating the crop plant to allow expression of the iRNA molecule by the polynucleotide, wherein expression of the iRNA molecule inhibits insect pest damage and/or growth, thereby reducing or eliminating yield loss due to pest infestation. In some embodiments, the iRNA molecule is a dsRNA molecule. In these and other embodiments, the dsRNA molecules may each comprise more than one polyribonucleotide that is specifically hybridizable to a nucleic acid molecule expressed in an insect pest cell. Thus, specific polyribonucleotides of a dsRNA molecule can be expressed by one or more nucleotide sequences within a polynucleotide of the invention.

In some embodiments, there is provided a method for modulating expression of a target gene in an insect pest, the method comprising: transforming a plant cell with a vector comprising a polynucleotide encoding at least one iRNA molecule of the invention, wherein said polynucleotide is operably linked to a promoter and a transcription termination element; culturing the transformed plant cell under conditions sufficient to allow development of a plant cell culture comprising a plurality of transgenic plant cells; selecting a transgenic plant cell that has integrated the polynucleotide into its genome; screening the transgenic plant cell for expression of an iRNA molecule encoded by the integrated polynucleotide; selecting a transgenic plant cell expressing an iRNA molecule; the selected transgenic plant cells are then used to feed the insect pest. Plants can also be regenerated from transgenic plant cells expressing iRNA molecules encoded by the integrated polynucleotides. In some embodiments, the iRNA molecule is a dsRNA molecule comprising a polyribonucleotide that is specifically hybridizable to a transcript of a target gene in an insect pest. In these and other embodiments, the dsRNA molecule comprises more than one polyribonucleotide transcribed from a nucleotide sequence within the polynucleotide encoding the dsRNA molecule.

The iRNA molecules of the invention can be incorporated into seeds of a plant species (e.g., brassica) as the expression product of a heterologous polynucleotide incorporated into the genome of a plant cell, or incorporated into a coating or seed treatment applied to the seed prior to planting. A plant cell comprising a polynucleotide of the invention is considered to comprise a transgenic event. Also included in embodiments of the invention are delivery systems for delivering iRNA molecules to insect pests. For example, an iRNA molecule of the invention can be introduced directly into a cell of one or more pests. Methods for introduction can include directly mixing iRNA with plant tissue from one or more insect pest hosts and applying to the host plant tissue a composition comprising an iRNA molecule of the invention. For example, the iRNA molecule can be sprayed onto the surface of a plant. Alternatively, the iRNA molecule may be expressed by a microorganism, which may then be applied to the surface of a plant, or introduced into the root or stem by physical means such as injection. As discussed previously, the transgenic plant can also be genetically engineered to express at least one iRNA molecule in an amount sufficient to kill an insect pest that infests the plant. iRNA molecules produced by chemical or enzymatic synthesis may also be formulated in a manner consistent with common agricultural practice and used as spray products or bait products to control plant damage by insect pests. The formulation may contain suitable adjuvants (e.g., sticking and wetting agents) necessary for effective leaf coverage, as well as UV protectants that protect the iRNA molecule from UV damage. Such additives are commonly used in the biopesticide industry and are well known to those skilled in the art. Such applications may be combined with other spray insecticide applications (biologically or otherwise based) to enhance plant protection against the pests.

All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein, to the same extent as if each reference were individually and specifically indicated to be incorporated by reference. The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

The following embodiments are provided to illustrate certain specific features and/or aspects. These embodiments should not be construed as limiting the disclosure to the particular features or aspects described.