TW201823459A - Pre-mrna processing factor 8 (prp8) nucleic acid molecules to control insect pests - Google Patents

Abstract

This disclosure concerns nucleic acid molecules and methods of use thereof for control of insect pests through RNA interference-mediated inhibition of target coding and transcribed non-coding sequences in insect pests, including coleopteran pests. The disclosure also concerns methods for making transgenic plants that express nucleic acid molecules useful for the control of insect pests, and the plant cells and plants obtained thereby.

Description

Pre-mRNA processing factor 8 (PRP8) nucleic acid molecule controlling insect pests

RELATED APPLICATIONS This application is based on the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the disclosure.

TECHNICAL FIELD OF THE INVENTION The present invention relates generally to genetic control of plant damage caused by insect pests (e.g., coleopteran pests). In a specific embodiment, the invention relates to the identification of targeted coding and non-coding polynucleotides, and the use of recombinant DNA techniques for post-transcriptional suppression or inhibition of targeted coding and non-coding polynucleotides in insect pest cells. Performance to provide the effect of plant protection.

BACKGROUND OF THE INVENTION Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is one of the most destructive corn rootworm species in North America and is grown in the Midwestern United States. The district is a particularly important thing. The northern corn rootworm (NCR), the northern corn rootworm ( Diabrotica barberi Smith and Lawrence), is a closely related species that co-exists in many of the same ranges of WCR. There are several other related species of Diabrotica in the Americas that are major pests: Mexican corn rootworm (MCR), Mexican corn root beetle ( D. virgifera zeae Krysan and Smith); Southern corn rootworm (SCR), D. undecimpunctata howardi Barber; Brazilian corn rootworm ( D. balteata LeConte); Cucumber eleventh leaf beetle ( D. Undecimpunctata tenella ); D. speciosa Germar; and Duundecimpunctata Mannerheim. The US Department of Agriculture has estimated that corn rootworms cause $1 billion in annual losses, including $800 million in production losses and $200 million in processing costs.

Eggs of both WCR and NCR are deposited in the soil during the summer. The insects are still in the egg stage throughout the winter. These eggs are oval, white, and less than 0.004 inches in length. The larvae hatch at the end of May or early June, and the precise time course of egg hatching varies from year to year due to temperature differences and location. The newly hatched larvae are white worms less than 0.125 inches in length. After hatching, the larvae begin to feed on the roots of the corn. Corn rootworms have passed through three larval ages. After several weeks of feeding, the larvae molt enters the stage of sputum. They become pupa in the soil and then emerge from the soil as adults in July and August. Adult rootworms are approximately 0.25 inches in length.

Corn rootworm larvae develop on maize and several other grass species. The larvae raised on yellow foxtail appear later and the size of the adult's head shell is smaller than that of larvae raised on corn. Ellsbury et al. (2005) Environ. Entomol. 34: 627-34. WCR adults feed on corn silk, pollen, and corn kernels on exposed ear tips. If WCR adults appear before the presence of maize reproductive tissues, they may feed on leaf tissue, thereby slowing plant growth and occasionally killing host plants. However, when the desired whiskers are available, the adults will move quickly to the pollen. Adult NCRs also feed on the reproductive tissues of corn plants, but conversely, corn leaves are rarely fed.

Most of the rootworm damage in corn is caused by feeding by larvae. The newly hatched rootworm initially feeds on the fine corn root hair and drills into the root tip. When the larvae grow larger, they feed on the main root and drill into the main root. When the amount of corn rootworm is large, larvae feeding often leads to root reduction until the base of the corn stem. Severe root damage interferes with the ability of the roots to transport moisture and nutrients to plants, reduces plant growth, and leads to reduced grain production, often resulting in a significant reduction in overall yield. Severe root damage also often causes the lodging of corn plants, which makes harvesting more difficult and further reduces yield. Furthermore, the feeding of adults on the reproductive tissues of corn can lead to the reduction of the tip of the ear. If such "silk clipping" is sufficiently severe during loosening, pollination may be interrupted.

Try to control through crop rotation, chemical pesticides, bio-pesticides (for example, spore-forming Gram-positive bacteria, Bacillus thuringiensis ), Bt toxin-expressing gene-transplanting plants, or combinations thereof. Corn rootworm. Crop rotation in the use of arable land suffers from significant disadvantages of undesired placement. Furthermore, spawning of some rootworm species may occur in soybean fields, thus reducing the effectiveness of rotation of corn and soybeans.

The most reliant strategy for achieving control of corn rootworms is chemical pesticides. Despite this, the use of chemical pesticides is an imperfect corn rootworm control strategy; despite the use of pesticides, when the cost of chemical pesticides is added to the cost of corn rootworm damage that may occur, the United States The loss of corn rootworm may exceed $1 billion. High population larvae, heavy rain and improper application of pesticides may all lead to insufficient control of corn rootworms. Furthermore, continued use of pesticides may select insecticide resistant rootworm strains and increase significant environmental concerns because of toxicity to non-target species.

European pollen beetles (PB) are serious pests of oilseed rape, and both larvae and adults feed on flowers and pollen. Damage to crops by pollen beetles can result in 20-40% yield loss. The main pest species is Meligethes aeneus . At present, the control of pollen beetles in European rapeseed mainly relies on pyrethroids, which are expected to be quickly eliminated because of their environmental and regulatory profiles. Furthermore, the resistance of pollen beetles to existing insecticides has been reported. Therefore, the pollen beetle control solution that urgently needs environmental protection is similar to the novel mode of action.

In nature, pollen beetles live in adults in the soil or under litter. In the spring, adults emerge from hibernation and begin to feed weeds and migrate to flowering Brassica plants. Spawning in the European rape buds. The larvae feed and develop on the flowers and buds. Late larvae look for pupation sites in the soil. The second generation of adults appeared in July or early August, and they ate on various flowering plants and looked for places to spend the winter.

RNA interference (RNAi) is a method of utilizing an endogenous cellular pathway whereby an interfering RNA (iRNA) molecule (eg, a dsRNA molecule) specific for all or any portion of an appropriately sized gene of a targeted gene Will cause degradation of the mRNA encoded thereby. In recent years, RNAi has been used in many species and experimental systems to perform gene "knockdown"; for example, Caenorhabditis elegans , plants, insect embryos, and cells in tissue culture. See, for example, 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 penetrates the endogenous pathway including the DICER protein complex to achieve mRNA degradation. DICER cleaves long dsRNA molecules into short fragments of approximately 20 nucleotides, designated small interfering RNA (siRNA). The siRNA is decomposed into two single-stranded RNAs: a passenger strand and a guide strand. The passenger strands are degraded and the guide strands are incorporated into the RNA-induced silent complex (RISC). Microribonucleic acid (miRNA) is a very similarly structured molecule that can be cleaved from a "loop" containing a polynucleotide that is linked to a hybrid passenger strand and a guide strand, and the like can be similar Into the RISC. Post-transcriptional gene silencing occurs when the leader strand specifically binds to a complementary mRNA molecule and induces cleavage by the Argoline family (Argonaute) as a catalyst component of the RISC complex. Although initial concentrations of siRNA and/or miRNA are limited in some eukaryotes such as plants, nematodes, and some insects, this process is known to be systematically spread throughout the organism.

Only transcripts complementary to siRNA and/or miRNA are cleaved and degraded, and thus the knock-down of mRNA expression is sequence specific. In plants, there are several functional groups in the DICER gene. The gene silencing effect of this RNAi persists for several days and, under experimental conditions, can result in a 90% or greater decrease in the abundance of the targeted transcript, with subsequent reduction in the corresponding protein level. In terms of insects, there are at least two DICER genes, and DICER1 promotes miRNA degradation guided by Argonaute1. Lee et al. (2004) Cell 117 (1): 69-81. DICER2 promotes the degradation of siRNA by the aggrecin family 2 .

U.S. Patent No. 7,612,194 and U.S. Patent Publication Nos. 2007/0050860, 2010/0192265 and 2011/0154545 disclose the separation from the cockroach of D. v. virgifera LeConte . A sequence library of 9112 performance sequence tags (ESTs). It is proposed in US Patent No. 7,612,194 and U.S. Patent Publication No. 2007/0050860 to operatively link a promoter to a nucleic acid molecule which is disclosed herein with D. v. virgifera One of several specific partial sequences of the vacuolar H ⁺ -ATPase (V-ATPase) is complementary for expression of antisense RNA in plant cells. U.S. Patent Publication No. 2010/0192265 suggests operatively linking a promoter to a nucleic acid molecule that is complementary to a specific portion of a functional gene that is unknown and not revealed by D. v. virgifera. The sequence (this part of the sequence is declared to be 58% identical to the gene product of C56C10.3 in C. elegans ) for expression of antisense RNA in plant cells. U.S. Patent Publication No. 2011/0154545 proposes to operatively link a promoter to a nucleic acid molecule complementary to two of the exogenous beta subunit genes of D. v. virgifera. A specific partial sequence for the expression of antisense RNA in plant cells. Further, U.S. Patent No. 7,943,819 discloses a sequence library of 906 performance sequence tags (ESTs) that are isolated from the larvae of the D. v. virgifera LeConte, and the cut midgut, and It is proposed to operably link a promoter to a nucleotide sequence that is complementary to a particular partial sequence of the charged polysomal protein 4b gene of D. v. virgifera . Used to express double-stranded RNA in plant cells.

In the U.S. Patent No. 7,612,194 and U.S. Patent Publication Nos. 2007/0050860, 2010/0192265 and 2011/0154545, except for a specific partial sequence of a V-ATPase and a specific part of a gene of unknown function. Outside the sequence, no further advice is provided regarding the use of any particular sequence in which more than 9000 sequences are listed for RNA interference. Further, none of the U.S. Patent No. 7,612,194 and U.S. Patent Publication Nos. 2007/0050860, 2010/0192265 and 2011/0154545 provide for the use of more than 9,000 sequences in corn rootworm species. When it is dsRNA or siRNA, which will be fatal or even useful, provide any guidance. U.S. Patent No. 7,943,819, in addition to the specific partial sequence of the charged polysomal protein 4b gene, provides no suggestion for the use of any particular sequence in which more than 900 sequences are listed for RNA interference. Furthermore, U.S. Patent No. 7,943,819 does not provide any guidance as to whether more than 900 sequences are provided for use as a dsRNA or siRNA in a corn rootworm species, which would be fatal or even useful. The use of a sequence derived from the Diabrotica virgifera Snf7 gene for maize interference of maize is described in US Patent Publication No. 2013/040173 and PCT Patent Publication No. WO 2013/169923. (also described in Bolognesi et al. (2012) PLOS ONE 7(10): e47534. doi: 10.1371/ journal.pone. 0047534).

When used as dsRNA or siRNA, the overwhelming majority of the sequences complementary to the corn rootworm DNAs (eg, the foregoing) do not provide protection from the damage of the corn rootworm species. For example, Baum et al. (2007) Nature Biotechnology 25: 1322-1326, describes the effect of inhibiting several WCR gene targets by RNAi. These authors recorded that 8 of the 26 targeted genes they tested did not provide experimentally significant coleopteran mortality at a very high iRNA (eg, dsRNA) concentration of over 520 ng/cm ² .

The authors of U.S. Patent No. 7,612,194 and U.S. Patent Publication No. 2007/0050860 first report on plant-bound RNAi targeting western corn rootworm in corn plants. Baum et al. (2007) Nat. Biotechnol. 25(11): 1322-6. These authors describe a high-output in vivo dietary RNAi system used to screen for possible target genes for development of gene-transferred RNAi maize. Of the initial 290 target gene pools, only 14 showed potential for larval control. One of the most potent double-stranded RNAs (dsRNAs) targets a vacuolar ATPase subunit A (V-ATPase) that causes rapid inhibition of the corresponding endogenous mRNA with a low concentration of dsRNA and triggers specificity RNAi reaction. Thus, for the first time, these authors have used sufficient evidence to demonstrate the potential of plant-bound RNAi as a possible pest management tool, while at the same time demonstrating that valid targets are not correctly a priori identified, even by relatively small candidate genomes.

SUMMARY OF THE INVENTION Disclosed herein are methods of using nucleic acid molecules (eg, targeting genes, DNAs, dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs) and the like for controlling insect pests, including, for example, coleopteran pests, such as corn. D. v. virgifera LeConte (Western Corn Rootworm, "WCR"); Northern Corn Rootworm ( D. barberi Smith and Lawrence) (Northern Corn Rootworm, "NCR"); Cucumber Eleven D. u. howardi Barber (Southern corn rootworm, "SCR"); Mexican corn root leaf ( D. v. zeae Krysan and Smith) (Mexico corn rootworm, "MCR") D. balteata LeConte; D. u. tenella ; D. speciosa Germar; Cucumber eleven-leaf sweet potato leaf beetle ( D. u Undecimpunctata Mannerheim), and Meligethes aeneus Fabricius (pollen beetle, "PB"). Exemplary nucleic acid molecules are disclosed in particular examples that may be homologous to at least a portion of one or more natural nucleic acids within an insect pest.

In this and further examples, the native nucleic acid sequence can be a target gene, which can be, for example but not limited to, a metabolic process involved; or a product involved in larval development. In some instances, transcription of a nucleic acid molecule comprising a polynucleotide homologous to a target gene inhibits the performance of the target gene, may be fatal in insect pests, or cause insect pest growth and / or development is reduced. In a particular example, pre- mRNA processing factor 8 ( prp8 ) or prp8 homolog can be selected as a target gene for post-transcriptional silence. In a specific example, a method for the inhibition of transcription of a given target gene is selected from the group of genes in Prp8 consisting of the following: Diabrotica Prp8 (e.g., sequence identification number: 1 (prp8-1) and sequence identification numbers: 3 (prp8-2)) and beetle (Meligethes) prp8 (e.g., SEQ ID. No: 5). Thus disclosed herein is an isolated nucleic acid molecule comprising the following polynucleotide: sequence number: 1; sequence identification number: 1 complement; sequence number: 3; sequence number: 3 complement; sequence Identification number: 5; sequence identification number: complement of 5; and/or fragments of any of the foregoing (eg, sequence identification number: 7-12 and its complement).

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 targeted gene product (for example, the product of the prp8 gene). For example, one nucleic acid molecule may encode a polypeptide comprising a polynucleotide, the polypeptide with at least 85% identity to the following: Diabrotica Prp8 (e.g., SEQ ID. No: 2 SEQ ID. No: 4); Nitidulidae Prp8 (eg, sequence ID: 6); and/or the amino acid sequence within the prp8 gene product. Further disclosed are nucleic acid molecules comprising a polynucleotide, wherein the polynucleotide is a reverse complement of a polynucleotide encoding a polypeptide, wherein the polypeptide is at least 85% identical to an amino acid in a targeted gene product sequence.

Also disclosed are cDNA polynucleotides that can be used to produce iRNA (eg, dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecules that are complementary to all or part of an insect pest target gene, such as a prp8 gene. In a particular embodiment, dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs can be produced in vitro or in vivo by a genetically engineered organism, such as a plant or a bacterium. In a specific example, cDNA molecules that can be used to produce iRNA molecules that are complementary to a prp8 gene (eg, Sequence ID: 1, Sequence ID: 3, and Sequence ID: 5) are disclosed . Or part.

Further exposure of the member for suppressing gene expression in the one kind prp8 Diabrotica insects, pests and Diabrotica prp8 medium for providing a protective member to the plant. A member for inhibiting the expression of a prp8 gene in a leaf pest is a double-stranded RNA molecule, wherein a strand of the molecule is selected from the group consisting of sequence identification number: 86-90; and its complement It consists of multiple ribonucleotides. Functional equivalents of a construct for inhibiting the expression of a prp8 gene in a leaf pest include a double stranded RNA molecule comprising a polyribonucleotide substantially homologous to all or part of a leaf prp8 gene, the leaf prp8 The gene comprises a nucleotide sequence selected from the group consisting of sequence identification numbers: 7-11. A method for providing media prp8 Diabrotica insects protective member to a plant-based one DNA molecule, the DNA molecule comprising a operably linked to a promoter to a polynucleotide of, in one lobe of the encoding polynucleotide for inhibiting A component of the prp8 gene expressed in a pest, wherein the DNA molecule is capable of integrating into the genome of the plant.

Also disclosed for inhibiting gene expression of one kind of member prp8 the Nitidulidae pests, and the pests Meligethes prp8 medium for providing a protective member to the plant. A method for inhibiting the pests Meligethes prp8 one kind of gene expression as a means double-stranded RNA molecule, wherein the molecule is an a-based sequence identification number: 92; and many of the complement consisting of ribonucleotides. Means for inhibiting the functional equivalents of the one kind of the pests Meligethes prp8 gene expression include double-stranded RNA molecule comprising one kind substantially homologous genes Nitidulidae prp8 all or part of a ribonucleotide much, the exposed The tail prp8 gene contains the sequence identification number: 12. An exposed plant for providing a prp8 vector, a pest-protecting member of a plant, a DNA molecule comprising a polynucleotide operably linked to a promoter, the polynucleotide encoding for inhibition in a A component of the prp8 gene expressed in a pest, wherein the DNA molecule is capable of integrating into the genome of the plant.

Further disclosed is a method for controlling a population of insect pests (eg, coleopteran pests) comprising providing an iRNA (eg, dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecules to an insect pest (eg, a coleopteran pest), The iRNA molecule acts upon being taken up by the pest to inhibit the biological function within the pest.

In some embodiments, a method for controlling a coleopteran pest population comprises providing an iRNA molecule to the coleopteran pest, the iRNA molecule comprising all or part of a polynucleotide selected from the group consisting of: a sequence Identification number: 84; sequence identification number: complement of 84; sequence identification number: 85; sequence identification number: complement of 85; sequence identification number: 86; sequence identification number: complement of 86; sequence identification number: 87; Sequence identification number: complement of 87; sequence identification number: 88; sequence identification number: complement of 88; sequence identification number: 89; sequence identification number: complement of 89; sequence identification number: 90; sequence identification number: 90 Complement; sequence identification number: 91; sequence identification number: complement of 91; sequence identification number: 92; sequence identification number: complement of 92; a natural code with a Diabrotica organism (eg WCR) polynucleotides hybridizing polynucleotide, the polynucleotide comprising the sequence naturally encoded identification number: 7-11 all or part of any one of: one green and Diabrotica The body naturally encoded polynucleotide hybridizing the complement of the polynucleotide, the polynucleotide comprising the sequence naturally encoded identification number: all or part of any one of 7-11; an organism with Nitidulidae (Meligethes) ( a polynucleotide encoding a natural coding polynucleotide hybridization, for example, of PB), the natural coding polynucleotide comprising all or part of the sequence identification number: 12; and a polynucleoside hybridizing to the native coding polynucleotide of the tail-tailed organism The complement of the acid, the native coding polynucleotide comprising all or part of the sequence number: 12.

In a specific embodiment, an iRNA molecule which acts upon inhibition by the insect pest to inhibit biological functions in the pest is transcribed from DNA, the DNA comprising a polynucleotide selected from the group consisting of All or part: sequence identification number: 1; sequence identification number: 1 complement; sequence identification number: 3; sequence identification number: 3 complement; sequence identification number: 5; sequence identification number: 5 complement; A naturally-coding polynucleotide of a Diabrotica organism (eg, WCR) comprising all or part of any one of Sequence Identification Numbers: 7-11; a naturally encoded polynucleus of a leaf-like organism A complement of a glycosidic acid comprising all or part of any one of Sequence Identification Numbers: 7-11; a native encoding polynucleotide of a Meligethes organism (eg, PB), naturally encoded polynucleotide comprising the sequence identification number: all or part of 12; and a natural organism encoding beetle dew complement of a polynucleotide, the polynucleotide comprising a sequence encoding a native resolution No: all or part of 12.

Also disclosed herein are methods for providing dsRNAs, siRNAs, shRNAs, miRNAs, and in genetically engineered plant cells that exhibit dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs in a diet-based assay. / or hpRNAs to an insect pest. In these and further examples, the dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs can be taken up by pests. Ingestion of the dsRNAs, siRNAs, shRNAs, miRNAs and/or hpRNAs of the invention may in turn lead to RNAi in the pest, which in turn may cause silent effects of genes necessary for pest viability and ultimately lead to death. In a particular example, the coleopteran pests controlled by the use of the nucleic acid molecules of the invention can be WCR, NCR, SCR and/or PB.

A number of embodiments described in detail below with reference made from the accompanying Figures 1-2, the features and other features will become more apparent.

Sequence Listing The nucleic acid sequences listed in the accompanying sequence listing are represented by standard letter abbreviations for nucleotide bases as defined in 37 CFR § 1.822. The listed nucleic acid and amino acid sequences define molecules having nucleotides and amino acid monomers configured in the manner described (i.e., polynucleotides and polypeptides, respectively). The listed nucleic acid and amino acid sequences also each define a class of polynucleotides or polypeptides comprising nucleotides and amino acid monomers configured in the manner described. In view of the redundancy of the genetic code, it will be appreciated that a nucleotide sequence comprising a coding sequence also describes a polynucleotide of this class which encodes the same polypeptide as the polynucleotide consisting of the reference sequence. It will further be appreciated that the monoamino acid sequence describes polynucleotide ORFs of the class encoding the polypeptide.

Each nucleic acid sequence shows only one share, but it will be understood that the complementary strands are included by reference to the presentation stock. Since the complement and the reverse complement of the primary nucleic acid sequence must be revealed by the primary sequence, the complementary sequence and the reverse complementary sequence of a nucleic acid sequence are included by reference to the nucleic acid sequence unless otherwise specified The statement (or its clarity from the context in which the sequence appears). Furthermore, it is known in the art that the nucleotide sequence of an RNA strand is determined by the sequence of the DNA transcribed into the RNA (but the uracil (U) nucleobase replaces thymine (T)), so an RNA sequence is It is included by reference to the DNA sequence encoding the RNA. In the accompanying sequence listing:

Sequence Identification Number: 1 shows a fragment contig containing exemplary WCR prp8 DNA, referred to as WCR prp8 or WCR prp8-1 in some places herein:

Sequence ID: 2 shows an amino acid sequence of a PRP8 polypeptide encoded by an exemplary WCR prp8 DNA, also referred to as WCR PRP8 or WCR PRP8-1 in some places herein:

Sequence ID: 3 shows a fragment contig containing additional exemplary WCR prp8 DNA, referred to as WCR prp8-2 in some places herein:

Sequence ID: 4 shows an additional amino acid sequence of the WCR PRP8 polypeptide encoded by the exemplary WCR prp8 DNA (i.e., prp8-2 ):

Sequence ID: 5 shows an exemplary PB prp8 DNA:

Sequence ID: 6 shows the amino acid sequence of a PB PRP8 polypeptide encoded by an exemplary PB prp8 DNA:

Sequence Identification Number: 7 shows an additional exemplary WCR prp8 DNA, referred to as WCR prp8-1 reg1 (region 1) in some places herein, which is used in producing a dsRNA some examples: caatttacaagatgtgtgggatgtgaatgaaggggagtgtaacgtgttactggaatctaagtttgaaaaactatatgaaaagatcgatttgactctacttaacagacttctccgattgatagtggaccacaacatagctgattacatgaccgctaagaataacgtcgttataaactacaaagatatgaatcacaccaacagttacggaattattcgaggattgcagtttgcctcgttcattactcagtattatggtctggttttggatctgctggtattgggtctgcagagagccagtgaaatggctgggccacctcaaatgcctaacgatttcttgacgttccaagatgttcaatccgaaacgtgccatcctattcggctttactgcagatatgtggacagaattcatatgtttttcagattttctgcagaagaagccaaagatttgatccaaagatacctaacagaacatccagatcctaataatg

SEQ ID. No: 8 shows an additional exemplary WCR prp8 DNA, referred to as WCR prp8-2 reg1 (region 1) in some places herein, which is used in producing a dsRNA some examples: cggcttaatccgcggcctccagttcagcagtttcatattccaatattatgctctggtcatagatcttctgattttagggctgacgcgagccaatgaacttgccggcagtataggtggcggcggaggcggaggtttcgctaatctcaaagatcgcgaaacggagataaaacatcccatccgcttgtattgccgatatatagatgaaatatggatctgcttcaaattcaccaaagaggagtctcgtagcttgattcaaaggtatttgacggagaatccaaccgctagtcagcagctctccactgaagaaggcatcgactaccccatcaaaaagtgttggcctaaagactgccgaatgagaaaaatgaaattcgacgttaatatcggacgagccgttttctgggagattcagaaacgtctaccgagaagtttagctgagctgagttggggcaaag

SEQ ID. No: 9 shows an additional exemplary WCR prp8 DNA, referred to as WCR prp8-3 reg1 (region 1) in some places herein, which is used in producing a dsRNA some examples: ctaagaataacgtcgttataaactacaaagatatgaatcacaccaacagttacggaattattcgaggattgcagtttgcctcgttcattactcagtattatggtctggttttggatctgctggtattgggtctgcagagagccagtgaaatggctgggccacctcaaatgcctaacgatttcttgacgttccaagatgttcaatccgaaacgtgccatcctattcggctttactgcagatatgtggacagaattcatatgtttttcagattttctgcagaagaagccaaagatttgatccaaagatacctaacagaacatccagatcctaataatg

Sequence ID: 10 shows an additional exemplary WCR prp8 DNA, referred to herein as WCR prp8-3 v1 (version 1), which is used in some instances to produce a dsRNA: Ctaagaataacgtcgttataaactacaaagatatgaatcacaccaacagttacggaattattcgaggattgcagtttgcctcgttcattactcagtattatgcctcgttcattactcagtattatggtctggttttggatctgc

Sequence Identification Number: 11 shows an additional exemplary WCR prp8 DNA, referred to herein as WCR prp8-3 v2 (version 2), which is used in some instances to produce a dsRNA: Tggctgggccacctcaaatgcctaacgatttcttgacgttccaagatgttcaatccgaaacgtcccccctattcggctttactgcagtagtgtggacagaattcatatgtttttcagattttctgcagaagaagccaaagatttgatccaaagatacctaacagaacatccagatcctaataatg

SEQ ID. No: 12 shows an additional exemplary PB prp8 DNA, referred to as PB prp8 reg1 (region 1) in some places herein, which is used in producing a dsRNA some examples: AAATGGAAGACCGCCGAAGAAGTAGCCGCTCTAATTCGTTCGCTTCCCGTAGAAGAACAACCCAAACAAATCATCGTTACCAGAAAAGGAATGTTGGATCCTCTTGAAGTGCATCTTCTTGATTTCCCCAACATCGTTATCAAAGGATCTGAACTACAACTGCCTTTCCAGGCTTGCCTCAAGATAGAAAAGTTCGGCGATTTGATTTTGAAAGCTACCGAACCTCAGATGGTTCTGTTCAATCTTTACGACGATTGGTTGAAGACCATTTCGTCTTACACCGCCTTCTCCAGGTTGATTTTGATATTAAGGGCATTACACGTCAATACAGAAAGAACTAAGGTTATTCTTAAACCCGACAAAACCACAATTACCGAGCCGCATC

Sequence ID: 13 shows the nucleotide sequence of a T7 phage promoter.

Sequence Identification Number: 14 shows a fragment of an exemplary YFP coding region.

Sequence Identification Number: 15-26 show primers used to amplify the portion of an exemplary Prp8 sequence, which sequence comprises Prp8 WCR prp8-1 reg1, WCR prp8-2 reg1, WCR prp8-3 reg1, WCR prp8 -3 v1, WCR Prp8-3 v2 and PB prp8 reg1, used in some instances to produce dsRNA.

Sequence ID: 27 shows an exemplary YFP gene.

Sequence ID: 28 shows the DNA sequence of a annexin region 1.

Sequence ID: 29 shows the DNA sequence of an annexin region 2.

Sequence Identification Number: 30 shows the DNA sequence of a region 1 of the erythrocyte membrane protein ( spectrin ) 2 .

Sequence Identification Number: 31 shows the DNA sequence of a region 2 of the β-erythrocyte membrane protein 2 .

Sequence Identification Number: 32 shows a DNA sequence of mtRP-L4 region 1.

Sequence Identification Number: 33 shows a DNA sequence of mtRP-L4 region 2.

Sequence Identification Number: 34-61 shows primers that are used to amplify the gene regions of annexin, β-erythrocytic membrane protein 2, mtRP-L4, and YFP for dsRNA synthesis.

Sequence ID: 62 shows a maize DNA sequence encoding a TIP41-like protein.

Sequence ID: 63 shows the nucleotide sequence of a T20VN primer oligonucleotide.

Sequence Identification Number: 64-68 shows the expression analysis of dsRNA transcripts used in maize, such as primers and probes.

Sequence Identification Number: 69 shows the nucleotide sequence of a portion of a SpecR coding region that is used for binary vector backbone detection.

Sequence ID: 70 shows the nucleotide sequence of an AAD1 coding region for genomic copy number analysis.

Sequence Identification Number: 71 shows the DNA sequence of a maize magnifying enzyme gene.

Sequence Identification Number: 72-80 shows the nucleotide sequence of the DNA oligonucleotide, which is used for gene copy number determination and binary vector stem detection.

Sequence Identification Number: 81-83 shows primers and probes, which are used for the analysis of the dsRNA transcript maize expression.

Sequence ID: 84-92 shows exemplary RNA transcribed from nucleic acids comprising exemplary prp8 polynucleotides and fragments thereof. Detailed description I. Overview of several specific examples

We have developed RNA interference (RNAi) as a tool for insect pest management, using one of the most likely target pest species for gene transfer plants expressing dsRNA; western corn rootworm. Until now, most of the genes proposed as RNAi targets in rootworm larvae have not actually achieved their purpose. Here, we describe the reduction of prp8 by RNAi vectors in exemplary insect pests: Western corn rootworm and pollen beetles, for example, when iRNA molecules are delivered by ingesting or injecting prp8 dsRNA, showing a fatal phenotype . In this particular example, the ability to deliver prp8 dsRNA to insects by feeding confers a very useful RNAi effect on insect (eg, coleopteran) pest management. By combining the prp8 -mediated RNAi with other useful RNAi targets, for example, the potential of multiple target sequences in larval rootworms and pollen beetles can increase the development of insect pest management sustainability methods involving RNAi technology. opportunity.

Disclosed herein are methods and compositions for genetically controlling insect (eg, coleoptera) pest infestation. Methods for identifying one or more genes necessary for the life cycle of an insect pest are also provided to use a target gene for insect pest population control as an RNAi vector. A DNA plastid vector encoding an RNA molecule can be designed to clamp one or more targeted genes necessary for growth, survival, and/or development. In some embodiments, the RNA molecule may be capable of forming a dsRNA molecule. In some embodiments, a method of suppressing or inhibiting the post-transcriptional expression of a targeted gene is provided via a nucleic acid molecule that is complementary to a coding or non-coding sequence of a target gene in an insect pest. In these and further embodiments, a pest may ingest one or more dsRNA, siRNA, shRNA, miRNA, and/or hpRNA molecules from all of the nucleic acid molecules complementary to a coding or non-coding sequence of a target gene. Transcription in part or in part to provide plant protection.

Thus, some specific examples relate to sequence-specific inhibition of the expression of a targeted gene product using dsRNA, siRNA, shRNA, miRNA and/or hpRNA complementary to the coding and/or non-coding sequence of the target gene to effect insects ( For example, coleoptera) is at least partially controlled by pests. Disclosed are a group of isolated and purified nucleic acid molecules comprising a polynucleotide, for example, as listed in the Sequence Identification Numbers: 1, 3, 5, and the like. In some embodiments, a stable dsRNA molecule can be expressed from such polynucleotides, fragments thereof, or genes comprising one or more of these polynucleotides for post-transcriptional targeting of a target gene Silence or suppression. In certain embodiments, the isolated and purified nucleic acid molecule comprises all or part of any of: sequence identification numbers: 1, 3, and 5 (eg, sequence ID: 7-12), and/or Complementary.

Some specific examples relate to a recombinant host cell (e.g., a plant cell) having at least one recombinant DNA in its genome that encodes at least one iRNA (e.g., dsRNA) molecule. In a particular embodiment, when ingested by an insect (e.g., coleoptera) pest, an encoded dsRNA molecule can be provided to silence or inhibit the expression of a target gene in the pest after transcription. The recombinant DNA may comprise, for example, any one of sequence identification numbers: 1, 3, 5, and 7-12; a sequence identification number: a fragment of any of 1, 3, 5, and 7-12; A polynucleotide consisting of a partial sequence of a gene comprising one or more of sequence identification numbers: 1, 3, 5, and 7-12; and/or complements thereof.

Some specific examples relate to a recombinant host cell having at least one recombinant DNA in its genome encoding at least one iRNA (eg, dsRNA) molecule, which comprises all or part of: sequence identification number: 84, sequence identification Number: 85 or sequence identification number: 91 (eg, selected from at least one polyribonucleotide comprising the group of sequence identification numbers: 86-90 and 92). When ingested by an insect (e.g., coleoptera) pest, the iRNA molecule can silence or inhibit a target prp8 DNA (for example, a DNA comprising all or part of a polynucleotide, the polynucleotide selection From the group consisting of: sequence identification number: 1, 3, 5, and 7-12) performance in the pest, and thereby causing the growth, development, and/or cessation of feeding of the pest.

In some embodiments, a recombinant host cell can be a transformed plant cell having at least one recombinant DNA encoding at least one RNA molecule in its genome that is capable of forming a dsRNA molecule. Some specific examples relate to genetically transformed plants comprising such transformed plant cells. In addition to such genetically transgenic plants, the products of progeny plants, gene transfer seeds, and gene transfer plants of any gene transfer plant generation are provided, each of which contains recombinant DNA. In a specific embodiment, an RNA molecule capable of forming a dsRNA molecule can be expressed in a gene transfer plant cell. Therefore, in these and other specific examples, a dsRNA molecule can be isolated from a gene transfer plant cell. In a specific embodiment, the genetically modified plant is selected from the group consisting of corn ( Zea mays ), soybean ( Glycine max ), cotton, Poaceae plants, and rapeseed (芸A plant of the group of Brassica sp.).

Some specific examples relate to a method for modulating the expression of a targeted gene in an insect (e.g., coleopteran) pest cell. In these and other embodiments, a nucleic acid molecule can be provided, wherein the nucleic acid molecule comprises a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule. In a particular embodiment, 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 a specific embodiment, a method for regulating the expression of a targeted gene in an insect pest cell can comprise: (a) transforming a plant cell with a vector comprising a multinuclear encoding an RNA molecule capable of forming a dsRNA molecule (b) cultivating 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 that has integrated the vector into its genome And (d) determining that the selected transformed plant cell comprises an RNA molecule encoding a dsRNA molecule encoded by a polynucleotide of the vector. A plant can be regenerated from a plant cell having an integrated vector in its genome and comprising the dsRNA molecule encoded by the polynucleotide of the vector.

Thus, a gene transfer plant comprising a vector integrated into its genome having a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule, wherein the gene transfer plant comprises a polynucleoside from the vector, is also disclosed The dsRNA molecule encoded by the acid. In a particular embodiment, the expression of an RNA molecule capable of forming a dsRNA molecule in a plant is sufficient to modulate contact with the transformed plant or plant cell (for example, by feeding the transformed plant, a portion of the plant ( The expression of a target gene in a cell of an insect (e.g., coleoptera) of a root, such as a plant or a plant cell, such that the growth and/or survival of the pest is inhibited. The genetically transformed plants disclosed herein may exhibit resistance to and/or enhanced tolerance to insect pest infestation. Specific gene transfer plants may exhibit resistance and/or enhanced protection against one or more coleopteran pests selected from the group consisting of: WCR; NCR; SCR; MCR; Brazilian corn rootworm ( D. balteata LeConte); D. u. tenella ; Meligethes aeneus Fabricius; and Cucumber eleven deciduous beetle ( D. u. undecimpunctata Mannerheim).

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

In some embodiments, a composition (eg, a topical composition) is provided that comprises an iRNA (eg, dsRNA) molecule for use in a plant, animal, and/or plant or animal environment to achieve an insect (eg, coleopteran) ) Elimination or reduction of pest infestation. In a particular embodiment, the composition may be a nutritional composition or a food source for feeding the insect pest. Some specific examples include the nutritional composition or food source available for making the pest. Ingestion of a composition comprising an iRNA molecule may cause the molecule to be taken up by one or more cells of the pest, which in turn may result in inhibition of the performance of at least one target gene in the pest cell. By providing one or more compositions comprising iRNA molecules in the host of the pest, it is possible to limit or eliminate the uptake or damage of plants or plant cells infested by insect pests in or near any host tissue or environment present in the pest. .

The compositions and methods disclosed herein can be used in combination with other methods and compositions for controlling pest damage in insects, such as coleoptera. For example, an iRNA molecule for protecting a plant from insect pests as described herein may be used in a method comprising the additional use of one or more chemicals effective against insect pests, A pest-effective biopesticide, crop rotation, recombinant gene technology exhibits characteristics different from those of RNAi-mediated methods and RNAi compositions (eg, recombinant production of proteins harmful to insect pests in plants (eg, Bt toxins and PIP-) 1 polypeptide (see US Patent Publication No. 2014/0007292 Al), and/or other recombinant expression of iRNA molecules. II. Abbreviations

dsRNA double-stranded ribonucleic acid

EST performance sequence label

GI growth inhibition

NCBI National Biotechnology Information Center

gDNA genomic DNA

iRNA inhibitory ribonucleic acid

ORF open reading frame

RNAi RNA interference

miRNA microRNA

shRNA small hairpin ribonucleic acid

siRNA small inhibitory ribonucleic acid

hpRNA hairpin ribonucleic acid

UTR non-translated area

WCR Western corn rootworm ( Diabrotica virgifera virgifera LeConte)

NCR Northern Corn Rootworm ( Diabrotica barberi Smith and Lawrence)

MCR Mexican corn rootworm ( Diabrotica virgifera zeae Krysan and Smith)

PB Pollen beetle ( Meligethes aeneus Fabricius)

PCR polymerase chain reaction

qPCR quantitative polymerase chain reaction

RISC RNA-induced silent complex

SCR Southern corn rootworm ( diabrotica undecimpunctata howardi Barber)

SEM mean standard error

YFP Yellow Fluorescent Protein III. Terminology

In the following descriptions and tables, many terms are used. In order to provide this specification and claims, including the scope given by these terms, a clear and consistent understanding, the following definitions are provided:

Coleoptera pests: As used herein, the term "Coleoptera" means Coleoptera (order Coleoptera) of insect pests, encompasses the genus Diabrotica (genus Diabrotica) insect pests, such crops and crop insects feeding products Including corn and other real grasses. In a specific example, a coleopteran pest is selected from the list consisting of: D. v. virgifera LeConte (WCR); D. barberi Smith and Lawrence (NCR); D. u. howardi (SCR); Mexican corn root beetle ( D. v. zeae ) (MCR); Brazilian corn rootworm ( D. balteata LeConte); cucumber ten D. u. tenella ; D. u. undecimpunctata Mannerheim; D. speciosa Germar; and Meligethes aeneus Fabricius ) (PB).

Contact (an organism): As used herein, the term "contacting" an organism (eg, a coleopteran pest) or "uptake" by an organism, when in the case of a nucleic acid molecule, includes internalizing the nucleic acid molecule ( Internalization) to the organism, for example but not limited to: ingesting the molecule by the organism (eg, by feeding); contacting the organism with a composition comprising the nucleic acid molecule; and soaking the organism A solution comprising the nucleic acid molecule.

Contig: As used herein, the term "fragment contig" means a DNA sequence that is reconstituted from a set of overlapping DNA segments derived from a single genetic source.

Corn plant: As used herein, the term "corn plant" means a species, a plant of Zea mays (maize).

Performance: As used herein, "express" of a coding polynucleotide (for example, a gene or a transgene) means a process in which a nucleic acid transcription unit (including, for example, gDNA or cDNA) is encoded. Information is converted into operational, non-operating or structural parts of the cell, usually including the synthesis of proteins. External signals can affect gene expression; for example, exposing cells, tissues, or organisms to one 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, through transcription, translation, RNA transport and processing, control of intermediate molecules such as mRNA degradation, or activation of specific protein molecules after they are manufactured, Activation, compartmentalization or degradation, or a combination thereof. Gene expression can be measured at the RNA level 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 Analysis of protein activity in vivo.

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, when used to describe an effect on a coding polynucleotide (for example, a gene), the term "inhibiting" means transcribed from the mRNA of the encoding polynucleotide and/or the A measurable decrease in cell level of a peptide, polypeptide or protein product encoding a polynucleotide. In some instances, the performance of a coding polynucleotide can be inhibited thereby thereby approximating the performance. "Specific inhibition" means inhibition of a targeted coding polynucleotide, and does not necessarily affect the performance of other coding polynucleotides (eg, genes) in the cell, wherein specific inhibition is achieved in the cell.

Insects: As used herein with respect to pests, the term "insect pests" specifically includes coleopteran insect pests.

Isolating: An "isolated" biological component (such as a nucleic acid or protein) has substantially been associated with other biological components in the naturally occurring region of the living organism (ie, other chromosomes and extrachromosomal DNA). And RNA and protein are separated, separately manufactured or purified to leave, while affecting the chemical or functional changes of the component (for example, a nucleic acid can be isolated from the chromosome by breaking the chemical bond linking the nucleic acid to the remaining DNA in the chromosome) go away). Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules, proteins and peptides.

Nucleic acid molecule: As used herein, the term "nucleic acid molecule" may refer to a polymeric form of a nucleotide, which may include both the sense strand of the RNA and the antisense strand, cDNA, gDNA, and the synthetic forms described above. Mix the polymer. A nucleotide or nucleobase may mean a ribonucleotide, a deoxyribonucleotide or a modified form of any type of nucleotide. A "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise indicated, a nucleic acid molecule is typically at least 10 bases in length. Conventionally, the nucleotide sequence of a nucleic acid molecule is read from the 5' end of the molecule to the 3' end. A "complement" of a nucleic acid molecule means a polynucleotide having a nucleobase that can form a base pair (ie, A-T/U, and G-C) with the nucleobase of the nucleic acid molecule.

Some specific examples include nucleic acids containing a template DNA that is transcribed into an RNA molecule that is a complement of an mRNA molecule. In these embodiments, the complement of the nucleic acid transcribed into an mRNA molecule is presented in a 5' to 3' orientation, whereby the RNA polymerase (which transcribes the DNA in the 5' to 3' direction) will The substance transcribes a nucleic acid that can hybridize to the mRNA molecule. Unless specifically stated otherwise or clear from this context, the term "complement" thus means a polynucleotide having a nucleobase from 5' to 3' which is compatible with a nucleobase of a reference nucleic acid. The base forms a base pair. Likewise, a "reverse complement" of a nucleic acid means a complement oriented in the opposite direction unless explicitly stated otherwise (or clear from the context). The foregoing is explained in the following diagram: ATGATGATG polynucleotide "complement" of the TACTACTAC polynucleotide "reverse complement" of the CATCATCAT polynucleotide

Some specific examples of the invention may include hairpin RNA to form an RNAi molecule. In these RNAi molecules, both the complement of the nucleic acid targeted by the RNA interference and the reverse complement may be found in the same molecule, whereby the single stranded RNA molecule can be "fold over" and hybridized to The region itself contains the complementary and reverse complementary polynucleotides.

"Nucleic acid molecule" includes all polynucleotides, for example, single-stranded and double-stranded forms of DNA; single-stranded forms of RNA; and double-stranded forms of RNA (dsRNA). The term "nucleotide sequence" or "nucleic acid sequence" means both a sense strand and an antisense strand of a nucleic acid in either a single strand or in a doublet. 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 or not loaded with the corresponding deuterated amino acid), and cRNA (complementary RNA). The term "deoxyribonucleic acid" (DNA) includes cDNA, gDNA, and DNA-RNA hybrids. The terms "polynucleotide" and "nucleic acid" and "fragment thereof" will be understood by the person of ordinary skill in the art to include a term that includes two gDNA; ribosomal RNA; transfer RNA; messenger RNA; operon; An engineered polynucleotide that encodes or may be suitable for encoding a peptide, polypeptide or protein.

Oligonucleotide: An oligonucleotide is a short nucleic acid polymer. Oligonucleotides can be formed by cleavage of longer nucleic acid segments or by polymerization of individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to hundreds of bases in length. Because oligonucleotides can bind to a complementary nucleic acid, they can be used as probes for detecting DNA or RNA. Oligonucleotides (oligodeoxyribonucleotides) composed of DNA can be used in PCR, and PCR is a technique for amplifying DNA. In terms of PCR, an oligonucleotide is typically referred to as an "introduction" that allows the DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

A nucleic acid molecule can include any or both of the modified nucleotides that are naturally occurring and linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. Nucleic acid molecules can be chemically or biochemically modified, or can contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, by way of example, labeling, methylation, substitution of one or more naturally occurring nucleotides with an analog, internucleotide modification (eg, an uncharged linkage: for example, phosphine) Acid methyl esters, phosphate triesters, phosphoramidates, urethanes, etc.; charged links: for example, phosphorothioates, phosphorodithioates, etc.; Pendent): for example, a peptide; an intercalator: for example, acridine, psoralen, etc.; a chelating agent; an alkylating agent; and a modified chain: In terms of α-alpha anomeric nucleic acid, etc.). The term "nucleic acid molecule" also includes any topological configuration, including single stranded, double stranded, partially duplexed, triplet, hairpin, round, and padlocked configurations.

As used herein with respect to DNA, the terms "encoding polynucleotide", "structural polynucleotide" or "structural nucleic acid molecule" mean a polynucleotide that is passed under the control of appropriate regulatory elements, via a polynucleotide Transcription is ultimately translated into a polypeptide, with mRNA. In the context of RNA, the term "coding polynucleotide" means a polynucleotide that is translated into a peptide, polypeptide or protein. The borderline of a coding polynucleotide is determined by one of the 5'-end translation start codon and one of the 3'-end translation stop codon. Encoding polynucleotides include, but are not limited to, gDNA; cDNA; EST; and recombinant polynucleotides.

As used herein, "transcribed non-coding polynucleotide" means a segment of an mRNA molecule, such as a 5' UTR, 3' UTR, and an intron segment that has not been translated into a peptide, polypeptide or protein. Further, "transcribed non-coding polynucleotide" means a nucleic acid which is transcribed into an intracellular RNA, for example, structural RNAs (such as ribosomal RNA (rRNA), for example, 5S rRNA, 5.8S rRNA, 16S rRNA, 18S rRNA, 23S rRNA and 28S rRNA and analogs; transfer RNA (tRNA); and snRNAs such as U4, U5, U6 and the like. Non-coding polynucleotides for transcription also include, by way of example and not limitation, small RNAs (sRNA), which are commonly used to describe small bacterial non-coding RNAs; small nucleolar RNAs (snoRNA); microRNAs (miRNAs); Small interfering RNAs (siRNA); Piwi-interacting RNAs (piRNA); and long non-coding RNAs. Still further, "transcribed non-coding polynucleotide" means a polynucleotide which may be natively present in a nucleic acid as a "gap" within the gene, and which is transcribed into an RNA molecule.

Fatal RNA interference: As used herein, the term "lethal RNA interference" means that RNA interferes with the death or activity of a subject who causes delivery, for example, dsRNA, miRNA, siRNA, shRNA, and/or hpRNA. .

Genome: As used herein, the term "genome" means chromosomal DNA found within the nucleus of a cell and also means organelle DNA found within the secondary cell component of the cell. In some embodiments of the invention, a DNA molecule may be introduced into a plant cell whereby the DNA molecule is integrated into the genome of the plant cell. In these and further embodiments, the DNA molecule may be integrated into the nuclear DNA of the plant cell or integrated into the chloroplast or mitochondrial DNA of the plant cell. The term "genome", when applied to a bacterium, means both a chromosome and a plastid within the bacterial cell. In some embodiments of the invention, a DNA molecule may be introduced into a bacterium whereby the DNA molecule is integrated into the genome of the bacterium. In these and further embodiments, the DNA molecule may be integrated into a chromosome or located as a stable plastid or in a stable plastid.

Sequence identity: The term "sequence identity" or "identity" of two polynucleotides or polypeptides, as used in this context, means that when aligned across a particular comparison window for maximum correspondence The same residues in the sequences of the two molecules when aligned.

As used herein, the term "percent sequence identity" may mean a value determined by comparing two optimal alignment sequences (eg, a nucleic acid sequence or a polypeptide sequence) of a molecule across a comparison window, wherein The optimal alignment of the partial sequences in the comparison window for the two sequences may include additions or deletions (ie, gaps) when compared to the reference sequence (the reference sequence does not contain additions or deletions). The percentage is calculated by determining the number of positions in which the same nucleotide or amino acid residue occurs in both sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and This result is multiplied by 100 to produce a percentage of sequence identity. A sequence that is identical to a reference sequence at each position is said to be 100% identical to the reference sequence and vice versa.

Methods for comparing alignment sequences are well known in the art. Various programs and alignment algorithms are described, for example: 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. USA 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) FEMS Microbiol. 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 Biotechnology Information Center (NCBI) Basic Local Alignment Search Tool (BLAST ^TM; Altschul et al. (1990)) can be obtained from several sources, including the National Biotechnology Information Center (Bethesda, MD), and on the Internet at, Used in conjunction with several sequence analysis programs. Instructions on how to determine sequence identity using this program are available on the Internet at the BLAST ^TM "help" section. For the comparison of nucleic acid sequences may be utilized "Blast 2 sequences" function BLAST ^TM (Blastn) program, which is to use the default BLOSUM62 mode is set to default parameters. Nucleic acids with greater sequence similarity to a reference polynucleotide sequence will exhibit an increased percent identity when evaluated by this method.

Specific hybridization/specific complementation: As used herein, the terms "specific hybridization" and "specific complementation" are terms that indicate a sufficient degree of complementarity whereby a nucleic acid molecule and a target nucleic acid molecule are utilized. Stable and specific binding occurs between them. Hybridization between two nucleic acid molecules involves the formation of an anti-parallel alignment between the nucleobases of the two nucleic acid molecules. The two molecules can then form a hydrogen bond with the corresponding base on the opposite strand to form a doublet molecule which, if sufficiently stable, can be detected using methods well known in the art. A polynucleotide does not require 100% of a target nucleic acid that is complementary to its specific hybridization. However, there must be a function such that the number of hybridizations to specific complementarity is a function of the hybridization conditions used.

Hybridization conditions that result in a certain degree of stringency will vary depending on the nature of the hybridization method chosen and the composition and length of the hybridizing nucleic acid. In general, although the time of washing also affects stringency, the hybridization temperature and the ionic strength of the hybridization buffer (especially Na ⁺ and / or Mg ⁺⁺ concentrations) will determine the stringency of hybridization. The calculation of hybridization conditions required to achieve a certain degree of stringency is known to those of ordinary skill in the art and is discussed, for example, by Sambrook et al. (ed.) Molecular Cloning: A Laboratory Manual , 2 ^nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Chapters 9 and 11; and Hames and Higgins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985. Further detailed teaching and guidance on nucleic acid hybridization may be found below, for example, Tijssen, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," in Laboratory Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, NY, 1993; and Ausubel et al, Eds., Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-Interscience, NY, 1995.

As used herein, "stringent conditions", including hybridization, will only occur if the mismatch between the hybrid molecule sequence and a homologous polynucleotide within the target nucleic acid molecule is less than 20%. "Stringent conditions" include the stringency of further specific levels. Thus, as used herein, "medium stringency" conditions are those in which more than 20% of the molecules of the molecule do not hybridize; "high stringency" conditions are those in which more than 10% of the mismatched sequences do not hybridize. Those conditions; and "very high stringency" conditions are those conditions in which more than 5% of the mismatched sequences do not hybridize.

The following are representative, non-limiting hybridization conditions. High stringency conditions (detection of polynucleotides sharing at least 90% sequence identity): Hybridization in 5X SSC buffer at 65 °C for 16 hours; washing twice in 2X SSC buffer at room temperature , 15 minutes each time; and wash twice in 0.5 × SSC buffer at 65 ° C for 20 minutes each time. Medium stringency conditions (detection of polynucleotides sharing at least 80% sequence identity): 5x-6x SSC buffer, hybridization at 65-70 °C for 16-20 hours; in 2 x SSC buffer at room temperature Wash twice, 5-20 minutes each time; and wash twice with 30 x 70 °C in 1 x SSC buffer for 30 minutes each time. Non-stringent control conditions (polynucleotides sharing at least 50% sequence identity will hybridize): hybridization in 6x SSC buffer at room temperature to 55 °C for 16-20 hours; in 2x-3x SSC buffer at room temperature to 55 °C is washed at least twice for 20-30 minutes each time.

As used herein, the term "substantially homologous," "substantially identical," or "substantially homologous" when referring to a nucleic acid, refers to a contiguous nucleobase possessed by a polynucleotide, which Hybridization to a reference nucleic acid under stringent conditions. For example, a nucleic acid that is substantially homologous to a reference nucleic acid of any one of Sequence Identification Numbers: 1, 3, 5, and 7-12 is a hybridization under stringent conditions (eg, stringency conditions in the foregoing statements) To those nucleic acids of the reference nucleic acid. A substantially homologous polynucleotide may have at least 80% sequence identity. For example, a substantially homologous polynucleotide may have from about 80% to 100% sequence identity, such as 79%; 80%; about 81%; about 82%; about 83%; about 84%; 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 nature of substantial homology is closely related to specific hybridization. For example, when there is a sufficient degree of complementarity, a nucleic acid molecule specifically hybridizes to avoid nucleic acid and non-targeted polynucleotides under conditions of desirable hybridization, for example under stringent hybridization conditions. Non-specific binding.

As used herein, the term "ortholog" means that in two or more species, a gene has evolved from a common ancestral nucleic acid and may be retained in the two or more species. The same function.

As used herein, each nucleotide of a polynucleotide read in the 5' to 3' direction is complementary to each nucleoside read from another polynucleotide in the 3' to 5' direction. In the case of an acid, two nucleic acid molecules are considered to exhibit "complete complementarity". A polynucleotide complementary to a reference polynucleotide will display a sequence that is identical to the reverse complement of the reference polynucleotide. These terms and descriptions are well defined in the art and will be readily understood by those of ordinary skill in the art.

Manipulably linked: when the first polynucleotide is in a functional relationship with the second polynucleotide, the first polynucleotide is operably linked to the second polynucleotide. When recombinantly produced, the operably linked polynucleotides are generally contiguous and, where necessary, link the two protein coding regions in the same reading frame (e.g., in a translational fusion ORF). However, nucleic acids do not have to be manipulated in a continuous manner.

The term "operably linked" when used with reference to a regulatory genetic element and a coding polynucleotide means that the regulatory element affects the performance of the linked coding polynucleotide. "Regulatory element" or "control element" means a polynucleotide that affects the timing and level/quantity of transcription of the associated coding polynucleotide, RNA processing or stability or translation. Regulatory elements can include promoters; translational leader; intron; enhancer; stem loop structure; suppressor binding polynucleotide; polynucleotide with termination sequence; polynucleotide with polyadenylation recognition sequence, etc. . A particular regulatory element may be located upstream and/or downstream of the encoding polynucleotide operably linked thereto. Also, a particular regulatory element operably linked to a coding polynucleotide may be located on an associated complementary strand of a double stranded nucleic acid molecule.

Promoter: As used herein, the term "promoter" means a region of DNA that may be upstream of the initiation of transcription and may involve recognition and binding of RNA polymerase with other proteins to initiate transcription. A promoter may be operably linked to a coding polynucleotide for expression in a cell, or a promoter may be operably linked to a polynucleotide encoding a signal peptide, wherein the signal peptide may be operably Link to a coding polynucleotide for expression in a cell. A "plant promoter" can be a promoter capable of eliciting transcription in a plant cell. Examples of promoters under developmental control include promoters which preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or thick-walled tissues. This type of promoter is called "organizational priority." Promoters that initiate transcription only in certain tissues are referred to as "tissue specificity." A "cell type specific" promoter drives expression primarily in certain cell types in one or more organs, for example, vascular bundle cells in roots or leaves. An "inducible" promoter can be a promoter 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, tissue-preferred, cell-type-specific, and inducible promoters constitute a "non-sustained" type of promoter. A "sustained phenotype" promoter is a promoter that is active under most environmental conditions, or in most tissues or cell types.

Any inducible promoter can be used in some embodiments of the invention. See Ward et al. (1993) Plant Mol. Biol. 22:361-366. With an inducible promoter, the response rate of transcription to an inducer is increased. Exemplary inducible promoters include, but are not limited to, a promoter derived from the ACEI system that responds to copper; an In2 gene derived from maize in response to the sulfonamide herbicide safener; derived from Tn10 a Tet repressor; and an inducible promoter derived from a steroid hormone gene whose transcriptional activity can be induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad Sci. USA 88:0421).

Exemplary sustained phenotype promoters include, but are not limited to, promoters from plant viruses, such as the 35S promoter from Cauliflower Mosaic Virus (CaMV); promoters from the rice actin gene; Ubiquitin promoter; pEMU; MAS; maize H3 tissue protein promoter; and ALS promoter, Xba1/NcoI fragment 5' to Brassica napus ALS3 structural gene (or similar to Xba1 /NcoI fragment) A polynucleotide) (International PCT Publication No. WO 96/30530).

Furthermore, any tissue-specific or tissue-preferred promoter can be utilized in some embodiments of the invention. A plant comprising a nucleic acid molecule operably linked to one of a tissue-specific promoter encoding a polynucleotide can be produced exclusively or preferentially in a particular tissue. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a sub-preferred promoter, such as the one derived from the phaseolin gene; a leaf-specific and light-inducible promoter, such as the's cab or from ribulose bisphosphate carboxylase (Rubisco) of; an anther-specific promoter, such as that from LAT52 of persons; a pollen-specific promoter, such as that from Zm13 of persons; and A spore-preferred promoter, such as the one derived from apg .

Transmorphism: As used herein, the term "transformation" or "transduction" means the transfer of one or more nucleic acid molecules into a cell. By transducing a nucleic acid molecule to the cell, either by incorporating the nucleic acid molecule into the genome of the cell, or by replicating the free genome, the nucleic acid molecule is stabilized and replicated by the cell, then a cell It is "transformed". 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; transformation with plastid vectors; electroporation (Fromm et al. (1986) Nature 319:791-3); lipofection (lipgfection) (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); Microinjection (Mueller et al. (1978) Cell 15: 579-85); Transfer of Agrobacterium media (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

Transgene: An exogenous nucleic acid sequence. In some examples, a transgene can be a DNA encoding one or two strands of RNA capable of forming a dsRNA molecule comprising a polyribonucleotide complementary to a nucleic acid molecule found in a coleopteran pest. In a further example, a transgene may be a gene (eg, 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, a transgene may contain a regulatory element that operably links to the transgene encoding polynucleotide (eg, a promoter).

Vector: A nucleic acid molecule which, when introduced into a cell, produces, for example, a transformed cell. A vector may include genetic elements that permit its replication in the host cell, such as an origin of replication. Examples of vectors include, but are not limited to, a plastid; a plastid; a bacteriophage; or a virus that carries foreign DNA into a cell. A vector may also include one or more genes, including those that produce antisense molecules, and/or selectable marker genes, as well as other genetic elements known in the art. A vector may transduce, transform or infect a cell such that the cell exhibits a nucleic acid molecule and/or protein encoded by the vector. A vector optionally includes a substance (eg, a liposome, a protein coating, etc.) that assists in the entry of the nucleic acid molecule into the cell.

Yield: Stable yield of about 100% or greater relative to the check variety grown at the same growth location, at the same time and under the same conditions. In a particular embodiment, "improving yield" or "improving yield" means a cultivar having a stable yield of 105% or greater, relative to growing at the same time and under the same conditions, containing significant density at the same growth location. The yield of the test species of the coleopteran pest that harms the crop, which is targeted by the compositions and methods herein.

The terms "a", "an" and "the" are used to mean "at least one", unless otherwise specifically indicated or implied.

Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Definitions of commonly used terms in molecular biology can be found, for example, in Lewin's Genes X, Jones & Bartlett Publishers, 2009 (ISBN 10 0763766321); Krebs et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd. , 1994 (ISBN 0-632-02182-9); and Meyers RA (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). All percentages are by weight and all solvent mixtures are by volume unless otherwise indicated. All temperatures are in degrees Celsius. IV. Nucleic Acid Molecules Containing Insect Pest Sequences A. Overview

Described herein are nucleic acid molecules useful for controlling insect pests. In some examples, the insect pest is a coleopteran insect pest. Nucleic acid molecules described include targeted polynucleotides (eg, native and non-coding polynucleotides), dsRNAs, siRNAs, shRNAs, hpRNAs, and miRNAs. For example, dsRNAs, siRNA, miRNA, shRNA, and/or hpRNA molecules are described in some specific examples that may specifically complement all or part of one or more natural nucleic acids in a coleopteran pest. In these and further embodiments, the natural nucleic acid may be one or more targeted genes, the products of which may, for example but not limited to, involve metabolic processes or involve larval development. A nucleic acid molecule as described herein, when introduced into a cell comprising at least one natural nucleic acid that is specifically complementary to the nucleic acid molecule, may elicit RNAi in the cell and thereby reduce or eliminate the performance of the natural nucleic acid. In some instances, a targeting gene causes the growth, development, and/or withdrawal of the coleopteran pest to be stopped by a nucleic acid molecule that is specifically complementary thereto.

In some embodiments, at least one targeting gene of an insect pest can be selected, wherein the targeting gene comprises a coleopteran prp8 polynucleotide. In a specific example, select a prp8 comprising a coleopteran gene targeting polynucleotide, wherein the targeted gene sequence identification number selected from the group comprising: 1, 3, and 7-11 among Diabrotica polynucleotide. In a specific example, a targeting gene comprising a coleopteran prp8 polynucleotide, wherein the targeting gene comprises an off- tail alpha polynucleotide selected from the sequence identification numbers 5 and 12, is selected.

In some embodiments, a targeting gene can be a nucleic acid molecule comprising a polynucleotide that can be reverse translated into a polypeptide by in silico , the polypeptide comprising at least a contiguous amino acid sequence About 85% of a prp8 multinucleus identical (eg, at least 84%, 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or 100% identical) The amino acid sequence of the protein product of the nucleotide. One targeted gene may be any of the prp8 polynucleotides in an insect pest, the post -transcriptional inhibition of which has a detrimental effect on the growth and/or survival of the pest, for example, providing plant protection benefits against pests. In a specific example, a targeting gene is a nucleic acid molecule comprising a polynucleotide which can be inverted in silico into a polypeptide comprising a contiguous amino acid sequence, the amino group The acid sequence 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 the amino acid sequence selected from the group consisting of: sequence identification numbers: 2, 4 and 6.

Providing a DNA according to the invention, the performance of which will result in an RNA molecule comprising a polynucleotide which is specifically complementary to a polynucleotide encoding an insect (eg, Coleoptera) pest All or part of the encoded natural RNA molecule. In some embodiments, down-regulation of the encoding polynucleotide can be obtained in the pest cell after the insect pest ingests the expressed RNA molecule. In a particular embodiment, down-regulation of the encoding polynucleotide in the insect pest cell results in an adverse effect on the growth and/or development of the pest.

In some embodiments, the targeting polynucleotide comprises a transcribed non-coding RNA sequence, such as a 5' UTRs; a 3' UTRs; a splicing leader; an intron; a terminal intron (eg, subsequent trans-splicing) a modified 5'UTR RNA); a donor (donatron) (eg, a non-coding RNA required to provide a donor sequence for trans-splicing); and other non-coding transcribed RNAs that target the insect pest gene. Such polynucleotides may be derived from both mono-cistronic and poly-cistronic genes.

Thus, iRNA molecules (eg, dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs) are also described herein in connection with specific examples, which comprise specifically complementary to all of a target nucleic acid in an insect (eg, coleopteran) pest or Part of at least one polynucleotide. In some embodiments, an iRNA molecule may comprise a polynucleotide that is complementary to all or part of a plurality of targeted nucleic acids; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 More or more targeted nucleic acids. In a particular embodiment, the iRNA molecule may be made in vitro or produced in vivo by a genetically modified organism, such as a plant or a bacterium. Also disclosed is cDNA, which may be used in the manufacture of dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules that are specifically complementary to all or part of a target nucleic acid in an insect pest. Further described are recombinant DNA constructs for achieving stable transformation of a particular host target. The transduced host target may represent an effective level of dsRNA, siRNA, miRNA, shRNA and/or hpRNA molecules from the recombinant DNA construct. Thus, a plant-transformed vector comprising at least one polynucleotide operably linked to a functional heterologous promoter in a plant cell is also described, wherein the expression of the polynucleotide results in an RNA molecule, the RNA The molecule comprises a sequence of contiguous nucleobases that are specifically complementary to all or part of the target nucleic acid in an insect pest.

In a specific example, the control of insects (e.g., Coleoptera) nucleic acid molecule may be useful pests include: from beetle (Diabrotica) as a single whole or in part from the native nucleic acid comprising a polynucleotide prp8 (e.g., SEQ ID. No.: 1, 3, and 7-11); DNAs, when expressed, result in an RNA molecule comprising a polynucleotide that specifically complements the native RNA molecule encoded by the leaf prp8 All or part; iRNA molecules (eg, dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs) comprising at least one polynucleotide that is specifically complementary to all or part of the genus prp8 ; cDNA, which can be used in production dsRNA molecules, siRNA molecules, miRNA molecules, shRNA designed molecules and / or hpRNA molecule, such molecule specifically complementary to all or part of Diabrotica prp8; Nitidulidae from all or part of the native single isolated nucleic comprising Prp8 polynucleotides (eg, sequence number: 5 and 12); DNAs, when expressed, result in an RNA molecule comprising a polynucleotide that specifically complements the native RNA encoded by the late- tailed prp8 All of the molecules Or a portion; iRNA molecules (eg, dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs) comprising at least one polynucleotide that is specifically complementary to all or part of the genus prp8 ; cDNA, which can be used in production a dsRNA molecule, an siRNA molecule, a miRNA molecule, a shRNA molecule, and/or an hpRNA molecule that specifically complements all or part of the prp8 ; and recombinant DNA used to achieve stable transformation of a particular host target A construct, wherein a transformed host target comprises one or more of the aforementioned nucleic acid molecules. B. Nucleic acid molecules

The present invention provides, in addition to other things, iRNA (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules that inhibit the performance of a targeted gene in cells, tissues, or organs of an insect (eg, coleopteran) pest. And a DNA molecule that can be expressed as an iRNA molecule in a cell or microorganism to inhibit the performance of the target gene in cells, tissues or organs of insect pests.

Some specific embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (eg, one, two, three or more) polynucleotides selected from the group consisting of: Sequence identification number: 1, 3 and 5; sequence identification number: complement of any of 1, 3 and 5; sequence identification number: fragment of at least 15 contiguous nucleotides of any of 1, 3 and 5 (eg, sequence identification number: 7-12); sequence identification number: complement of at least 15 contiguous nucleotide fragments of any of 1, 3, and 5; a type of Diabrotica organism A natural coding polynucleotide (eg, WCR) comprising any one of sequence identification numbers: 7-11; a complement of a native coding polynucleotide of a leaf beetle organism, the native coding polynucleotide comprising a sequence identification number : any one of 7-11; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a leaf beetle organism, the native coding polynucleotide comprising any one of sequence identification numbers: 7-11; Diabrotica organism naturally encodes a plurality of at least 15 contiguous nucleotides Nucleotide complement fragment, which comprises a polynucleotide sequence encoding a native identification number: any one of 7-11; Nitidulidae one kind of organism (e.g., PB) of naturally encoded polynucleotide comprising the sequence identification numbers: 12; Lu one kind of organism naturally encoded beetles complement of a polynucleotide, the polynucleotide comprising the sequence naturally encoded identification number: 12; tail exposed one kind of organism naturally A polynucleotide encoding at least 15 contiguous nucleotides of acid fragment, which comprises a polynucleotide sequence encoding a native identification number: 12; and encodes a naturally Nitidulidae organism of polynucleotide fragments of at least 15 consecutive nucleotides of the complement of the polynucleotide encoding the native Contains serial identification number: 12.

In a particular embodiment, an insect (eg, coleoptera) pest is contacted or ingested iRNA transcribed from the isolated polynucleotide inhibits growth, development, and/or feeding of the pest. In some embodiments, insect contact or ingestion occurs via feeding of plant material comprising the iRNA. In some embodiments, insect contact or ingestion occurs by spraying a plant containing the insect with a composition comprising the iRNA.

In some embodiments, the isolated nucleic acid molecules of the invention may comprise at least one (eg, one, two, three or more) polynucleotides selected from the group consisting of: sequence identification No.: 84; sequence identification number: complement of 84; sequence identification number: 85; sequence identification number: complement of 85; sequence identification number: 86; sequence identification number: complement of 86; sequence identification number: 87; sequence Identification number: complement of 87; sequence identification number: 88; sequence identification number: complement of 88; sequence identification number: 89; sequence identification number: complement of 89; sequence identification number: 90; sequence identification number: 90 Complement; sequence identification number: 91; sequence identification number: complement of 91; sequence identification number: 92; sequence identification number: complement of 92; sequence identification number: at least 15 of any of 84, 85 and 91 Fragment of contiguous nucleotide; sequence identification number: complement of at least 15 contiguous nucleotide fragments of any of 84, 85, and 91; a natural coding polynucleotide of a leaf beetle organism, the day The coding polynucleotide comprises a sequence identification number: 86-90; a complement of a native coding polynucleotide of a leaf beetle organism, the natural coding polynucleotide comprising a sequence identification number: 86-90 persons; naturally encodes a plurality of Diabrotica organism of at least 15 contiguous nucleotides, a nucleotide, a polynucleotide comprising the sequence naturally encoded identification number of any one of 86-90; and one kind of natural organism of Diabrotica A complement encoding a fragment of at least 15 contiguous nucleotides of a polynucleotide comprising a sequence identification number: 86-90; a native coding polynucleotide of an exposed tail organism, the naturally encoded polynucleotide comprising the sequence identification number: 92; A polynucleotide complement of the exposed organism naturally encoded beetles of the polynucleotide comprising the sequence naturally encoded identification number: 92; a natural organism beetles dew polynucleotides encoding at least 15 contiguous nucleotides of a fragment of the native encoding polynucleotide comprising the sequence identification number: 92; and encodes a naturally Nitidulidae polynucleotide of the organism At least 15 contiguous nucleotides of the complement fragment, the native encoding polynucleotide comprising the sequence identification number: 92.

In a particular embodiment, contacting or ingesting the isolated polynucleotide with a coleopteran pest inhibits growth, development, and/or feeding of the pest. In some embodiments, insect contact or ingestion occurs via feeding of plant material or bait containing the iRNA. In some embodiments, the contact or ingestion of a coleopteran pest occurs by spraying a plant containing the insect with a composition comprising the iRNA.

In certain embodiments, the dsRNA molecules provided herein comprise a polynucleotide complementary to a transcript from a target gene comprising sequence identification numbers: 1, 3, 5, and 7. Any of -12, and fragments thereof, the inhibition of the target gene in an insect pest results in the reduction or removal of a polypeptide or polynucleotide agent necessary for growth, development or other biological function of the pest. A selected polynucleotide may exhibit from about 80% to about 100% sequence identity for any of the following: sequence identification number: 1, 3, 5, and 7-12; sequence identification number: a continuous segment of any of 1, 3, 5, and 7-12; and the complement of any of the foregoing. For example, a selected polynucleotide can exhibit 79%; 80%; about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; 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: sequence identification number: 1, 3, 5, and 7-12; sequence identification number: 1, 3, 5, and 7-12 a continuous segment; and the complement of any of the foregoing. In some embodiments, a dsRNA molecule is transcribed from a polynucleotide comprising a sense nucleotide sequence that is substantially identical or identical to the sequence identification number: 1, 3, 5 And a contiguous fragment of any of 7-12; an antisense nucleotide sequence which is at least substantially the reverse complement of the nucleotide sequence of the sense; and a fall between the sense and the antisense sequence Interacting a nucleotide sequence whereby the meaning of transcription from the respective nucleotide sequence and the antisense polyribonucleotide hybridize to form a "stem" structure of the dsRNA, and the polyribonucleoside transcribed from the intervening sequence The acid will form a "ring". This dsRNA molecule can be referred to as a hairpin RNA (hpRNA) molecule.

In some embodiments, a DNA molecule capable of acting as an iRNA molecule in a cell or microorganism to inhibit expression of a targeted gene can comprise a single polynucleotide that is specifically complementary to one or more targeted insect pest species All or part of a natural polynucleotide found in (for example, a coleopteran pest species), or the DNA molecule can be constructed as a chimera from a plurality of such specifically complementary polynucleotides.

In some embodiments, a nucleic acid molecule can comprise first and second polynucleotides separated by a "gap". A spacer can be a region between the first and second polynucleotides, which when desired, comprises any nucleotide sequence that facilitates the formation of a secondary structure. In one embodiment, the gap is a portion of the mRNA or a portion of the antisense encoding polynucleotide. The spacer may optionally comprise any combination of nucleotides capable of covalent linkage to a nucleic acid molecule or homologs thereof. In some embodiments, the spacer can be an intron (eg, an ST-LS1 intron or an RTM1 intron).

For example, in some embodiments, the DNA molecule may comprise a polynucleotide encoding one or more different iRNA molecules, wherein the different iRNA molecules each comprise a first polyribonucleotide and a A dipolyribonucleotide, wherein the first and second polyribonucleotides are complementary to each other. The first and second polyribonucleotides can be joined within an RNA molecule by a spacer. The spacer may constitute part of the first polyribonucleotide or the second polyribonucleotide. Expression of an RNA molecule comprising the first and second nucleotide polyribonucleotides can result in dsRNA molecules by specific intramolecular base pairing of the first and second nucleotide polyribonucleotides form. The first polyribonucleotide or the second polyribonucleotide may be a polyribonucleotide substantially identical to an insect pest (eg, a target gene transcript or a transcribed non-coding polynucleoside) An acid, a derivative thereof, or a polynucleotide complementary thereto.

The dsRNA nucleic acid molecule comprises a double-stranded polymeric ribonucleotide and may include modifications to either of the phosphate sugar backbone or nucleoside. Modifications in the RNA structure can be made to allow for specific inhibition. In one embodiment, the dsRNA molecule can be modified by a ubiquitous enzymatic process so that siRNA molecules can be generated. This enzymatic process may be carried out in vitro or in vivo using a ribonuclease III enzyme, such as DICER in eukaryotes. See Elbashir et al. (2001) Nature 411: 494-8; and Hamilton and Baulcombe (1999) Science 286 (5441): 950-2. DICER or a functionally equivalent ribonuclease III enzyme cleaves larger dsRNA strands and/or hpRNA molecules into smaller oligonucleotides (eg, siRNA), each of which is about 19-25 nucleotides in length . The siRNA molecules produced by these enzymes have a 3' overhang of 2 to 3 nucleotides, and a 5' phosphate end and a 3' hydroxyl terminus. The siRNA molecule generated by the ribonuclease III enzyme is unfolded in the cell and separated into single-stranded RNA. The siRNA molecule then specifically hybridizes to a target gene transcribed RNA, and the two RNA molecules are subsequently degraded by an intrinsic cellular RNA degradation mechanism. This process may result in efficient degradation or removal of the RNA encoded by the target gene in the target organism. The result is post-transcriptional silence of the targeted gene. In some embodiments, the siRNA molecule produced by the endogenous ribonuclease III enzyme from the heterologous nucleic acid molecule is effective to mediate down-regulation of the target gene in the insect pest.

In some embodiments, a nucleic acid molecule can include at least one non-naturally occurring polynucleotide that can be transcribed into a single strand of RNA molecule capable of forming a dsRNA molecule in vivo through intermolecular hybridization. Such dsRNAs are typically self-assembling and can be provided in an insect (eg, coleopteran) pest nutrient source to achieve post-transcriptional inhibition of a targeted gene. In these and further embodiments, a nucleic acid molecule can comprise two different non-naturally occurring polynucleotides, each of which is specifically complementary to a different target gene within an insect pest. When the nucleic acid molecule is provided as a dsRNA molecule, for example a coleopteran pest, the dsRNA molecule inhibits the performance of at least two different target genes in the pest. C. Obtaining nucleic acid molecules

Various polynucleotides in insect (eg, Coleoptera) pests can be used as targets for designing nucleic acid molecules, such as iRNAs and DNA molecules encoding iRNA. However, the choice of natural polynucleotides is not a straightforward process. For example, only a small number of natural polynucleotides in coleopteran pests would be effective targets. It is not possible to predict whether a particular natural polynucleotide can be effectively down-regulated by the nucleic acid molecule of the present invention, or whether down-regulation of a particular natural polynucleotide will be in the growth, vigor, feeding and/or of insect pests. Or have an adverse effect on survival. The vast majority of natural coleopteran pest polynucleotides, such as the EST thus isolated (for example, the coleopteran pest polynucleotides listed in U.S. Patent No. 7,612,194), on the growth and/or viability of pests Does not have adverse effects. Which of these natural polynucleotides may have an adverse effect on insect pests, can be used in recombinant techniques to display nucleic acid molecules complementary to such natural polynucleotides in a host plant, and rely on feeding to provide disadvantage to pests The effect is not harmful to the host plant, both of which are unpredictable.

In some embodiments, a nucleic acid molecule (eg, a dsRNA molecule provided in a host plant of an insect pest) targets a cDNA that encodes a protein or portion of a protein necessary for pest development and/or survival, such as a biochemical pathway involved in metabolism or decomposition. , cell division, energy metabolism, digestion, host plant recognition, and the like. As described herein, a targeted pest organism ingests a composition comprising one or more dsRNAs, wherein at least one of the one or more dsRNAs is specifically complementary to the target pest organism cell At least substantially identical RNA segments produced can cause death or other inhibition of the target. A polynucleotide derived from an insect pest, either DNA or RNA, can be used to construct plant cells that are protected from pest infestation. The host plant of the coleopteran pest (eg, Z. mays or Brassica sp.), for example, may be transformed to contain a Coleopteran pest as provided herein. One or more polynucleotides. A polynucleotide that is transformed into a host can encode one or more RNAs that form a dsRNA structure in a cell or biological fluid within the transgenic host, thus if/when the pest is When the gene transfer host forms a nutritional relationship, the dsRNA can be obtained. This may result in the pinching of one or more genes in the pest cell, and ultimately death or inhibition of its growth or development.

In a particular embodiment, a gene line that is essentially involved in the growth and development of an insect (eg, Coleoptera) pest is targeted. Other targeting genes for use in the present invention may include, for example, those who play an important role in the viability, movement, movement, migration, growth, development, infectivity, and establishment of feeding sites of pests. A targeted gene may thus be a housekeeping gene or a transcription factor. Furthermore, the natural insect pest polynucleotide used in the present invention may also be derived from a homolog of a plant, virus, bacterial or insect gene (for example, an ortholog), the function of which is familiar to The skilled artisan is aware that the polynucleotide of the individual specifically hybridizes to a targeted gene line in the genome of the targeted pest. Methods for identifying genetic homologues having known nucleotide sequences by hybridization are known to those skilled in the art.

In some embodiments, the invention provides methods for obtaining a nucleic acid molecule comprising a polynucleotide for use in the manufacture of a molecule of an iRNA (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA). One such specific example comprises: (a) analysing the performance, function, and phenotype of one or more target genes in an insect (eg, coleopteran) pest by dsRNA-mediated gene clamp; (b) A probe detects a cDNA or gDNA library, wherein the probe comprises all or part of a polynucleotide derived from a target pest or a homolog thereof, and all or part of the polynucleotide sequence or a homolog thereof is The growth or developmental phenotype of the altered (eg, reduced) display in the clamp analysis of the dsRNA-vector; (c) identification of the DNA colony that specifically hybridizes to the probe; (d) identification of the individual identified in step (b) The DNA selection; (e) sequencing comprises a cDNA or gDNA fragment of the clone isolated in step (d), wherein the sequenced nucleic acid molecule comprises all or most of the RNA sequence or the like And (f) chemically synthesize a gene or all or a majority of siRNA, miRNA, hpRNA, mRNA, shRNA or dsRNA.

In a further embodiment, a method for obtaining a nucleic acid fragment comprising a polynucleotide for producing a majority of iRNA (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules, the method comprising: a) synthesizing first and second oligonucleotide primers that are specifically complementary to a portion of a native polynucleotide derived from a pest of a targeted insect (eg, coleoptera); and (b) using step (a) The first and second oligonucleotide primers are used to amplify a cDNA or a gDNA insert present in a selection vector, wherein the amplified nucleic acid molecule comprises most of the siRNA, miRNA, hpRNA, mRNA, shRNA Or a dsRNA molecule.

Nucleic acids can be isolated, amplified or produced by a number of methods. For example, an iRNA (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule may amplify a targeted polynucleotide derived from a gDNA or cDNA library by PCR (eg, a target gene or a targeted transcription) Obtained from a non-coding polynucleotide), or a portion thereof. DNA or RNA may be extracted from a target organism and the nucleic acid library may be prepared using methods known to those skilled in the art. A gDNA or cDNA library generated from a target organism can be used for PCR amplification and sequencing of targeted genes. The confirmed PCR product can be used as a template for transcription in vitro with a minimal promoter to generate sense and antisense RNA. Alternatively, nucleic acid molecules may be synthesized by any of a number of techniques (see, for example, Ozaki et al. (1992) Nucleic Acids Research, 20: 5205-5214; and Agrawal et al. (1990) Nucleic Acids Research, 18: 5419-5423), including the use of an automated DNA synthesizer (for example, PEBiosystems (Foster City, CA) Model 392 or 394 DNA/RNA synthesizer) using standard chemicals such as phosphite ( Phosphoramidite) chemicals. See, for example, 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. Chemicals that lead to the optional choice of non-natural backbone groups, such as phosphorothioate, phosphoramidate, and the like, can 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 reaction, or in vivo in a cell comprising a nucleic acid molecule, A nucleic acid molecule comprises a polynucleotide encoding the RNA, dsRNA, siRNA, miRNA, shRNA or hpRNA molecule. RNA can also be produced by partial or total organic synthesis - any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. An RNA molecule can be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (eg, T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA polymerase). Expression constructs useful for the selection and expression of 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. Chemically synthesized or RNA molecules synthesized by in vitro enzymatic synthesis may be purified prior to introduction into a cell. For example, the RNA molecule can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, RNA molecules chemically synthesized or enzymatically synthesized by in vitro may be used without purification or minimal purification, for example, to avoid loss of sample processing. The RNA molecule may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote adhesion and/or stabilization of the dsRNA molecule doublet strands.

In a specific example, a dsRNA molecule may be formed from a single self-complementary RNA strand or from two complementary RNA strands. The dsRNA molecule may be synthesized in vivo or in vitro. A cellular endogenous RNA polymerase may mediate transcription of one or both RNA strands in vivo, or may use a cloned RNA polymerase to mediate transcription in vivo or in vitro. Post-transcriptional inhibition of a targeted gene in an insect pest may be host-targeted by specific transcription in an organ, tissue or cell type of the host (eg, by using a tissue-specific promoter) Stimulation of environmental conditions in the host (eg, by using an inducible promoter that responds to infection, stress, temperature, and/or chemical inducer); and/or genetic engineering transcription at the developmental stage or age of the host (eg by using a developmental stage-specific promoter). Whether transcribed in vitro or in vivo, RNA strands that form dsRNA molecules may or may not be polyadenylated and may or may not be translated into polypeptides by cell translational devices. D. Recombinant vector and host cell transformation

In some embodiments, the invention also provides a DNA molecule for introduction into a cell (eg, a bacterial cell, a yeast cell, or a plant cell), wherein the DNA molecule comprises a polynucleotide that is expressed in After RNA is ingested by an insect (for example, Coleoptera and/or Hemiptera) pests, the targeting gene is clamped in the cells, tissues or organs of the pest. Thus, some specific examples provide a recombinant nucleic acid molecule comprising a polynucleotide capable of acting as an iRNA (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule in a plant cell to inhibit a targeted gene in an insect pest. Performance. To elicit or enhance performance, such recombinant nucleic acid molecules may comprise one or more regulatory elements that may be operably linked to a polynucleotide capable of acting as an iRNA. Methods for expressing a gene-clamping molecule in plants are known and may be used to represent a polynucleotide of the invention. See, for example, International PCT Publication No. WO06/073727; and U.S. Patent Publication No. 2006/0200878 Al.

In a particular embodiment, a recombinant DNA molecule of the invention may comprise a polynucleotide encoding an RNA that may form a dsRNA molecule. Such a recombinant DNA molecule can encode an RNA that forms a dsRNA molecule that, upon ingestion, is capable of inhibiting the expression of an endogenous target gene in an insect (eg, coleopteran) pest cell. In many embodiments, the transcribed RNA can form a dsRNA molecule that can be provided in a stable form; for example, in a hairpin and stem-loop structure.

In some embodiments, a strand of a dsRNA molecule can be formed by transcription from a polynucleotide that is substantially homologous to a polynucleotide selected from the group consisting of: sequence identification No.: 1, 3 and 5; sequence identification number: complements of 1, 3 and 5; sequence identification number: fragments of at least 15 contiguous nucleotides of any of 1, 3 and 5 (eg sequence identification number: 7-12); sequence identification number: a complement of a fragment of at least 15 contiguous nucleotides of any of 1, 3, and 5; a native coding polynucleotide of a leaf beetle organism (eg, WCR), comprising Sequence identification number: any of 7-11; a complement of a natural coding polynucleotide of a toad organism comprising a sequence identification number: 7-11; a leaf beetle A fragment of at least 15 contiguous nucleotides of a native coding polynucleotide comprising at least one of sequence identification numbers: 7-11; at least one of the native coding polynucleotides of a leaf beetle organism Complement of fragments of 15 contiguous nucleotides, the natural The coding polynucleotide comprises a sequence identification number: any one of 7 to 11; a natural coding polynucleotide of an appendix organism (eg, PB) comprising a sequence identification number: 12; a natural species of an exposed tailworm organism Encoding a complement of a polynucleotide comprising a sequence ID: 12; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of an appendix organism, the native coding polynucleotide Included in sequence identification number: 12; and a complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a tail-tailed organism comprising a sequence identification number: 12.

In some embodiments, a strand of a dsRNA molecule can be formed by transcription from a polynucleotide that is substantially homologous to a polynucleotide selected from the group consisting of: sequence identification ID: 7-12; sequence identification number: complement of any of 7-12; sequence identification number: fragment of at least 15 contiguous nucleotides of any of 7-12; and sequence identification number: 7- A complement of a fragment of at least 15 contiguous nucleotides of any of 12.

In a specific embodiment, a recombinant DNA molecule encoding an RNA capable of forming a dsRNA molecule can comprise a coding region, wherein at least two polynucleotides are configured such that one polynucleotide is at least one promoter relative to at least one promoter a sense orientation, and another polynucleotide sequence is in an antisense orientation, wherein the sense polynucleotide and the antisense polynucleotide are, for example, from about five (~5) to about one thousand ( The ~1000) nucleotide spacers are linked or linked. The spacer can form a loop between the sense and the antisense polynucleotide. The polynucleotide or the antisense polynucleotide may be substantially homologous to a target gene (eg, a prp8 gene comprising any one of sequence identification numbers: 1, 3, 5, and 7-12), or Wait for the fragment. However, in some embodiments, a recombinant DNA molecule can encode an RNA that forms a dsRNA molecule without a gap. In a specific example, the length of a sense encoding polynucleotide and an antisense encoding polynucleotide may vary.

A polynucleotide identified as having an adverse effect on an insect pest or having a plant protective effect on the pest can be easily incorporated by creating an appropriate expression cassette in the recombinant nucleic acid molecule of the present invention. Is expressed within the dsRNA molecule. For example, such a polynucleotide can be expressed as a hairpin having a stem-loop structure by obtaining a first segment corresponding to a targeting gene polynucleotide (eg, a sequence containing a sequence number: sequence Identification number: any of 1, 3, 5, and 7-12, and the prp8 gene of any of the foregoing fragments; linking the polynucleotide to a different source or not complementary to the second region of the first segment a segment gap sub-region; and linking this to a third segment, wherein at least a portion of the third segment is substantially complementary to the first segment. The transcript of the construct forms a stem-loop structure by intramolecular base pairing of the polyribonucleotide encoded by the first segment with the polyribonucleotide encoded by the third segment, wherein the loop The structure forms a polyribonucleotide comprising the second segment encoding. See, for example, U.S. Patent Publication Nos. 2002/0048814 and 2003/0018993; and International PCT Patent Publication Nos. WO94/01550 and WO98/05770. A dsRNA molecule may be generated, for example, in a double-stranded structural form, such as a stem-loop structure (eg, a hairpin), such that a target of a target gene is co-presented, such as on an additional plant performance cassette, to target a natural The production of siRNAs for insect (eg, Coleoptera) pest polynucleotides is increased, which results in enhanced siRNA production, or reduced methylation, to prevent transcriptional gene silencing of the dsRNA hairpin promoter.

Some specific examples of the invention include the introduction of a recombinant nucleic acid molecule of the invention into a plant (i.e., transformed) to achieve the level of pest inhibition of insect (e.g., coleoptera) exhibited by one or more iRNA molecules. A recombinant DNA molecule can be, for example, a vector such as a linear or a circular closed plastid. The vector system may be a single vector or plastid, or two or more vectors or plastids that together contain the total DNA to be introduced into the host genome. Furthermore, the vector may be a performance vector. The polynucleotide of the present invention may, for example, be suitably inserted into a vector under the control of a suitable promoter, wherein the promoter functions in one or more hosts to drive the linked polynucleotide encoding Or the performance of other DNA elements. A number of vectors can be used for this purpose, and the choice of a suitable vector will primarily depend on the size of the nucleic acid to be inserted into the vector and the particular host cell into which the vector is to be transformed. Each vector contains various components depending on its function (for example, amplifying DNA or expressing DNA) and its compatible specific host cells.

In order to deliver a coleopteran pest pest to a genetically transgenic plant, a recombinant DNA can be transcribed into an iRNA molecule (eg, an RNA molecule that forms a dsRNA molecule) in a tissue or fluid of a recombinant plant, for example. An iRNA molecule can comprise a polyribonucleotide that substantially homologously and specifically hybridizes to a corresponding transcribed polyribonucleotide within an insect pest that may cause damage to the host plant species. The pest can, for example, be exposed to an iRNA molecule transcribed in the gene transfer host plant cell by ingesting a cell or fluid of the host plant that contains the iRNA molecule. Thus, in a particular example, the targeted gene expression within an insect pest that invades the gene to the host plant is clamped by the iRNA molecule. In some embodiments, the use of a targeting gene in the targeted coleopteran pest may result in the plant being protected from attack by the pest. .

In order to be able to deliver an iRNA molecule to an insect pest in a nutritional relationship with a plant cell that has been transformed into a recombinant nucleic acid molecule of the invention, it is necessary to be able to express (i.e., transcribe) the iRNA molecule in the plant cell. Thus, a recombinant nucleic acid molecule can comprise a polynucleotide of the invention operably linked to one or more regulatory elements that act in a host cell, such as a heterologous promoter element that acts in a host cell, such as a bacterial cell. Wherein the nucleic acid molecule is amplified and the nucleic acid molecule is expressed in a plant cell.

Promoters suitable for use in the nucleic acid molecules of the invention include those inducible, viral, synthetic or sustained phenotypes, all of which are well known in the art. Non-limiting examples of such promoters include U.S. Patent No. 6,437,217 (Maize RS81 promoter); No. 5,641,876 (rice actin promoter); No. 6,426,446 (maize RS324 promoter) ; No. 6,429,362 (maize PR-1 promoter); No. 6,232,526 (maize A3 promoter); No. 6,177,611 (maintaining type maize promoter); Nos. 5,322,938, 5,352,605 No. 5,359,142 and 5,530,196 (CaMV 35S promoter); No. 6,433,252 (maize L3 oil membrane protein (oleosin) promoter); No. 6,429,357 (rice actin 2 promoter and rice actin) 2 intron); 6, 294, 714 (photoinducible promoter); 6, 140, 078 (salt-inducible promoter); 6, 252, 138 (pathogenic-inducible promoter); No. 6, 175, 060 (phosphorus-inducible promoter) No. 6,388,170 (bidirectional promoter); No. 6,635,806 (gamma-coilin promoter); and US 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. USA 84(16): 5745-9) and octopine synthase (OCS). Promoters (these are implemented on tumor-inducing plastids of Agrobacterium tumefaciens ); cauliimovirus promoters, such as cauliflower mosaic virus (CaMV) 19S (Lawton et al. (1987) Plant Mol. Biol. 9: 315-24); CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-2); figwort mosaic virus (figwort mosaic) Virus) 35S-promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84(19): 6624-8); sucrose synthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-8); R gene complex promoter (Chandler et al. (1989) Plant Cell 1:1175-83); chlorophyll a/b binding protein gene promoter; CaMV 35S (US patent) Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196); FMV 35S (U.S. Patent Nos. 6,051,753 and 5,378,619); PC1SV promoter (USA) Patent No. 5,850,019); SCP1 promoter (U.S. Patent No. 6,677,503); and AGRtu.nos promoter (GenBankTM accession number V00087; Deepick 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. Root-specific promoters specifically or preferentially drive manipulation of linker-encoding polynucleotides in root tissue. Examples of root-specific promoters are known in the art. See, for example, U.S. Patent No. 5,110,732; 5,459,252 and 5,837,848; and Opperman et al. (1994) Science 263:221-3; and Hirel et al. (1992) Plant Mol. Biol. 20:207-18 . In some embodiments, polynucleotides or fragments for coleopteran pest control according to the invention can be cloned between two root-specific promoters, wherein the two promoters are oriented relative to the polynucleotide or fragment In the opposite direction of transcription, and the polynucleotide or fragment is operably and expressed in the gene transfer plant cell to produce an RNA molecule in the gene transfer plant cell which may subsequently form a dsRNA molecule, as previously described Described. The iRNA molecules expressed in plant tissues may be taken up by an insect pest, thereby achieving the immobilization of the targeted gene expression.

Additional regulatory elements that can be selectively manipulated to link to a nucleic acid include 5' UTRs, which are located between a promoter element and a coding polynucleotide, acting as a translation leader element. The translational leader element is present in the fully processed mRNA and may affect the processing of the primary transcript and/or the stability of the RNA. Examples of translational leader elements include maize and petunia heat shock protein leader (U.S. Patent No. 5,362,865), plant virus coat protein leader, plant ribulose bisphosphate carboxylase leader, and other. See, for example, Turner and Foster (1995) Molecular Biotech. 3(3): 225-36. Non-limiting examples of 5' UTRs include GmHsp (U.S. Patent No. 5,659,122); PhDnaK (U.S. Patent No. 5,362,865); AtAnt1 TEV (Carrington and Freed (1990) J. Virol. 64: 1590-7); and AGRtunos (GenBankTM Accession No. V00087; and Beva et al. (1983) Nature 304: 184-7).

Additional regulatory sequences that are selectively manipulated to link to a nucleic acid also include a 3' non-translated element, a 3' transcription termination region, or a polyadenylation region. These are genetic elements downstream of a polynucleotide and include polynucleotides that provide polyadenylation signals, and/or other regulatory signals that can affect transcription or mRNA processing. The polyadenylation signal acts in the plant to cause the addition of a polyadenylation nucleotide at the 3' end of the mRNA precursor. The polyadenylation element can be derived from various plant genes or derived from the T-DNA gene. 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. USA 80:4803-7). An example of the use of different 3' non-translated regions is provided in Ingelbrecht et al. (1989) Plant Cell 1:671-80. Non-limiting examples of polyadenylation signals include one derived from the pea ( Pisum sativum ) RbcS2 gene (Ps. RbcS2-E9; Coruzz et al. (1984) EMBO J. 3: 1671-9) and AGRtu. Nos (GenBankTM accession number E01312).

Some specific examples can include a plant-transformed vector comprising an isolated and purified DNA molecule comprising at least one regulatory element described above operably linked to one or more polynucleotides of the invention. When expressed, the one or more polynucleotides result in one or more iRNA molecules comprising polyribonucleotides that are specifically complementary to all or part of a native RNA molecule in an insect pest. Thus, the polynucleotide may comprise a segment encoding all or part of a polyribonucleotide present in a targeted RNA transcript within an insect pest and may comprise all or part of a targeted pest transcript Reverse repeat. A plant-transformed vector may contain a polynucleotide that is specifically complementary to more than one targeting polynucleotide, thereby allowing the production of more than one dsRNA for use in inhibiting one or more ethnic groups or species of cells that target insect pests. Or the performance of multiple genes. Polynucleotide segments that are specifically complementary to polynucleotides present in different genes can be combined into a single composite nucleic acid molecule for expression in a genetically transgenic plant. These sections may be continuous or separated by a gap.

In some embodiments, a plastid of the invention that already contains at least one polynucleotide of the invention can be modified by sequentially inserting additional polynucleotides in the same plastid, wherein the additional polynucleotide is as initial At least one polynucleotide is operably linked to the same regulatory element. In some embodiments, a nucleic acid molecule may be designed to inhibit multiple targeting genes. In some embodiments, the inhibited multiplex gene can be obtained from the same insect (eg, coleopteran) pest species, which may enhance the effectiveness of the nucleic acid molecule. In other specific examples, the gene may be derived from different insect pests, which may extend the range of the agent to be an effective pest. When a multiple gene line is targeted for a combination of clamping or expression and clamping, a polycistronic DNA element can be genetically engineered.

The recombinant nucleic acid molecule or vector of the invention may comprise a selectable marker which confers a transforming cell, such as a plant cell, a selectable phenotype. A selectable marker can also be used to select a plant or plant cell comprising a recombinant nucleic acid molecule of the invention. The marker may encode biocide resistance, antibiotic resistance (eg, kanamycin, geneticin (G418), bleomycin, hygromycin, etc. ) or herbicide tolerance (eg glyphosate, etc.). Examples of selectable markers include, but are not limited to, the neo gene, which encodes kanamycin resistance and can be selected using kanamycin, G418, etc.; bar gene, which encodes bialaphos resistance; a mutant EPSP synthase gene, by encoding the phosphorus Ka plug (glyphosate) resistance; a nitrile synthase (nitrilase) gene, which confers resistance to bromoxynil by (bromoxynil); and a mutant b A lactic acid synthase ( ALS ) gene that confers imidazolinone or sulforaphane resistance; and an anti-aminomethyl folate (methotrexate) DHFR gene. Multiple selectable markers are available which confer resistance to: ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin ( Hygromycin), kanamycin, lincomycin, amine methyl folate, phosphinothricin, puromycin, spectinomycin, rifampicin ), streptomycin and tetracycline and the like. Examples of such a selectable marker are exemplified by, for example, U.S. Patent 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 also comprise a selectable marker. Filterable markers can be used to monitor performance. Exemplary selectable markers include beta-glucuronidase or uidA gene (GUS), which encodes a variety of chromogenic matrix systems known as enzymes (Jefferson et al. (1987) Plant Mol. Biol. Rep. 5 : 387-405); R-locus gene, which encodes a product that regulates the production of anthocyanin pigment (red) in plant tissues (Dellaporta et al. (1988) "Molecular cloning of the maize R- Nj allele by transposon tagging with Ac ." In 18 ^th Stadler Genetics Symposium, P. Gustafson and R. Appels, eds. (New York: Plenum), pp. 263-82); β-endoprostanase gene (Sutcliffe et al) Human (1978) Proc. Natl. Acad. Sci. USA 75:3737-41); a gene encoding various chromogenic substrates known as enzymes (eg, PADAC, a cephalosporin) a luciferase gene (Ow et al. (1986) Science 234: 856-9); a xylE gene encoding a catechol dioxygenase that converts catechol (Zukowski et al. 1983) Gene 46(2-3): 247-55); an amylase gene (Ikatu et al. (1990) Bio/Technol. 8:241-2); a tyrosinase group , which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses into melanin (Katz et al. (1983) J. Gen. Microbiol. 129: 2703-14); - Galactosidase.

In some embodiments, a recombinant nucleic acid molecule, as described above, can be used in a method of creating a genetically transgenic plant and expressing a heterologous nucleic acid in a plant to produce a reduced display of insects (eg, coleoptera). The susceptibility of the gene to the plant. Plant transformation vectors can be prepared, for example, by inserting nucleic acid molecules encoding iRNA molecules into plant transformation vectors and introducing them into plants.

Suitable methods for transforming host cells include any method by which DNA can be introduced into a cell, such as by protoplast transformation (see, e.g., U.S. Patent No. 5,508,184), by drying/inhibiting ( Desiccation/inhibition) DNA uptake by the media (see, for example, Potrykus et al. (1985) Mol. Gen. Genet. 199: 183-8) by electroporation (see, e.g., U.S. Patent No. 5,384,253) Stirring with cerium carbide fibers (see, for example, U.S. Patent Nos. 5,302,523 and 5,464,765), which are incorporated by Agrobacterium (see, for example, U.S. Patent No. 5,563,055; 5,591,616; 5,693,512; 5,824,877; No. 5,981,840; and No. 6,384,301, and the use of accelerated DNA-coated particles (see, for example, U.S. Patent Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865) . Techniques that are particularly useful for the transformation of corn are described, for example, in U.S. Patent Nos. 7,060,876 and 5,591,616; and International PCT Patent Publication No. WO 95/06722. Through the application of these technologies, it is almost possible to stably transform cells of any species. In some embodiments, the transformed DNA line is integrated into the genome of the host cell. In the case of multicellular species, gene transfer cells can regenerate a gene transfer organism. Any of these techniques can be used to make a genetically transformed plant, for example, comprising one or more nucleic acid sequences encoding one or more iRNA molecules in the genome of the gene transfer plant.

The most widely used method for introducing a performance vector into plants is based on the natural transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria, which cause plant cell genes to be transformed. The Ti and Ri system of A. tumefaciens and A. rhizogenes carry genes responsible for plant gene transformation. Ti (tumor-inducing)-plastids contain a large segment called T-DNA, which is transferred to transformed plants. Another segment of the Ti plastid, the Vir region, is responsible for the transfer of T-DNA. The T-DNA region is repeatedly bordered by the ends. In the modified binary vector, the tumor-inducing gene has been deleted, and the function of the Vir region is utilized to transfer foreign DNA bordering the T-DNA junction element. The T-region may also contain a selectable marker for efficient recovery of gene transfer cells and plants, as well as a multiplex destination for insertion of a transferred polynucleotide, such as a dsRNA encoding a nucleic acid.

In a specific embodiment, a plant-transformed carrier is derived from a Ti-plast of Agrobacterium tumefaciens (see, for example, U.S. Patent Nos. 4,536,475, 4,693,977, 4,886,937 and 5,501,967; and European Patent No. EP 0 No. 122 791) or Ri plastid derived from Rhizoctonia solani. Additional plant-transformed vectors include, for example but are not limited to, those described below: Herrera-Estrella 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 European Patent No. EP 0 120 516, and those derived from any of the foregoing. Other bacteria that naturally interact with plants, such as Sinorhizobium , Rhizobium , and Mesorhizobium , can be modified to mediate gene transfer in many diverse plants. These plant-associated commensal bacteria can be competent for gene transfer by obtaining both harmless Ti plastids and a suitable binary vector.

After providing exogenous DNA to the recipient cells, the transforming cells are typically identified for further culture and plant regeneration. In order to improve the ability to identify transformed cells, it may be desirable to employ a selectable or screenable marker gene, as previously stated, plus a transforming vector used to generate the transform. Where a selectable marker is used, the transformed cell line within the population of potentially transformed cells is identified by exposing the cell to a selective agent or agent. Where a selectable marker is used, the cells can be screened for the desired marker gene trait.

Cells that survive the exposure to the selection agent, or cells that have been scored positive in the screening assay, can be cultured in a medium that supports plant regeneration. In some embodiments, any suitable plant tissue culture medium (for example, MS and N6 medium) can be modified by the inclusion of additional materials, such as growth regulators. The tissue can be maintained on a basal medium with a growth regulator until sufficient tissue is available to initiate plant regeneration, or a manual selection of repeated cycles until the morphology of the tissue is suitable for regeneration (for example, at least 2 weeks) Then, it is transferred to a medium that helps stem formation. The culture is periodically transferred until sufficient stem formation has occurred. After the stem is formed, it is transferred to a medium that facilitates root formation. After sufficient root formation, the plant can be transferred to the soil for further growth and maturation.

To confirm that there is a nucleic acid molecule of interest in the regenerated plant (for example, a DNA encoding one or more iRNA molecules that inhibit the targeting gene from being expressed in a coleopteran pest), a wide variety of assays can be performed. Such analyses include, by way of example: molecular biological analysis, such as Southern and Northern blots, PCR, and nucleic acid sequencing; biochemical analysis, for example, by immunological means (ELISA and/or Western blotting) Or through the function of enzymes to detect the presence of protein products; analysis of plant parts, such as leaf or root analysis; and analysis of the phenotype of whole plant regenerated plants.

Integration events can be analyzed, for example, by PCR amplification, for example using oligonucleotide primers specific for the nucleic acid molecule of interest. PCR genotyping is understood to include, but is not limited to, polymerase chain reaction (PCR) amplification of gDNA derived from an isolated host plant callus, wherein the callus is predicted to contain integration into the genome. One of the nucleic acid molecules of interest, followed by standard selection and sequencing analysis of the PCR amplification product. PCR genotyping methods are well described (for example, Rios, G et al. (2002) Plant J. 32: 243-53) and may be applied to any plant species (eg, Z. mays , cotton). , soy and big rape ( B. napus ) or tissue type gDNA, including cell culture.

A gene transfer plant formed using a method dependent on Agrobacterium transformation typically contains a single recombinant DNA inserted into a chromosome. The single recombinant DNA polynucleotide is referred to as a "gene transfer product" or "integrated article." Such a genetically transgenic plant is heterozygous because of the inserted exogenous polynucleotide. In some embodiments, a gene-transferred plant that is homozygous for a transgene can be obtained by sexually mating (self-crossing) an independent isolate gene containing a single exogenous gene into a plant. for example, one kind of T ₀ plants to produce T ₁ seed. Generated a quarter of species T ₁ seed in respect of gene transfer in terms of engagement will be the same type. T ₁ of seed germination induced plants, which can be used for zygote divergent profile of the test person, typically used to allow the same and the difference between homozygous zygotic profile (meaning, zygotes (zygosity) analysis) Analysis SNP analysis or thermal amplification .

In a particular embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different plant cells are produced in a plant cell having an insect (eg, coleopteran) pest protective effect. iRNA molecule. The iRNA molecule (eg, a dsRNA molecule) may be represented by multiple nucleic acids introduced in different transformed articles, or from a single nucleic acid introduced in a single transformed article. In some embodiments, several iRNA molecules are expressed under the control of a single promoter. In other embodiments, several iRNA molecules are expressed under the control of multiple promoters. A single iRNA molecule comprising multiple polynucleotides can be represented, each of which is homologous to a different locus within one or more insect pests (for example, by sequence identification number: 1, 3, and 5 The defined loci, both in different ethnic groups of the same insect pest species, or in insect pests of different species.

In addition to directly transforming a plant with a recombinant nucleic acid molecule, the genetically transformed plant can be prepared by crossing a first plant having at least one gene-transferred product with a second plant lacking such a product. For example, a recombinant nucleic acid molecule comprising a polynucleotide encoding an iRNA molecule can be introduced into a first plant line that conforms to a transformation to produce a gene transfer plant that may be associated with a second plant line Hybridization is performed to introgress the polynucleotide gene encoding the iRNA molecule into the second plant line.

In some aspects, the seed and commercial product produced by a genetically transformed plant derived from a transformed plant cell, wherein the seed or commercial product comprises a detectable amount of a nucleic acid of the invention. In some embodiments, such commercial products may be made, for example, by obtaining genetically transgenic plants and preparing food or feed from the individual. Commercial products comprising one or more polynucleotides of the invention include, by way of example and not limitation, a plant meal, oil, comminuted or whole grain or seed, and any meal, oil or comminuted comprising a recombinant plant or seed Or any food product of whole grains, wherein the recombinant plant or seed contains one or more nucleic acid molecules of the invention. In a particular example, a commercial product is a bait composition or formulation comprising one or more nucleic acid molecules of the invention. Detection of one or more polynucleotides of the invention in one or more commercial or commercial products is a de facto evidence that the commercial or commercial product is produced from one or more iRNAs designed to represent the invention. Molecular gene transfer plants in order to achieve the purpose of controlling insect pests.

In some embodiments, a genetically transgenic plant or seed comprising a nucleic acid molecule of the invention may also comprise at least one other genetically-transferred article in its genome, including but not limited to: a genetically-transferred article, Transcription of an iRNA molecule that targets a locus in a coleopteran pest that is not identified by sequence identification number: 1, sequence identification number: 3, and sequence identification number: 5, for example, selected from One or more loci in the following group: Caf1-180 (U.S. Patent Publication No. 2012/0174258), VatpaseC (U.S. Patent Publication No. 2012/0174259), Rho1 (U.S. Patent Publication No. No. 2012/0174260), VatpaseH (U.S. Patent Publication No. 2012/0198586), PPI-87B (U.S. Patent Publication No. 2013/0091600), RPA70 (U.S. Patent Publication No. 2013/0091601), RPS6 (U.S. Patent Publication No. 2013/0097730), ROP (U.S. Patent Publication No. 14/577,811), RNA polymerase I1 (U.S. Patent Publication No. 62/133,214), and RNA polymerase II140 (U.S. Patent Publication No. 14) /577 , No. 854), RNA polymerase II215 (U.S. Patent Publication No. 62/133,202), RNA polymerase II33 (US Patent Application No. 62/133,210), transcription elongation factor spt5 (US Patent Application No. 62/168,613) No.), transcription elongation factor spt6 (US Patent Application No. 62/168,606), ncm (US Patent Application No. 62/095487), dre4 (US Patent Application No. 14/705,807), COPI α (US Patent) Application Nos. 62/063,199), COPI β (US Patent Application No. 62/063,203), COPI γ (US Patent Application No. 62/063,192), and COPI δ (US Patent Application No. 62/063,216) A gene-transferred product that transcribes an iRNA molecule that targets a gene in a non-coleoptera pest organism (eg, a plant-parasitic nematode); a gene encoding a pesticidal protein (eg, Suri a gene for Bacillus thuringiensis ), a herbicide tolerance gene (eg, a gene that provides tolerance to glyphosate); and a gene that facilitates the gene transfer The desired phenotype of the plant, such as increased yield Quantity, altered fatty acid metabolism or repair of cytoplasmic male sterility. In a particular embodiment, the polynucleotide encoding the iRNA molecule of the invention may be combined with other insect control and disease traits in a plant to achieve the desired trait for enhancing control of plant diseases and insect damage. In some embodiments, the genes encoding the pesticidal proteins can be combined, including, by way of example and not limitation, a single or recombinant nucleic acid molecule encoding Alcaligenes Insecticidal Protein-1A and Alcaligenes Insecticidal Protein - 1B (AfIP-1A and AfIP-1B) polypeptide (U.S. Patent Publication No. 2014/0033361); or a PIP polypeptide encoded by a single or recombinant nucleic acid molecule (WO 2015038734). Combining insect control traits using a distinct mode of action can provide superior endurance of protected gene transfer plants beyond plants with a single control trait, for example, because of the chance of developing this trait in the field Will be reduced. V. Clamping of Targeted Genes in Insect Pests A. Overview

In some embodiments of the invention, at least one nucleic acid molecule useful for insect (eg, coleopteran) pest control can be provided to an insect pest, wherein the nucleic acid molecule causes gene silencing of the RNAi vector in the pest. In a specific embodiment, an iRNA molecule (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) can be provided to a coleopteran pest. In some embodiments, a nucleic acid molecule useful for controlling insect pests can be provided to the pest by contacting the nucleic acid molecule with a pest. In these and further embodiments, a nucleic acid molecule useful for controlling insect pests can be provided in the feed matrix of the pest, for example, a nutritional composition. In these and further embodiments, a nucleic acid molecule useful for controlling insect pests may be provided by ingesting a plant material comprising the nucleic acid molecule, wherein the nucleic acid molecule is taken up by the pest. In some embodiments, the nucleic acid molecule is present in the plant material by expression of a recombinant nucleic acid introduced into the plant material, for example, by transforming a plant cell with a vector comprising the recombinant nucleic acid, And regenerating a plant material or whole plant from the transformed plant cell.

In some embodiments, a pest contacts the nucleic acid molecule via contact with a topical composition (e.g., by applying a composition by spraying) or an RNAi decoy, which results in gene silencing of the RNAi vector within the pest. The RNAi bait is formed when the dsRNA is mixed with food or an attractant or both. When pests eat bait, they also eat dsRNA. The bait can take the form of granules, gels, fluid powders, liquids or solids. In a particular embodiment, an iRNA molecule that targets a prp8 can be incorporated into a bait formulation, such as described in U.S. Patent No. 8,530,440, incorporated herein by reference. In general, by the bait, the bait is placed in or near the environment of, for example, insect pests, whereby the insect pests come into contact with the bait and/or are attracted by the bait. B. RNAi- mediated target gene clamp

In some embodiments, the invention provides iRNA molecules (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) that can be designed to target an insect pest (eg, coleoptera (eg, WCR, SCR, NCR, and PB) A natural polynucleotide (eg, a necessary gene) necessary for transcriptome of a pest, for example, by designing an iRNA molecule comprising at least one strand, the strand comprising specific complementarity The polynucleotide of the targeted polynucleotide. The sequence of the iRNA molecule thus designed may be identical to the sequence of the target polynucleotide, or may be incorporated without interfering with the iRNA molecule and its target polynucleotide. Specific hybridization mismatch.

The iRNA molecule of the present invention can be used in a method of genetically clamping an insect (e.g., coleopteran) pest, thereby reducing the pest on a plant (for example, a protected plant comprising an iRNA molecule) The level or incidence of damage caused. As used herein, the term "gene-clamping" means any well-known method for reducing the level of protein produced by transcription of a gene into mRNA and subsequent translation of the mRNA, including reducing the protein from a gene or a polynucleotide encoding a polynucleoside. The performance of acid, including post-transcriptional inhibition and transcriptional clampation of performance. Post-transcriptional inhibition is mediated by specific homology between all or part of the mRNA transcribed from the target gene for immobilization and the corresponding iRNA molecule used for immobilization. Furthermore, post-transcriptional inhibition means a large and measurable reduction in the amount of mRNA available for ribosome binding in this cell.

In some specific examples where the iRNA molecule is a dsRNA molecule, the dsRNA molecule may be cleaved by the enzyme, DICER, into a short siRNA molecule (approximately 20 nucleotides in length). The double-stranded siRNA molecules generated on the dsRNA molecule by DICER activity may be separated into two single-stranded siRNAs; "passenger strands" and "guide strands". The passenger shares may be degraded and the lead shares may be incorporated into the RISC. Post-transcriptional inhibition occurs by specific hybridization of the leader strand with a specific complementary polynucleotide of an mRNA molecule, and is subsequently cleaved by the enzyme, the aggrecin family (the catalytic component of the RISC complex).

In other embodiments of the invention, any form of iRNA molecule can be used. It will be understood by those skilled in the art that dsRNA molecules are typically more stable during preparation and during the step of providing the iRNA molecule to a cell, and are typically more stable in the cell than single-stranded RNA molecules. Thus, although siRNA and miRNA molecules, for example, may be equally effective in some specific examples, dsRNA molecules may be selected for stability of the dsRNA molecule.

In a specific embodiment, a nucleic acid molecule comprising a polynucleotide which may be expressed in vitro to produce an iRNA molecule comprising a polyribonucleotide, the polyribonucleoside The acid is substantially homologous to the polyribonucleotide of the RNA molecule encoded by the polynucleotide within the genome of an insect pest. In certain embodiments, an in vitro transcribed iRNA molecule may be a stable dsRNA molecule comprising a stem-loop structure. After an insect pest contacts an in vitro transcribed iRNA molecule, post-transcriptional inhibition of the target gene (for example, a necessary gene) in the pest may occur.

In some embodiments of the invention, the expression of a nucleic acid molecule is for use in a method of post-transcriptionally inhibiting a target gene of an insect (eg, a coleoptera) pest comprising at least 15 polynucleotides a contiguous nucleotide (eg, at least 19 contiguous nucleotides), wherein the polynucleotide is selected from the group consisting of: sequence identification number: 1; sequence identification number: 1 complement; sequence identification No.: 3; sequence identification number: complement of 3; sequence identification number: 5; sequence identification number: complement of 5; sequence identification number: 7; sequence identification number: complement of 7; sequence identification number: 8; sequence Identification number: complement of 8; sequence identification number: 9; sequence identification number: complement of 9; sequence identification number: 10; sequence identification number: complement of 10; sequence identification number: 11; sequence identification number: 11 Complement; sequence identification number: 12; sequence identification number: 12 complement; sequence identification number: fragment of at least 15 contiguous nucleotides of 1; sequence identification number: at least 15 linkages of 1 The complement of the nucleotide fragment; SEQ ID. No: 3 of at least 15 contiguous nucleotides of the fragment; SEQ ID. No: 3 of the complement of at least 15 contiguous nucleotides of the fragment; Diabrotica one kind of organism A naturally-coding polynucleotide comprising a sequence number: 7-11; a complement of a native coding polynucleotide of a leaf- bean organism comprising a sequence identification number: 7-11 any one; Diabrotica organism naturally encodes a polynucleotide of at least 15 contiguous nucleotides of a fragment of the native encoding polynucleotide comprising the sequence identification number: any one of 7-11; Diabrotica one kind of organism A complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide comprising at least one of sequence identification number: 7-11; sequence identification number: at least 15 contiguous cores of 5 a nucleotide fragment; SEQ ID. No: 5 was complementary to fragments of at least 15 consecutive nucleotides; naturally encoded tail exposed one kind of organism a polynucleotide comprising the sequence identification number: 12; biological one kind Meligethes The naturally encoded the complement of a polynucleotide, the polynucleotide comprising the sequence naturally encoded identification number: 12; tail exposed one kind of organism naturally A polynucleotide encoding at least 15 contiguous nucleotides of the fragment, which naturally encoded multicore The nucleoside comprises a sequence identity number: 12; and a complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a tail-tailed organism comprising a sequence identification number: 12. In certain embodiments, a nucleic acid molecule exhibits at least about 80% identity to any of the foregoing (eg, 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%) can be used. In these and further embodiments, a nucleic acid molecule can be expressed that specifically hybridizes to an RNA molecule present in at least one cell of a coleopteran insect (e.g., a leaf and an appendix ) pest.

An important feature of some specific examples in this paper is that the RNAi post-transcriptional inhibition system can tolerate sequence changes in the target gene, which is attributed to gene mutation, strain polymorphism or evolutionary divergence. of. The introduced nucleic acid molecule may not need to be absolutely homologous to either a primary transcript of a targeted gene or a fully processed mRNA, as long as the introduced nucleic acid molecule specifically hybridizes to the primary transcript of the target gene or Any of the fully processed mRNAs. Furthermore, the introduced nucleic acid molecule may not need to be full length, relative to either the primary transcript of the target gene or the fully processed mRNA.

The use of the iRNA technology of the invention inhibits sequence specificity of a target gene; that is, a polynucleotide line substantially homologous to the iRNA molecule is targeted for gene suppression. In some embodiments, an RNA molecule comprising a polynucleotide can be used for inhibition, the nucleotide sequence carried by the polynucleotide being identical to the nucleotide sequence of a portion of the target gene. In these and further embodiments, an RNA molecule comprising a polynucleotide having one or more insertions, deletions, and/or point mutations relative to a target polynucleotide can be used. In a particular embodiment, an iRNA molecule may be shared with a portion of a target gene, 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. Optionally, a duplex region of a dsRNA molecule may specifically hybridize to a portion of a targeted gene transcript. Among the molecules that specifically hybridize, a polynucleotide that exhibits greater homology than the full length will compensate for a longer, more diverse source sequence. A polynucleotide sequence length of a dsRNA molecule duplex region that is identical to a portion of a targeted gene transcript, possibly at least about 25, 50, 100, 200, 300, 400, 500, or at least about 1000 Base. In some embodiments, polynucleotides greater than 20-100 nucleotides can be used. In particular embodiments, polynucleotides greater than about 200-300 nucleotides can be used. In a particular embodiment, a polynucleotide greater than about 500-1000 nucleotides can be used depending on the size of the target gene.

In certain embodiments, the performance of a targeting gene in a pest (eg, coleopteran) pest can be inhibited by at least 10% in the cell of the pest; at least 33%; at least 50%; or at least 80%, Significant inhibition occurs by this. Significant inhibition means that the inhibition exceeds a threshold that results in a detectable phenotype (eg, stopping growth, stopping feeding, stopping development, causing death, etc.), or corresponding to the inhibited target gene, There is a detectable decline in RNA and/or gene products. Although in some embodiments of the invention, inhibition occurs in all cells of a substantial pest, in other embodiments, inhibition occurs only in a subset of cells expressing the target gene.

In some embodiments, transcriptional clampation is mediated by the appearance of a dsRNA molecule in a cell that exhibits substantial sequence identity to a promoter DNA or its complement, thereby inducing a "promoter reversal""promoter trans suppression". Gene immobilization may be effective for targeting genes of an insect pest that may ingest or contact such dsRNA molecules, for example, by ingesting or contacting a plant material containing the dsRNA molecule. The dsRNA molecules used in the reverse clamp of the promoter can be specifically designed to inhibit or clamp the expression of one or more homologous or complementary polynucleotides in the insect pest cell. Post-transcriptional gene clampation of antisense or sense-directed RNA to modulate gene expression in plant cells is disclosed in U.S. Patent Nos. 5,107,065; 5,759,829; 5,283,184; and 5,231,020. C. Performance of iRNA molecules provided to insect pests

Gene suppression of RNAi vectors for expression of iRNA molecules in an insect (e.g., coleopteran) pest can be performed in any of a number of in vitro or in vivo formats. The iRNA molecule can then be provided to an insect pest, for example, by contacting the iRNA molecule with the pest or by ingesting or otherwise internalizing the iRNA molecule. Some specific examples include host plants of the coleopteran pest transformation, transgenic plant cells, and progeny of the transformed plant. Transformed plant cells and transformed plants can be genetically engineered, for example, to exhibit one or more iRNA molecules under the control of a heterologous promoter to provide pest protection. Therefore, when an insect pest consumes a gene transfer plant or plant cell during feeding, the pest can ingest the iRNA molecule expressed in the gene transfer 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 can include partial or complete immobilization of such performance. In another embodiment, a method for clamping gene expression in an insect (eg, Coleoptera) pest comprises providing a gene-clamped amount of at least one dsRNA molecule in the tissue of the pest host, the dsRNA molecule being in this context The described polynucleotide is formed after transcription, and at least a segment of the polynucleotide is complementary to an mRNA within the insect pest cell. A dsRNA molecule that is ingested by an insect pest, including a modified form thereof, such as an siRNA, miRNA, shRNA or hpRNA molecule, can be at least from 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 a prp8 An RNA molecule transcribed by a DNA molecule, the molecule comprising, by way of example, a polynucleotide selected from the group consisting of sequence identification numbers 1, 3, 5, and 7-12. Thus providing isolated and substantially purified nucleic acid molecules including, but not limited to, non-naturally occurring polynucleotides and recombinant DNA constructs providing dsRNA molecules which, when introduced therein, will clamp or inhibit endogenous in insect pests The expression of a polynucleotide encoding a polynucleotide or a polynucleotide encoding a polynucleotide.

A specific embodiment provides a delivery system for delivering an iRNA molecule for use in post-transcriptional inhibition of one or more target genes in an insect (eg, Coleoptera) plant pest and controlling the population of the plant pest. In some embodiments, the delivery system comprises ingesting or ingesting a host gene transgenic plant cell, the inclusion comprising an RNA molecule transcribed in the host cell. In these and further embodiments, a gene transfer plant cell or a gene transfer plant line is created which contains a recombinant DNA construct which provides a stable dsRNA molecule of the invention. Gene transfer plant cells and gene transfer plants comprising a nucleic acid encoding a particular iRNA molecule can be produced by recombinant DNA techniques, which are well known in the art, to construct a polynucleotide comprising a polynucleotide. Plant transformation vector encoding an iRNA molecule of the invention (eg, a stable dsRNA molecule); transforming a plant cell or plant; and producing a gene transfer plant cell or gene transgene containing a transcriptional iRNA molecule Colonized plants.

To deliver insect pest protection to a genetically transgenic plant, a recombinant DNA molecule can, for example, be transcribed into an iRNA molecule, such as a dsRNA molecule, siRNA molecule, miRNA molecule, shRNA molecule, or hpRNA molecule. In some embodiments, an RNA molecule transcribed from a recombinant DNA molecule may form a dsRNA molecule within the tissue or fluid of the recombinant plant. Such a dsRNA molecule may to some extent comprise a polyribonucleotide which is identical to a corresponding polyribonucleotide from which the corresponding polyribonucleotide may be infested The host plant is transcribed by DNA within the insect pest type. The expression of the target gene in the pest is clamped by the dsRNA molecule, and the immobilization of the target gene in the pest causes the gene transfer plant to be protected against the pest. The regulatory effects of dsRNA molecules have been shown to be applicable to a variety of genes that are expressed in pests, including, for example, endogenous genes responsible for cellular metabolism or cell transformation, including housekeeping genes; transcription factors; molting-related genes; and other coding A gene involved in the metabolism of a cell or a polypeptide that normally grows and develops.

In order to transcribe from a living organism or a transgene expressing a construct, in some embodiments a regulatory region (eg, a promoter, an enhancer, a silencer, and a polyadenylation signal) can be used to transcribe the RNA strand (or Wait). Thus, in some embodiments, as set forth above, a polynucleotide for use in the production of an iRNA molecule may be operably linked to one or more promoter elements that function in a plant host cell. The promoter may be an endogenous promoter, usually resident in the host genome. The polynucleotides of the present invention, under the control of the promoter elements of the manipulation linkage, may further flank additional elements that advantageously affect their transcription and/or stability of the resulting transcript. Such an element may be located upstream of the manipulative link promoter, downstream of the 3' end of the display construct, and may occur both upstream of the promoter and downstream of the 3' end of the performance construct.

Some embodiments provide a method for reducing damage to a host plant (eg, a corn plant) caused by a pest of a feeding plant (eg, a coleoptera), wherein the method comprises providing a host plant in the host plant a transgenic plant cell of at least one nucleic acid molecule, wherein the nucleic acid molecule acts to inhibit the performance of a targeted polynucleotide in the pest after being taken by the pest, the expression inhibiting the mortality of the pest and / or reduced growth, thereby reducing the damage caused by the pest to the host plant. In some embodiments, the nucleic acid molecule comprises a dsRNA molecule. In these and further embodiments, the nucleic acid molecule comprises a dsRNA molecule, wherein each of the dsRNA molecules comprises more than one polyribonucleotide that specifically hybridizes to a nucleic acid molecule expressed in a coleopteran pest cell. In some embodiments, the nucleic acid molecule consists of a polynucleotide, wherein the polynucleotide specifically hybridizes to a nucleic acid molecule expressed in an insect pest cell.

In some embodiments, a method for increasing the yield of a crop, such as a corn crop and a Brassica napus crop, is provided, wherein the method comprises introducing at least one polynucleotide-containing nucleic acid molecule of the invention into a plant; cultivating the plant An iRNA molecule expression from the polynucleotide is allowed, wherein the 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 further embodiments, the dsRNA molecules can each comprise more than one polyribonucleotide that specifically hybridizes to a nucleic acid molecule expressed in an insect pest cell. Thus, one or more nucleotide sequences within a polynucleotide of the invention may exhibit a specific polyribonucleotide of a dsRNA molecule.

In some embodiments, a method for regulating the performance of a target gene in an insect pest is provided, the method comprising: transforming a plant cell with a vector comprising a polynucleotide, wherein the polynucleotide encodes At least one iRNA molecule of the invention, wherein the polynucleotide is operably linked to a promoter and a transcription termination element; the transformation is cultured under conditions sufficient to permit development of a plant cell culture comprising a plurality of transformed plant cells a plant cell; selecting a transformed plant cell that has integrated the polynucleotide into its genome; screening the transformed plant cell that exhibits the iRNA molecule encoded by the integration polynucleotide; selecting a gene that expresses the iRNA molecule Plant cells; and feeding the selected gene transfer plant cells to the insect pest. The plant may also be regenerated from a gene transfer plant cell that expresses the iRNA molecule encoded by the integrated nucleic acid molecule. In some embodiments, the iRNA is a dsRNA molecule comprising a polyribonucleotide that specifically hybridizes to a target gene within an insect pest cell. In these and further embodiments, the dsRNA molecules each comprise more than one polyribonucleotide transcribed from a nucleotide sequence within a polynucleotide encoding the dsRNA molecule.

The iRNA molecules of the invention may be incorporated into the seeds of a plant species (eg, corn and rapeseed), either as a product derived from the expression of a recombinant gene incorporated into the genome of a plant cell, or incorporated prior to planting Paint or seed treatment applied to the seed. A plant cell line comprising a recombinant gene is considered a genetically modified product. Also included in specific embodiments of the invention are delivery systems for delivering iRNA molecules to insect (e.g., coleopteran) pests. For example, the iRNA molecules of the invention can be introduced directly into the cells of a pest. The introduced method may comprise directly mixing the iRNA to a plant tissue derived from an insect pest host, and administering a composition comprising the iRNA molecule of the invention to the host plant tissue. For example, iRNA molecules can be sprayed onto the surface of plants. Alternatively, an iRNA molecule may be represented by a microorganism and the microorganism may be applied to the surface of the plant or introduced into the root or stem by physical means such as injection. As discussed above, a genetically transformed plant can also be genetically engineered to exhibit at least one iRNA molecule sufficient to kill the number of insect pests known to infest the plant. IRNA molecules produced by chemical or enzymatic synthesis may also be formulated in a manner consistent with general agricultural practices and used as a spray or bait product for controlling plant damage caused by insect pests. The formulation may include appropriate adjuvants (eg, stickers and moisturizers) required for effective foliar coverage, as well as UV protectants to protect iRNA molecules (eg, dsRNA molecules) from UV damage. Such additives are common in the biopesticide industry and are well known to those skilled in the art. This application can be combined with other spray insecticide applications (based on biology or other means) to enhance plant protection from pest damage.

All of the references, including publications, patents, and patent applications, are hereby incorporated by reference in their entireties in the entireties in The documents pointed out are fully incorporated into the case for reference. The references discussed herein are provided solely for disclosure prior to the filing date of the present application. The disclosure herein is not to be construed as limiting the invention by the present invention.

The following examples are provided to illustrate certain features and/or aspects. The examples are not to be construed as limiting the invention to the particular features or aspects described. EXAMPLES Example 1 : Materials and Methods

Many dsRNA molecules (including those corresponding to prp8-1 reg1 (SEQ ID NO: 7), prp8-2 reg1 (SEQ ID NO: 8), prp8-3 reg1 (SEQ ID NO: 9), prp8-3 v1 (sequence Identification number: 10) and prp8-3 v2 (SEQ ID NO: 11), and using the MEGASCRIPT ^® T7 RNAi kit (LIFE TECHNOLOGIES, Carlsbad, CA) or T7 rapid high yield RNA synthesis kit (NEW ENGLAND BIOLABS, Whitby, Purified and purified. The purified dsRNA molecule was prepared in TE buffer and all bioassays contained a control consisting of this buffer, which served as WCR ( Diabrotica virgifera virgifera LeConte) Background check for mortality or growth inhibition. The concentration of dsRNA molecules in this bioassay buffer was measured using a NANODROPTM 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington, DE).

The insect activity of the samples was tested in a bioassay with fresh insect larvae on an artificial insect diet. The egg line of WCR was obtained from CROP CHARACTERISTICS, INC. (Farmington, MN).

Bioanalysis was performed in a 128 well plastic tray designed specifically for insect bioanalysis (CD INTERNATIONAL, Pitman, NJ). Each well contains approximately 1.0 mL of an artificial diet designed for the growth of coleopteran insects. A 60 μL aliquot of the dsRNA sample was delivered by pipette to the diet surface of each well (40 μL/cm ² ). The dsRNA sample concentration was calculated as the number of dsRNA per square centimeter of surface area (1.5 cm ² ) in the well (ng/cm ² ). The treated well plate is maintained in a fume hood until the liquid on the surface of the diet evaporates or is absorbed into the diet.

Within a few hours of emergence, individual larvae are picked up with a wet camel hair brush and placed on a treated diet (one or two larvae per well). Then, the transparent plastic bonding sheet is used to seal the intrusion hole of the 128-well plastic disk, and the hole is opened to allow gas exchange. The bioassay panel was maintained under controlled environmental conditions (28 °C, ~40% relative humidity, 16:8 (light: dark)) for 9 days, after which the total number of insects exposed to each sample was recorded and died. The number of insects and the weight of surviving insects. The mean percentage of mortality and average growth inhibition for each treatment were calculated. Growth inhibition (GI) is calculated as follows: GI = [1 - (TWIT/TNIT) / (TWIBC/TNIBC)], where TWIT is the total weight of live insects in the treatment; TNIT is the insect in the treatment Total; TWIBC is the total weight of live insects in the background check (buffer control); and TNIBC is the total number of insects in the background check (buffer control).

Statistical analysis using the JMP ^TM system software (SAS, Cary, NC) were.

LC ₅₀ (lethal concentration) is defined as the dose at which lines are killed 50% of the test insects. GI ₅₀ (growth inhibition) is defined as the average growth (e.g., live weight) of the test insect is the dose of 50% of the average value seen by the background test sample.

Repeated bioassays demonstrated that ingestion of specific samples resulted in surprising and unexpected mortality and growth inhibition of corn rootworm larvae. Example 2 : Identification of Candidate Targeted Genes

Insect lines from multi-stage development of WCR ( Diabrotica virgifera virgifera LeConte) were selected for pooled transcriptome analysis to provide candidate target gene sequences for RNAi gene transfer plant insect protection Control of technology.

In one example, total RNA system from about 0.9 grams whole first-instar WCR larvae; (4-5 days after hatching, was maintained at 16 deg.] C) to be isolated, using the following phenol / TRIREAGENT ^® based approach (MOLECULAR RESEARCH CENTER, Cincinnati, OH) to be purified:

Larvae based at room temperature 15 mL of a homogenizer to 10 mL TRI REAGENT ^® homogenized until a uniform suspension. After incubation at room temperature for 5 minutes, the homogenate was dispensed into a 1.5 mL microcentrifuge tube (1 mL per tube), 200 μL of chloroform was added, and the mixture was shaken vigorously for 15 seconds. After allowing the extract to stand at room temperature for 10 minutes, the phases were separated by centrifugation at 12,000 x g at 4 °C. The upper phase (containing approximately 0.6 mL) was carefully transferred to another sterilized 1.5 mL tube and an equal volume of room temperature isopropanol was added. After incubation for 5 to 10 minutes at room temperature, the mixture was centrifuged at 12,000 x g for 8 minutes (4 ° C or 25 ° C).

The supernatant was carefully removed and discarded, while the RNA pellet was washed twice by vortexing with 75% ethanol and recovered by centrifugation at 7,500 xg for 5 minutes (4 ° C or 25 ° C) after each wash. The ethanol was carefully removed and the precipitate allowed to air dry for 3 to 5 minutes and then dissolved in nuclease-free sterile water. The RNA concentration was determined by measuring the absorbance (A) at 260 nm and 280 nm. A typical extraction yields more than 1 mg of total RNA from approximately 0.9 gm of larvae with an A ₂₆₀ /A ₂₈₀ ratio of 1.9. The RNA thus extracted was stored at -80 ° C until further processing.

RNA quality was determined by spreading an aliquot through a 1% agarose gel. The agarose gel solution was autoclaved with 10x TAE buffer (Tris-acetic acid EDTA; 1x concentration of 0.04 M Tris-acetic acid, 1 mM EDTA (sodium salt of ethylenediaminetetraacetic acid), pH 8.0), DEPC (diethyl pyrocarbonate)-treated water is prepared by dilution in a autoclaved vessel. Use 1x TAE as the expansion buffer. Prior to use, the electrophoresis groove and a hole formed in a comb-based ^{RNAseAway TM (INVITROGEN INC., Carlsbad} , CA) for cleaning. 2 μL of RNA sample with 8 μL of TE buffer (10 mM Tris HCl, pH 7.0; 1 mM EDTA) and 10 μL of RNA sample buffer (Novagen ^® Cat. No. 70606; EMD4 Bioscience, Gibbstown, NJ) mixing. The sample was heated at 70 ° C for 3 minutes, cooled to room temperature, and loaded with 5 μL per well (containing 1 μg to 2 μg of RNA). Commercially available RNA molecular weight markers are simultaneously deployed in separate wells for molecular size comparison. The gel was developed at 60 volts for 2 hours.

A standardized cDNA library was prepared from larval total RNA by a commercial service provider (EUROFINS MWG Operon, Huntsville, AL) using random priming. Normalized cDNA library based on larvae backsheet 1/2 scale (plate scale), by GS FLX EUROFINS MWG Operon sequencer 454 Titanium ^TM chemistry in series, by generating the read and more than 600,000 of average read length of 348 bp. The 350,000 reading system was assembled into a segment contig of more than 50,000. Both unassembled reads and fragment contigs are converted to BLASTable databases using publicly available programs, FORMATDB (available from NCBI).

Total RNA and standardized cDNA libraries were also prepared from materials harvested from other WCR developmental stages. A pool of transcriptomics libraries for targeted gene screening is constructed by combining cDNA library members representing various developmental stages.

RNAi-targeted candidate gene lines are assumed to be essential for the survival and growth of pest insects. Selected target gene homologs are identified in the transcript sequence database as described below. The full length or partial sequence of the target genes is amplified by PCR to prepare a template for the production of double stranded RNA (dsRNA).

The TBLASTN search using candidate protein coding sequences was performed on a BLASTable database containing unassembled Diabrotica sequence reads or assembled contigs. Significant hits on the sequence of the leaf (defined as a contig of contigs of the contig is better than e ^-20 , and reading homologs for unassembled sequences is better than e ^-10 ) using BLASTX for NCBI non-redundant Non-redundant database confirmation. The results of this BLASTX search confirmed that the sequence of the melon homolog candidate gene identified in the TBLASTN search did contain the leaf methylation or the best hit for the non-leaf candidate gene sequence for the leaf sequence. In most cases, the Tribolium candidate gene is annotated as a protein encoding a sequence, or a sequence or sequence in the transcript sequence of the leaf plexus. In a few cases, it is apparent that some of the phylum or unassembled sequence reads selected by homologous to a non-leaf candidate gene are overlapped, and the assembly of the contig is joined. These overlaps have failed. In such cases, using ^{Sequencher TM v4.9 (GENE CODES CORPORATION,} Ann Arbor, MI) to assemble fragments of such longer sequences become contigs.

Several candidate target gene lines encoding Diabrotica prp8 (SEQ ID NO: 1 and Sequence ID: 3) were identified as likely to cause coleopteran mortality, growth inhibition, developmental inhibition and/or The gene for food inhibition.

Fruit flies (Drosophila) prp8 transgenic lines consisting of: one kind of amine end NusG (NGN) and one C terminal art Kyprides-Onzonis-Woese (KOW) field, a dual function as a transcriptional regulator, which functions as a positive and negative The extension factor of the direction. The NGN domain of prp8 binds to RNAP, while the KOW domain complements additional transcriptional regulators to RNAP. The KOW field in eukaryotes is thought to complement a large number of transcription factors. In addition, prp8 can also be involved in the regulation of pre-mRNA processing due to its interaction with capping enzymes. Together with the small zinc finger protein SPT4 (inhibitor of Ty 4), prp8 constructs the heterodimeric complex DSIF (DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) ) sensitivity-inducing factor).

The sequence identification number: 1 and the sequence identification number: 3 sequence are novel. The sequence is not provided in the public database, and PCT International Patent Publication No. WO/2011/025860; U.S. Patent Application No. 20070124836; U.S. Patent Application No. 20090306189; U.S. Patent Application No. US20070050860; U.S. Patent Application No. No. 20,100, 192, 265; U.S. Patent No. 7,612,194; or U.S. Patent Application Serial No. 2013-192256. WCR prp8-1 (SEQ ID NO: 1) is somewhat related to sequence fragments derived from Tribolium castaneum (GENBANK Accession No. XM_961838.2). WCR prp8-2 (SEQ ID NO: 3) is somewhat related to sequence fragments derived from Oryctolagus cuniculus (GENBANK Accession No. NM_144353.4). The closest homolog of the WCR PRP8-1 amino acid sequence (SEQ ID NO: 2) is a pseudo-German protein with GENBANK accession number XP_966931.1 (99% similar in the homologous region; 98% identical) ). The closest homolog of the WCR PRP8-2 amino acid sequence (SEQ ID NO: 4) is a Gregarina niphandrodes protein with GENBANK accession number XP_011131272.1 (85% similar in the homologous region; 76) %same).

The Prp8 dsRNA transgene can be combined with other dsRNA molecules to provide redundant RNAi targeting as well as synergistic RNAi effects. Gene-transgenic maize products that exhibit dsRNA targeting prp8 are useful for preventing root feeding damage in corn rootworms. The Prp8 dsRNA transgene represents a new mode of action that combines the insecticidal protein technology of Bacillus thuringiensis with Insect Resistance Management gene pyramids to make these rootworm control techniques resistant. The development of the sexual insect population has eased. Example 3 : Amplification of Targeted Genes to Produce dsRNA

The leaf candidate gene sequence, referred to herein as prp8 , is used to generate PCR amplifications for dsRNA synthesis. The primer system is designed to amplify a coding region of a portion of each of the targeted genes by PCR. See Table 1 . If appropriate, a T7 phage promoter sequence (TTAATACGACTCACTATAGGGAGA; SEQ ID NO: 13) is incorporated into the sense of amplification or the 5' end of the antisense strand. See Table 1 . Total RNA was extracted from WCR using TRIzol ^® (Life Technologies, Grand Island, NY), and the first strand of cDNA was fabricated using the SuperScript III ^® First Synthesis System and the manufacturer's oligo dT priming protocol (Life Technologies, Grand Island, NY). . The first strand of cDNA was used as a template for a PCR reaction using an opposite position primer to amplify all or part of the naturally-targeted gene sequence. The dsRNA is also amplified from a DNA selection body comprising a coding region for yellow fluorescent protein (YFP) (SEQ ID NO: 14; Shagin et al. (2004) Mol. Biol. Evol. 21 (5) ): 841-50).

Table 1. Primers and primer pairs used to amplify portions of the coding region of the exemplary prp8 target gene and the YFP negative control gene. Example 4 : RNAi constructs

Templates and dsRNA synthesis were prepared by PCR. A strategy used to provide specific templates for the production of prp8 and YFP dsRNA is shown in Figure 1 . The template DNA intended for use in the synthesis of prp8 dsRNA was prepared by PCR using the primer pair in Table 1 , and the first cDNA line (as a PCR template) was from the WCR egg, the first instar larva or the adult. The isolated total RNA was prepared. For each selected prp8 and YFP target gene region, PCR amplification introduces a T7 promoter sequence at the 5' end of the amplified sense strand and the antisense strand ( YFP segment is a DNA clone from the YFP coding region) Amplify). Two PCR amplified fragments of each region of the targeted gene are mixed in approximately equal amounts, and the mixture is used as a template for dsRNA production. See Figure 1. The sequence of the amplified dsRNA template with a specific primer pair is: sequence identification number: 7 ( prp8-1 reg1), sequence identification number: 8 ( prp8-2 reg1), sequence identification number: 9 ( prp8-3 reg1), sequence Identification number: 10 ( prp8-3 v1), sequence identification number: 11 ( prp8-3 v2), and sequence identification number: 14 (YFP). Double-stranded RNA using the AMBION ^® MEGASCRIPT ^® RNAi kit, following the manufacturer's (INVITROGEN) protocol, or the HiScribe ^® T7 in vitro transcription kit, following the manufacturer's (New England Biolabs, Ipswich, MA) guidelines. It is synthesized and purified. dsRNA concentrations were measured using a NANODROPTM 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington; DE).

Construction of plant-transformed vectors. The input vector was assembled using a combination of chemically synthesized fragments ( DNA 2.0 , Menlo Park, CA) and standard molecular selection methods containing a segment containing prp8 (SEQ ID NO: 1 and Sequence ID: 3). Targeted gene constructs formed by hairpins. The intramolecular hairpin formation of the RNA primary transcript is facilitated by arranging the two copies of the prp8 target gene segments in opposite orientations (in a single transcription unit), the two segments being linked by a linker Polynucleotides (for example, the ST-LS1 intron; Vancanneyt et al. (1990) Mol. Gen. Genet. 220(2): 245-50) are separated. Thus, the primary mRNA transcript contains two sequences of prp8 gene segments separated by the intron sequences as large inverted repeats. The primary mRNA hairpin transcript is produced by a copy of the maize ubiquitin 1 promoter (U.S. Patent 5,510,474) and contains a 3' non-translated region from the maize peroxidase 5 gene. Fragments (ZmPer5 3' UTR v2; U.S. Patent No. 6,699,984) were used to terminate transcription of hairpin-RNA-expressing genes.

Negative control comprising a hairpin dsRNA YFP gene expression binary vector system with a typical binary input vector and the target vector, by standard GATEWAY ^® recombination reaction to construct.

The binary target vector comprises a herbicide tolerance gene (aryloxyalknoate dioxygenase; AAD-1 v3) (U.S. Patent 7,838,733 (B2), and Wright et al. (2010) Proc. Natl. Acad. Sci. USA 107:20240-5), which is in a sugarcane bacilliform badnavirus (ScBV) promoter (Schenk et al. (1999) Plant Molec. Biol. 39:1221- 30, under the regulation of a 5'UTR derived from the maize stripe virus (MSV) coat protein gene and a synthesis of the intron 6 sequence derived from the maize alcohol dehydrogenase 1 (ADH1) gene 5' a UTR sequence between the 3' end of the SCBV promoter segment and the start codon of the AAD-1 coding region. A fragment comprising a 3' non-translated region derived from the maize lipase gene (ZmLip 3'UTR; U.S. Patent 7,179,902) is used to terminate the transcription of AAD-1 mRNA.

One additional negative control binary vector comprising a gene expression YFP protein, with a typical binary-based targeting vector and the input vector, by standard GATEWAY ^® recombination reaction to construct. The binary target vector comprises a herbicide tolerance gene (aryloxyalknoate dioxygenase; AAD-1 v3) (described above) which is in maize ubiquitin 1 The promoter (as described above) and the expression derived from the fragment of the 3' non-translated region of the maize lipase gene (ZmLip 3'UTR; as described above) are regulated. The input vector comprises a YFP coding region (SEQ ID NO: 27) which is in the maize ubiquitin 1 promoter (described above) and 3' derived from the maize peroxidase 5 gene. The performance of the fragment of the non-translated region (as described above) is under control. Example 5 : Screening of candidate target genes

Synthetic dsRNA designed to inhibit the target gene sequence identified in Example 2, when administered to WCR in a diet-based assay, causes mortality and growth inhibition.

Repeated bioassays demonstrated that uptake of dsRNA preparations derived from prp8-2 reg1, prp8-2 v1 and prp8-2 v2 resulted in mortality and growth inhibition in western corn rootworm larvae. Table 2 shows the results of a diet-based feeding bioassay of WCR larvae following exposure to prp8-2 reg1, prp8-2 v1 and prp8-2 v2 dsRNA for 9 days, and from the yellow fluorescent protein (YFP) coding region ( Sequence Identification Number: 27) Results obtained for the negative control dsRNA sample prepared. Table 3 shows the results of LC ₅₀ and GI ₅₀ exposure to prp8-2 v1 and prp8-2 v2 dsRNA.

Table 2. Results of a feeding bioassay based on the prp8 dsRNA diet obtained from western corn rootworm after 9 days of feeding. ANOVA analysis found significant differences in mean mortality and mean growth inhibition (GI)%. The average value was isolated using the Tukey-Kramer test. *SEM = mean standard error. The letters in parentheses indicate the statistical level. The levels that were not joined by the same letter were significantly different (P < 0.05). **TE = Tris HCl (1 mM) plus EDTA (0.1 mM) buffer, pH 7.2. ***YFP = yellow fluorescent protein

Table 3. Summary of oral titers of prp8 dsRNA for WCR larvae (ng/cm ² ).

It has previously been suggested that certain genes of the genus Diabrotica spp. can be used for insect control of RNAi vectors. See U.S. Patent Publication No. 2007/0124836, which is incorporated herein by reference. However, it has been determined that many genes that are useful for insect control of RNAi-mediated are not effective in controlling Diabrotica . It was also determined that the sequences prp8-2 reg1, prp8-2 v1 and prp8-2 v2 dsRNA provided surprising and unexpected leaves compared to other genes that have utility for RNAi-mediated insect control. A superior control.

For example, it is suggested in U.S. Patent No. 7,612,194 that each of annexin, β-erythrocytic membranous protein 2 and mtRP-L4 is effective in insect control of RNAi vectors. Sequence identification number: 28 is the DNA sequence of annexin region 1 (Reg 1) and sequence identification number: 29 is the DNA sequence of annexin region 2 (Reg 2). Sequence identification number: 30 is the DNA sequence of β-erythrocyte globule skeleton protein 2 region 1 (Reg 1) and sequence identification number: 31 is the DNA sequence of β-erythrocyte globule skeleton protein 2 region 2 (Reg 2). Sequence identification number: 32 is the DNA sequence of mtRP-L4 region 1 (Reg 1) and sequence identification number: 33 is the DNA sequence of mtRP-L4 region 2 (Reg 2). A YFP sequence (SEQ ID NO: 14) was also used to make dsRNA as a negative control.

Each of the aforementioned sequences was used to produce dsRNA via the method of Example 3. The strategy used to provide specific templates for dsRNA production is shown in Figure 2 . The template DNA intended for use in dsRNA synthesis was prepared by PCR using the primer pairs in Table 4 , and (as a PCR template) the first cDNA was prepared from the total RNA isolated from the first instar larvae of WCR. . (YFP is amplified from DNA colonies.) Two separate PCR amplifications were performed for each selected target gene region. The first PCR amplification introduced a T7 promoter sequence at the 5' end of the amplified sense strand. The second reaction was incorporated into the T7 promoter sequence at the 5' end of the antisense strand. The two PCR amplified fragments of each region of the targeted gene are then mixed in approximately equal amounts, and the mixture is used as a transcription template for dsRNA production. See Figure 2. The double-stranded RNA was synthesized and purified using the AMBION ^® MEGAscript ^® RNAi kit according to the manufacturer's instructions (INVITROGEN). dsRNA concentrations were measured using a NANODROPTM 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington; DE), and dsRNAs were each tested using the same diet-based bioassay described above. Table 4 lists the use of annexin Reg 1 , annexin Reg 2, β-erythrocyte membrane protein 2 Reg 1, β-erythrocytic membrane protein 2 Reg 2, mtRP-L4 Reg 1, mtRP-L4 Reg 2 , and the primer sequence of the YFP dsRNA molecule. Table 5 presents the results of a diet-based feeding bioassay of WCR larvae following exposure to these dsRNAs for 9 days. Repeated bioassays demonstrated that the mortality or growth inhibition of western corn rootworm larvae caused by the uptake of these dsRNAs did not exceed the mortality of western corn rootworm larvae seen on control samples of TE buffer, water or YFP protein or Growth inhibition.

Table 4. Primers and primer pairs used to amplify a portion of the coding region of a gene.

Table 5. Dietary feeding analysis results obtained after 9 days of western corn rootworm larvae. *TE = Tris HCl (10 mM) plus EDTA (1 mM) buffer, pH 8. **YFP=Yellow Fluorescent Protein Example 6 : Production of a gene containing insecticidal dsRNA into a maize tissue

Transformation of Agrobacterium media. After transformation of the Agrobacterium vector, the gene is transformed into maize cells, tissues and plants by the expression of a chimeric gene stably integrated into the plant genome, and the gene is transformed into corn cells, tissues and plants to produce a Or a plurality of insecticidal dsRNA molecules (for example, at least one dsRNA molecule comprising a dsRNA molecule targeting a gene comprising prp8 (eg, Sequence ID: 1 and Sequence ID: 3)). U.S. Patent No. 8,304,604, the entire disclosure of which is incorporated herein by reference. The transformed tissue is selected by its ability to grow on haloxyfluoride-containing medium and is suitably screened for dsRNA production. A portion of such a transformed tissue culture can be presented to a newborn corn rootworm larva for bioanalysis, essentially as described in Example 1.

Agrobacterium culture was initiated. The glycerol stocks containing the Agrobacterium strain DAt13192 cells (PCT International Publication No. WO 2012/016222A2) containing the binary transformation vector as described above (Example 4) were streaked into AB minimal medium containing appropriate antibiotics. On a flat plate (Watson et al. (1975) J. Bacteriol. 123: 255-264) and grown at 20 °C for 3 days. The culture was then streaked into YEP plates (gm/L: yeast extract, 10; peptone, 10; NaCl, 5) containing the same antibiotic, and the plates were incubated for 1 day at 20 °C.

Agrobacterium culture. On the day of the experiment, a stock solution of the inoculum medium in the appropriate number of constructs and acetosyringone were prepared in the experiment and pipetted into a sterile, disposable 250 mL flask. Inoculation medium (Frame et al. (2011) Genetic Transformation Using Maize Immature Zygotic Embryos . IN Plant Embryo Culture Methods and Protocols: Methods in Molecular Biology. TA Thorpe and EC Yeung, (Eds), Springer Science and Business Media, LLC. pp 327-341) contains: 2.2 gm/L MS salt; 1X ISU modified MS vitamin (Frame et al., supra) 68.4 gm/L sucrose; 36 gm/L glucose; 115 mg/L L-proline; And 100 mg/L myo-inositol; at pH 5.4). Ethyl syringone in a 1 M stock solution of 100% dimethyl hydrazine was added to a flask containing the inoculating medium to a final concentration of 200 μM, and the solution was thoroughly mixed.

For each construct, agrobacterium from 1 or 2 inoculation loops from the YEP plate was suspended in a 15 mL inoculation medium/acetone syringone stock solution in a sterile, disposable 50 mL centrifuge tube. A spectrophotometer was used to measure the optical density (OD ₅₅₀ ) of the solution at 550 nm. The suspension mixture was then uses acetosyringone additional inoculation medium / acetyl, diluted to OD ₅₅₀ 0.3 to 0.4. The tubes of the Agrobacterium suspension were then placed horizontally on a platform shaker, set at room temperature for approximately 75 rpm, and shaken for 1 to 4 hours while performing embryo stripping.

Ear sterilization and embryo isolation. Maize immature embryo line is a Zea mays plant self-bred line B104 grown from a greenhouse and self-pollinated or sib-pollinated to produce ear (Hallauer et al. (1997) Crop Science 37:1405-1406) to get. Ears are harvested approximately 10 to 12 days after pollination. On the day of the experiment, the surface of the exfoliated ear was sterilized by immersing in 20% commercial sodium hypochlorite solution (ULTRA CLOROX® Germicidal Bleach, 6.15% sodium hypochlorite; plus two drops of TWEEN 20) and shaking for 20 to 30 minutes. It was then rinsed three times with sterile deionized water in a fume hood. Immature zygotic embryos (1.8 to 2.2 mm long) were aseptically stripped from each ear and randomly assigned to a microcentrifuge tube containing 2.0 mL of liquid inoculating medium and 200 μM acetosyringone A suitable Agrobacterium cell suspension was added to 2 μL of 10% BREAK-THRU ^® S233 surfactant (EVONIK INDUSTRIES; Essen, Germany) into the tube. In a specific set of experiments, embryos derived from pooled ears were used for each transformation.

Agrobacterium is co-cultured. After inoculation, the embryos were placed on a swaying platform for 5 minutes. The contents of the tube were then poured onto a co-cultivation medium containing 4.33 gm/L of MS salt; 1X ISU modified MS vitamin; 30 gm/L sucrose; 700 mg/L L-脯Acid; 3.3 mg/L of Dicamba in KOH (3,6-dichloro-o-arasic acid or 3,6-dichloro-2-methoxybenzoic acid); 100 mg /L of myoinositol; 100 mg/L of casein hydrolysate; 15 mg/L of AgNO ₃ ; 200 μM of acetal syringone in DMSO; and 3 gm/L of GELZAN ^TM at pH 5.8 . The liquid Agrobacterium suspension is moved using a sterile, disposable pipette. The embryos are then oriented with the help of sterile forceps and a microscope with the scutellum facing up. The plate was covered with a 3MTM MICROPORETM medical tape and placed in a continuous light of approximately 60 μmol m ^-2 s ^-1 of photosynthetically active radiation (PAR) in a 25 ° C incubator.

Screening and regeneration of healing tissue of genetically modified pieces. After the period of co-cultivation, the germ was transferred to a dormant medium containing 4.33 gm/L of MS salt; 1X ISU modified MS vitamin; 30 gm/L of sucrose; 700 mg/L of L-valine; 3.3 mg/L Dicamba in KOH; 100 mg/L myoinositol; 100 mg/L casein hydrolysate; 15 mg/L AgNO ₃ ; 0.5 gm/L MES (2- (N- morpholino) ethanesulfonic acid monohydrate; PHYTOTECHNOLOGIES LABR;. Lenexa, KS ); 250 mg / L of the card of the present amoxicillin (Carbenicillin); and 2.3 gm / L of GELZAN ^TM; pH 5.8. Move no more than 36 embryos to each flat. Place the plate in a clear plastic box and incubate at 27 °C for 7 to 10 days with a continuous light of approximately 50 μmol m ^-2 s ^-1 PAR. The healed tissue embryos were then transferred to selection medium I (<18 cells/flat) and the selection medium I was prepared from resting medium (above) with 100 nM R-chlorofluoric acid (0.0362 mg/L; The composition of the healing tissue containing the AAD-1 gene was selected. The plate was placed back in a clear box and incubated at 27 °C for 7 days with continuous light of approximately 50 μmol m ^-2 s ^-1 PAR. The healed tissue embryos were then transferred to selection medium II (<12 cells/flat) and the selection medium II consisted of resting medium (above) and 500 nM R-chlorofluoric acid (0.181 mg/L). The flat plate was placed back in a clear box and incubated at 27 °C for 14 days with continuous light of approximately 50 μmol m ^-2 s ^-1 PAR. This selection step allows the gene to be transferred to the healing tissue for further proliferation and differentiation.

The proliferating embryogenic healing tissue was transferred to pre-regeneration medium (<9/flat). Pre-regenerated medium containing 4.33 gm/L MS salt; 1X ISU modified MS vitamin; 45 gm/L sucrose; 350 mg/L L-valine; 100 mg/L myoinositol; 50 mg /L casein hydrolysate; 1.0 mg/L of AgNO ₃ ; 0.25 gm/L of MES; 0.5 mg/L of naphthaleneacetic acid in NaOH; 2.5 mg/L of absicic acid in ethanol ; 1 mg / L of 6-benzyladenine (6-benzylaminopurine); 250 mg / L of the card of the present amoxicillin (Carbenicillin); 2.5 gm / L of GELZAN ^TM; and 0.181 mg / L laminated chlorofluorinated acid; the pH It is 5.8. The plates were stored in clear plastic boxes and incubated at 27 °C for 7 days at a sustained light of approximately 50 μmol m ^-2 s ^-1 PAR. The regenerated healing tissue is then transferred (<6/flat) to the PHYTATRAYSTM (SIGMA-ALDRICH) regeneration medium at 28 ° C and 16 hours per day bright / 8 hours dark (at approximately 160 μmol m ^-2 s ^-1 PAR) was incubated for 14 days or until stems and roots developed. The regeneration medium contains 4.33 gm/L of MS salt; 1X ISU modified MS vitamin; 60 gm/L of sucrose; 100 mg/L of myoinositol; 125 mg/L of Carbenicillin; 3 gm/ L is GELZAN ^TM gum; and 0.181 mg / L of R- engagement chlorofluorinated acid; the pH was 5.8. The small shoots with primary roots are then isolated and transferred to elongation medium without selection. Elongation medium containing 4.33 gm / L of MS salts; 1X ISU modified MS Vitamins; 30 gm / L of sucrose; and 3.5 gm / L of GELZAN ^TM; pH 5.8.

The transformed plant buds selected by their ability to grow on chlorofluorocarbon-containing medium are transplanted from PHYTATRAYSTM to a small pot filled with growth medium (PROMIX BX; PREMIER TECH HORTICULTURE), covering the cup or HUMI-DOMES (ARCO) PLASTICS), then seedlings were hardened-off in a CONVIRON growth chamber (daytime 27 °C / night 24 °C, 16 hour photoperiod, 50-70% RH, 200 μmol m ^-2 s ^-1 PAR). In some instances, putative gene transfer seedlings were analyzed for the number of transgene relative replicates by real-time quantitative PCR analysis using primers designed to detect the AAD1 herbicide tolerance gene integrated into the maize genome. Furthermore, RNA qPCR analysis was used to detect the presence of linker sequences within the dsRNAs of the putative transforms. The selected transformed seedlings were then transferred to a greenhouse for further growth and testing.

Transfer and the establishment of T ₀ plants in the greenhouse for seed production and biological analysis. When the plants reach the V3-V4 stage, they are transplanted to the IE CUSTOM BLEND (PROFILE/METRO MIX 160) soil mixture and grown in the greenhouse for flowering (light exposure type: Photo or Assimilation; glare) High Light Limit: 1200 PAR; 16-hour day length; daytime 27 °C/night 24 °C).

Insect bioassay for the plant lines transplanted into small pots ^{TINUS TM 350-4 ROOTRAINERS ® (PENCER-} LEMAIRE INDUSTRIES, Acheson, Alberta, Canada;) ( one plant per ROOTRAINERS ^® products a member). After about four days transplanted into ROOTRAINERS ^®, infested plant lines used for bioassay.

Plants of the T ₁ generation are subjected to pollination of T ₀ gene-transferred plants by pollen collected from non-genetic elite (Elite) self-bred lines B104 plants or other appropriate pollen donors, and planted. Obtained by the seeds. Backflushing can be performed when possible. Example 7 : Molecular analysis of genetically transformed maize tissues

Molecular analysis of maize tissue (eg, RNA qPCR) was performed on samples derived from leaves and roots, and samples of leaves and roots were collected from greenhouse grown plants on the same day that root damage was assessed.

The results of RNA qPCR analysis of Per5 3'UTR were used to confirm the performance of the hairpin transgene. (Non-transformed maize plants are expected to detect low-level Per5 3'UTR because of the endogenous Per5 gene expression in the maize tissue.) Intervening sequences between repeats in the expressed RNAs (which are responsible for the formation of dsRNA) RNA qPCR analysis results in which the molecule is indispensable is used to confirm the presence of hairpin transcripts. The performance level of the transgenic RNA was measured compared to the RNA level of an endogenous maize gene.

DNA qPCR analysis to detect a portion of the AAD1 coding region in gDNA was used to estimate the number of inserted copies of the transgene. Samples of these analyses were collected from plants grown in the environmental chamber. The results were compared to DNA qPCR analysis designed to detect a portion of a single copy of the native gene, and simple artifacts (one or two copies of the prp8 transgene) were used for further greenhouse studies.

In addition, qPCR analysis designed to detect a portion of the spectinomycin resistance gene ( SpecR ; a binary vector plastid present outside the T-DNA) is used to determine whether the gene transfer plant contains Externally integrated plastid backbone sequence.

RNA transcript expression level: Per 5 3'UTR qPCR. Healing cell lines or gene transfer plants are analyzed by real-time quantitative PCR (qPCR) of the Per 5 3'UTR sequence to determine the relative expression level of full-length hairpin transcripts compared to an internal maize gene (example) For the transcript level of GENBANK Accession No. BT069734), the latter encodes a TIP41-like protein (ie, a maize homolog of GENBANK Accession No. AT4G34270; a tBLASTX score with 74% identity; Sequence ID: 62). RNA was isolated using the RNeasyTM 96 kit (QIAGEN, Valencia, CA). After rinsing, total RNA was subjected to DNAse1 treatment according to the protocol recommended by the kit. RNA was then quantified on a NANODROP 8000 spectrophotometer (THERMO SCIENTIFIC) and the concentration was normalized to 25 ng/μL. The first cDNA was prepared using a high capacity cDNA synthesis kit (INVITROGEN), essentially 5 μL of denatured RNA in a 10 μL reaction volume, according to the manufacturer's recommended protocol. The protocol was slightly modified to include the addition of 10 μL of 100 μM T20VN oligonucleotide (IDT) (TTTTTTTTTTTTTTTTTTTTVN, where V is A, C or G, and N is A, C, G or T; Sequence ID: 63) Into a 1 mL tube of random primer stock mixture, hydrazine was prepared to prepare a combined random primer and oligo dT working stock.

After cDNA synthesis, the samples were diluted 1 :3 with nuclease-free water and stored at -20 °C until analysis.

Per5 3 'UTR and independent real time PCR-based analysis of transcripts TIP41 based on ^{LIGHTCYCLER TM 480 (ROCHE DIAGNOSTICS, Indianapolis} , IN) , the reaction is performed in a 10 μL volume. For Per5 3' UTR analysis, the reaction uses primers P5U76S (F) (sequence identification number: 64) and P5U76A (R) (sequence identification number: 65), and ROCHE UNIVERSAL PROBETM (UPL76; catalog number 4889960001; with FAM Mark it). For the TIP41 reference gene analysis, the primers TIPmxF (SEQ ID NO: 66) and TIPmxR (SEQ ID NO: 67), and the probe HXTIP labeled with HEX (hexachlorofluorescein) were used (SEQ ID NO: 68) .

All analyses included a negative control group without template (mixed only). For the standard curve, a blank sample (water in the source well) is also included on the source plate to check for sample cross-contamination. The primers and probe sequences are listed in Table 6 . The reaction component formulations for detecting various transcripts are disclosed in Table 7 , and the PCR reaction conditions are summarized in Table 8 . The fluorescent fraction of FAM (6-Carboxy Fluorescein Amidite) is excited at 465 nm and the fluorescence at 510 nm is measured; the corresponding value of the fluorescent portion of HEX (hexachlorofluorescein) is 533 nm and 580 nm.

Table 6. Oligonucleotide sequences used for molecular analysis of transcript levels in the gene transfer maize. *Class TIP41 protein. **NAv sequences are not available from the supplier.

Table 7. PCR reaction recipe for transcript detection.

Table 8. Thermal cycler conditions for RNA qPCR.

The data was based on the supplier's recommendation using the LIGHTCYCLERTM software v1.5, which uses a second-order derivative maximum algorithm to calculate the Cq value. For performance analysis, performance values were calculated using the ΔΔCt method (ie, 2-(Cq Target-Cq REF)), which relies on two target Cq values and each cycle of the product in the optimized PCR reaction. Under the assumption of doubling, the comparison between the selected baseline values 2 is made.

Transcript size and integrity: Northern blot analysis. In some examples, the additional molecular characterization of the plant-based gene transfer colonization by using Northern blot blots (RNA ink blot) analysis was obtained to determine the performance prp8 prp8 hairpin hairpin dsRNA transgenic plants The molecular size of the dsRNA.

All materials and equipment are treated with RNaseZAP (AMBION/INVITROGEN) prior to use. Tissue samples (100 mg to 500 mg) was collected based on 2 ml SAFELOCK EPPENDORF tube to KLECKO ^TM tissue pulverizer (GARCIA MANUFACTURING, Visalia, CA) by three tungsten beads, the destruction in 1 ml TRIZOL (INVITROGEN) Delta After 5 minutes, it was incubated at room temperature (RT) for 10 minutes. Alternatively, the samples were centrifuged at 11,000 rpm for 10 minutes at 4 °C and the supernatant was transferred to a new 2 ml SAFELOCK EPPENDORF tube. After adding 200 μL of chloroform to the homogenate, the tubes were mixed by inversion for 2 to 5 minutes, incubated at RT for 10 minutes, and centrifuged at 12,000 xg for 15 minutes at 4 °C. Transfer the top phase to a sterile 1.5 mL EPPENDORF tube, add 600 μL of 100% isopropanol, then incubate at RT for 10 minutes to 2 hours, then centrifuge at 12,000 xg for 4 to 5 °C 10 minutes. The supernatant was discarded and the RNA pellet was washed twice with 1 ml of 70% ethanol, and centrifuged at 7,500 xg for 10 minutes between 4 ° C and 25 ° C with washing. Ethanol was discarded and the pellet was briefly air dried for 3 to 5 minutes and then resuspended in 50 μL of nuclease-free water.

Total RNA was quantified using NANODROP 8000 ^® (THERMO-FISHER) and the samples were normalized to 5 μg/10 μL. Then add 10 μL of glyoxal (AMBION/ INVITROGEN) to each sample. Five to 14 ng of DIG RNA standard marker mixture (ROCHE APPLIED SCIENCE, Indianapolis, IN) was dispensed and an equal volume of glyoxal was added. The sample and labeled RNA were denatured at 50 °C for 45 minutes and stored on ice until 1.25% SEAKEM GOLD agarose (LONZA, Allendale, NJ) loaded in NORTHERNMAX 10X Glyoxal Development Buffer (AMBION/INVITROGEN) ) So far in the gel. RNAs were separated by electrophoresis at 65 volts/30 mA for 2 hours and 15 minutes.

After electrophoresis, the gel was rinsed in 2X SSC for 5 minutes and imaged on a GEL DOC workstation (BIORAD, Hercules, CA), then the RNA was transferred overnight at RT for passive transfer to a nylon membrane (MILLIPORE) using 10X SSC As a transfer buffer (20X SSC consists of 3 sodium chloride and 300 mM trisodium citrate, pH 7.0). Following transfer, the membrane was rinsed in 2X SSC for 5 minutes, the RNA was UV cross-linked to the membrane (AGILENT/STRATAGENE) and the membrane was allowed to dry at room temperature for up to 2 days.

The membrane was pre-hybridized in ULTRAHYBTM buffer (AMBION/ INVITROGEN) for 1 to 2 hours. The probe consists of a PCR amplification product containing the sequence of interest (for example, when appropriate, sequence identification number: the antisense sequence portion of 7-11) which is extended by means of the ROCHE APPLIED SCIENCE DIG program. The leaf radix rehmannia is labeled. Hybridization was performed overnight in a hybridization tube at a temperature of 60 ° C in a recommended buffer. Following hybridization, the ink stains were subjected to DIG washing, wrapping, exposure to a film for 1 to 30 minutes, and then the film was developed, all using the method recommended by the DIG kit supplier.

The number of transgenic copies was determined. The maize leaf pieces, which are approximately equal to two leaf punches, are collected in a 96 well collection pan (QIAGEN). Tissue disruption was performed in a BIOSPRINT96 AP1 lysis buffer (provided from BIOSPRINT96 PLANT KIT; QIAGEN) using a KLECKOTM tissue pulverizer (GARCIA MANUFACTURING, Visalia, CA) plus a stainless steel bead. After tissue dissociation, gDNA was isolated in a high-output format using a BIOSPRINT96 PLANT KIT and BIOSPRINT96 extraction automata. The gDNA system was diluted with 2:3 DNA: water, and then the qPCR reaction was set.

qPCR analysis. Transgenic detection was performed using a LIGHTCYCLER ^® 480 system by real-time PCR and by hydrolysis probe analysis. Use LIGHTCYCLER® Probe Design Software 2.0 to design oligonucleotides for hydrolysis probe analysis to detect linker sequences or to detect a portion of the SpecR gene (ie, spectinomycin resistance gene, present) Binary vector plastid; sequence identification number: 69; SPC1 oligonucleotide in Table 9 ). Further, using PRIMER EXPRESS software (APPLIED BIOSYSTEMS) to design oligonucleotide probes used in the analysis of the hydrolyzed nucleotides, AAD-1 to detect the herbicide tolerance gene segment (SEQ ID. No: 70; Table 9 GAAD1 oligonucleotide). Table 9 shows the sequences of the primers and probes. The endogenous maize genomic gene multiplex and reagents were used for analysis (invertase (SEQ ID NO: 71; GENBANK Accession No. U16123, herein referred to as IVR1), which served as an internal reference sequence to ensure the presence of gDNA in each assay. In terms of amplification, the LIGHTCYCLER® 480 PROBES MASTER mixture (ROCHE APPLIED SCIENCE) was prepared as a 1x final concentration in a 10 μL volume multiplex reaction containing 0.4 μM of each primer and 0.2 μM of each probe ( Table 10) The two-step amplification reaction was performed as outlined in Table 11. The fluorescent activation and radioactivity of the FAM- and HEX-labeled probes were as described above; the CY5 conjugate was excited at a maximum of 650 nm, and Fluorescence is up to 670 nm.

The Cp score (where the fluorescent signal intersects the background threshold) is determined from real-time PCR data, using a fit points algorithm (LIGHTCYCLER ^® SOFTWARE issue 1.5), and a relative quantitation module (based on the ΔΔCt method). The data was processed as previously described (above; RNA qPCR).

Table 9. Primers and probe sequences (with fluorescent conjugates) for gene copy number determination and binary vector plastid stem detection. CY5 = Cyanine-5

Table 10. Analysis of the number of gene copies and the reaction components of plastid stem detection. *NA = not applicable **ND = not determined

Table 11. Thermal cycler conditions for DNA qPCR. Example 8 : Biological analysis of genetically transplanted maize

Biological analysis of insects. The biological activity of the dsRNA produced by the subject invention in plant cells is demonstrated by bioanalytical methods. See, for example, Baum et al. (2007) Nat. Biotechnol. 25(11): 1322-1326. For example, one can demonstrate effectiveness by feeding various plant tissues or tissue blocks to a targeted insect in a controlled feeding environment derived from a plant that produces insecticidal dsRNA. . Alternatively, the extract is prepared from various plant tissues derived from a plant that produces insecticidal dsRNA, and the extracted nucleic acid system is distributed to the top of an artificial diet for bioanalysis as previously described. The results of such a feeding analysis are compared to the results of a bioassay performed in a similar manner using host control plants derived from host plants that do not produce insecticidal dsRNA, or in comparison with other control samples. Compared to the growth and survival of the control group, the growth and survival of the targeted insects on the test diet were reduced.

Insect bioanalysis of genetically transformed maize varieties. Two western corn rootworm larvae (1 to 3 days old) hatched from washed eggs were selected and placed in each well of the bioassay tray. It was then covered with a "PULL N' PEEL" label cover (BIO-CV-16, BIO-SERV) and placed in an incubator at 28 ° C and 18 hours / 6 hours of light/dark cycle. The larval mortality was assessed nine days after the initial infestation, and the mortality was calculated as the percentage of insects that died from the total number of insects treated. The insect samples were frozen at -20 °C for two days and then the individual treated insect larvae were pooled and weighed. The percent growth inhibition was calculated as the average weight of the experimental treatment divided by the average weight of the two control well treatments. Data are expressed as (negative control) percent inhibition of growth. The average weight over the control average weight was normalized to zero.

Biological analysis of insects in the greenhouse. Eggs from western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) were obtained from soil derived from CROP CHARACTERISTICS (Farmington, MN). The WCR eggs were cultured at 28 °C for 10 to 11 days. The eggs were washed from the soil, placed in 0.15% agar solution, and the concentration was adjusted to approximately 75 to 100 eggs per 0.25 mL aliquot. A hatching plate was established in a petri dish with an aliquot of egg suspension to monitor hatchability.

The soil surrounding the maize plant grown in ROOTRAINERS ^® is infested with 150 to 200 WCR eggs. The insects were allowed to feed for 2 weeks, after which time each plant was given a "Root Rating". The node damage scale is used for grading, which is essentially according to Oleson et al. (2005) J. Econ. Entomol. 98:1-8. By this bioanalysis, plant lines showing reduced damage were transplanted to 5 gallon pots for seed production. The transplant is treated with an insecticide to prevent further rootworm damage and insect release in the greenhouse. The plants are pollinated by hand for seed production. Seed from these plant-based storage for assessing T ₁ and subsequent generation plants.

Greenhouse bioassays included two negative control plants. Gene-negative negative control plants were generated by transformation with a vector designed to produce a gene for yellow fluorescent protein (YFP) or YFP hairpin dsRNA (see Example 4). The non-transformed negative control plant line is produced from the seed of the parental maize variety producing the genetically transgenic plant. Bioanalysis was performed on two different dates, and each group of plant material included a negative control. Example 9 : Gene-transplanted maize ( Zea mays ) containing a sequence of coleopteran pests

10-20 strain T ₀ transgenic maize plant lines like generated as described in Example 6. Obtain additional 10-20 strain Construction of RNAi hairpin dsRNA expression of T ₁ of maize was independent lines, test for corn rootworm. The hairpin dsRNA contains the sequence identification number: 1 and/or sequence identification number: 3. Additional hairpin dsRNA lines are derived, for example, from coleopteran pest sequences such as, for example, Caf1-180 (U.S. Patent Publication No. 2012/0174258), VatpaseC (U.S. Patent Publication No. 2012/0174259), rho1 (U.S. Patent Publication No. 2012/0174260), VatpaseH (U.S. Patent Publication No. 2012/0198586), PPI-87B (U.S. Patent Publication No. 2013/0091600), RPA70 (U.S. Patent Publication No. 2013 / No. 0091601), RPS6 (U.S. Patent Publication No. 2013/0097730), ROP (U.S. Patent Publication No. 14/577,811), RNA polymerase I1 (U.S. Patent Publication No. 62/133,214), RNA polymerase II140 (U.S. Patent Publication No. 14/577,854), RNA polymerase II215 (U.S. Patent Publication No. 62/133,202), RNA polymerase II33 (US Patent Application No. 62/133,210), transcription elongation factor spt5 (USA) Patent Application No. 62/168,613), transcription elongation factor spt6 (US Patent Application No. 62/168,606), ncm (US Patent Application No. 62/095487), dre4 (US Patent Application No. 14/705,807) ), COPI α (US Patent Application) No. 62/063, 199), COPI β (US Patent Application No. 62/063, 203), COPI γ (US Patent Application No. 62/063, 192), and COPI δ (US Patent Application No. 62/063,216). These lines were confirmed by RT-PCR or other molecular analysis methods.

T ₁ is independently selected from the total RNA preparation lines selectively used based on RT-PCR, primer plus links designed to bind promoter hairpin construct expression cassette was in each of the RNAi. In addition, specific primers for each target gene in the RNAi construct are selectively used to amplify and confirm the production of pre-processed mRNA required for the production of siRNA from the plant kingdom. For each target gene, amplification of the desired electrophoresis band confirms the performance of the hairpin RNA in each gene-transplanted maize plant. RNA blotting hybridization was then selectively used in independent gene transfer lines, and the dsRNA hairpins of these target genes were confirmed to be siRNA.

Furthermore, RNAi molecules with sequences that target gene mismatches and more than 80% sequence identity are similar to those that affect the corn rootworm with RNAi molecules that have 100% sequence identity to the target gene. The mismatched sequence is paired with the native sequence to form a hairpin dsRNA in the same RNAi construct, delivering plant-processed siRNAs that can affect the growth, development, and viability of the feeding coleopteran pest.

The dsRNA, siRNA or miRNA corresponding to the target gene is delivered in the plant kingdom, and then the target gene of the coleopteran pest is down-regulated by the coleopteran pest through feed intake resulting in silencing of the target gene through the RNA vector gene. When the function of a target gene is important in one or more developmental stages, the growth and/or development of the coleopteran pest is affected, and in WCR, NCR, SCR, MCR, Brazilian corn rootworm ( D. balteata LeConte) ), D. speciosa Germar, D. u. tenella , and at least one of D. u. undecimpunctata Mannerheim In this case, it is impossible to successfully invade, feed and/or develop, or cause the death of the coleopteran pest. The target gene was selected and successfully applied to control the coleopteran pests.

Phenotypic comparison of gene-transferred RNAi lines and untransformed maize. The targeted coleopteran pest gene or sequence selected for the creation of hairpin dsRNA does not have similarity to any known plant gene sequence. Thus, (systemic) RNAi, which is produced or activated by targeting the constructs of these coleopteran pest genes or sequences, is not expected to have any adverse effects on the genetically transformed plants. However, the developmental and morphological characteristics of the gene-transgenic lines were compared to untransformed plants, and to gene-transferred lines that were transformed with vectors that were "empty" without a hairpin. Compare plant roots, shoots, leaf groups and reproductive characteristics. There were no observable differences in root length and growth patterns of genetically propagated and untransformed plants. The bud characteristics of the plants, such as height, number and size of leaves, flowering time, flower size and appearance are similar. In general, no morphological differences were observed between gene transfer lines and those who did not exhibit targeted iRNA molecules when cultured in vitro and in greenhouse soil. Example 10 : Gene-transplanting maize containing a coleopteran pest sequence and an additional RNAi construct

Comprising a heterologous gene coding sequences in its genome a transfer colonize maize plants, organisms other than the iRNA molecules endogenous heterologous coding sequence is transcribed into the targeted coleopteran pest, via Agrobacterium WHISKERS ^TM based methodology (see Petolino and Arnold (2009) Methods Mol. Biol. 526:59-67) are subjected to secondary transformation to produce one or more insecticidal dsRNA molecules (for example, at least one dsRNA molecule comprising a targeted inclusion sequence Identification number: 1 and/or sequence identification number: dsRNA molecule of the gene of 3). Example 4 lines substantially as described in the preparation of the plant plastid Transformation vectors as described, via Agrobacterium based WHISKERS ^TM - from a transgenic maize Hi II or B104 maize plants obtained cell suspension medium delivered to the methods Transformation Or an immature maize embryo, wherein the host plant contains a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets an organism other than a coleopteran pest. Example 11 : Gene-transplanting maize containing RNAi constructs and additional coleopteran pest control sequences

A gene-transplanted maize plant comprising a heterologous coding sequence in its genome, the heterologous coding sequence being transcribed into an iRNA molecule targeting a coleopteran pest organism (for example, at least one dsRNA molecule comprising a target comprising the sequence identification number: 1 and / or sequence identification number: 3 gene a dsRNA molecule), or via Agrobacterium WHISKERS ^TM secondary transfer methodology to be shaped (see Petolino and Arnold (2009) methods Mol Biol 526:.. 59- 67) A protein that produces one or more insecticidal protein molecules, for example, Cry3, Cry34, and Cry35. Substantially as described in Example 4 Preparation of Transgenic Plants shaped like a plastid vector, or a system via Agrobacterium WHISKERS ^TM - Transformation method of delivering media from one to transgenic maize suspension cells or plants obtained B104 maize immature In a maize embryo, the gene transgenic B104 maize plant contains a heterologous coding sequence in its genome, which is transcribed into an iRNA molecule that targets a coleopteran pest organism. A double-turned plant is obtained which produces iRNA molecules and insecticidal proteins for controlling coleopteran pests. Pollen beetles transcription Xueku: Example 12

insect. Larval and adult pollen beetles (Giessen, Germany) were collected from the rapeseed flowering field. Young adult beetles (each treatment group: n = 20; 3 replicates) with two different bacteria (Staphylococcus aureus and Pseudomonas aeruginosa ), a yeast (S. cerevisiae) ) and bacterial LPS are tested. The bacterial culture was grown at 37 °C with stirring and the optical density (OD600) at 600 nm was monitored. Cells of OD600 ∼1 were harvested by centrifugation and resuspended in phosphate buffered saline. Puncture the abdomen of the pollen beetle adult and introduce the mixture through the ventrolateral side by using an aqueous solution immersed in 10 mg/ml LPS (purified E. coli endotoxin; SIGMA, Taufkirchen, Germany) and an anatomic needle in the bacteria and yeast culture. . Immunologically tested beetles, naïve beetles and larvae were collected together at the same time point (n=20 each and 3 replicates each).

RNA is isolated. After 8 h of immunization, TriReagent (Molecular Research Centre, Cincinnati, OH, USA) was used to extract total RNA from frozen beetles and larvae in each case, and using the RNeasy mini-set (Qiagen, Hilden, Germany), following the manufacturer's The guidelines are purified. RNA integrity was verified using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano kit (Agilent Technologies, Palo Alto, Calif., USA). The quality of the RNA was determined using a Nanodrop ND-1000 spectrophotometer. RNA was extracted from the adult immune-induced treatment group, the adult control group, and the larval group, respectively, and then an equal amount of total RNA was combined into a pooled group of each sample (immunized adult, control adult, and larvae). (pool) is used for sequencing.

Transcript library information. RNA-Seq data generation and assembly Single-read 100-bp RNA-Seq lines were obtained from immunologically tested adult beetles, primary (control) adult beetles, and 5 μg of total RNA isolated from untreated larvae, respectively. get on. The sequencing is performed using the Eurofins MWG Operon on the Illumina HiSeq-2000 platform. This yielded 2,800,000 readings of adult control beetle samples, 21.5 million reads of LPS-tested adult beetle samples, and 25.1 million larval samples. Read. The collection of readings ( 67.5 million) is based on the Velvet/Oases assembly software (Schulz et al. (2012) Bioinformatics. 28:1086-92; Zerbino and Birney (2008) Genome Res. 18:821-9 ) to assemble. This transcript library contains 55,648 sequences.

Pollen beetle prp8 identification. A tBLASTn search of the transcript library was used to identify matching fragment contigs. The prp8 peptide sequence derived from the leaf was used as a query (Genbank NP_652610). Example 13: pollen beetle mortality after treatment prp8 iRNA

A gene-specific primer containing the T7 polymerase promoter sequence at the 5' end was used to generate a PCR product of approximately 424 bp by PCR (SEQ ID NO: 12). The PCR fragments were cloned into the pGEM T easy vector according to the manufacturer's protocol and sent to the sequencing company to verify the sequence. The dsRNA was then produced from the sequenced plastids, PCR constructs generated according to the manufacturer's protocol, and through T7 RNA polymerase (MEGAscript® RNAi kit, Applied Biosystems).

~100 nL dsRNA (1 μg/ul) was injected into the adult beetle system using a micromanipulator under an anatomical stereomicroscope (n=10, 3 biological replicates). The animals were anesthetized on ice and then fixed on a double-sided tape. The control received the same volume of water. All controls at all stages were untestable due to lack of animals. Controls were performed on different dates due to the limited availability of insects. Pollen beetles are maintained in petri dishes with dry pollen and wet wipes.

Table 12. Results of biological analysis of adult pollen beetle in oilseed rape (average percentage of survival ± standard deviation (SD), n = 3 groups, 10 per group).

Feeding bioassay: The beetle was kept in an empty falcon tube for 24 h before treatment. A small drop of dsRNA (~5 μL) was placed in a small Petri dish and 5 to 8 beetles were added to the Petri dish. The animals were observed under an anatomical stereomicroscope and selected for ingestion of the dsRNA containing the dietary solution for bioanalysis. Move the beetle to a petri dish with dry pollen and wet tissue. The control received the same volume of water. All controls at all stages were untestable due to lack of animals. Controls were performed on different dates due to the limited availability of insects.

Table 13. Results of adult feeding bioassay of rapeseed (Averaging percentage ± standard deviation (SD), n = 3 groups, 10 per group).

Table 14. Results of adult feeding bioassay of raped beetle (average percent of survival ± standard deviation (SD), n = 3 groups, 10 per group). Example 14 : Agrobacterium-mediated transformation of rapeseed hypocotyls

Agrobacterium preparation. The Agrobacterium strain containing the binary plastid was streaked into YEP medium containing streptomycin (100 mg/ml) and spectinomycin (50 mg/mL) (Bacto PeptoneTM 20.0 gm/L and yeast extract 10.0 gm) /L) and incubate at 28 °C for 2 days. The Agrobacterium strain containing the reproduction of the binary plastid was scraped off from a 2-day scribing pan using a sterile inoculating loop. The scraped Agrobacterium strain containing the binary plastid was inoculated into 150 mL of a sterile 500 mL baffled flask with streptomycin (100 mg/ml) and spectinomycin (50 mg/mL). The modified YEP liquid was shaken at 200 rpm at 28 °C. Cultures were centrifuged and resuspended in M-medium (LS salts, 3% glucose, modified B5 vitamins, 1 μM cytosin, 1 μM 2,4-D, pH 5.8) and diluted to appropriate Density (measured as 50 Klett units using a spectrophotometer) followed by transformation of the canola hypocotyls.

Rapeseed transformation. Seed germination : Rapeseed seeds (var. NEXERA 710TM) were surface sterilized in 10% CloroxTM for 10 minutes and rinsed three times with sterile distilled water (seeds were housed in stainless steel filters during this method). Seeds were planted on PhytatraysTM (25 seeds/PhytatrayTM) containing 1⁄2 MS rapeseed medium (1/2 MS, 2% sucrose, 0.8% agar) for germination, and placed in a PercivalTM growth chamber, and the growth system Set to 25 ° C, 16 hours light and 8 hours dark light period for 5 days of germination.

Pretreatment : On day 5, the hypocotyl segments approximately 3 mm in length were aseptically stripped and the remaining root and stem segments discarded (the hypocotyl segments were immersed in 10 mL of sterile milliQTM water during the excision method) Prevent hypocotyl segments from drying). The hypocotyl segments were placed horizontally on a sterile tissue-inducing medium, MSK1D1 (MS, 1 mg/L cytosin, 1 mg/L 2,4-D, 3.0% sucrose, 0.7% phytoagar). In the PercivalTM growth chamber, the growth regime was set at 22-23 ° C, and the 16-hour light, 8-hour dark illumination period lasted for 3 days.

Co-cultivation with Agrobacterium : Agrobacterium strain containing a binary plastid was inoculated into a flask containing YEP medium containing an appropriate antibiotic before co-cultivation with Agrobacterium . The hypocotyl segments were transferred from filter paper healing tissue induction medium, MSK1D1, to an empty 100 x 25 mm PetriTM dish containing 10 mL of liquid M-medium to prevent hypocotyl drying. At this stage, the spatula is used to dig out the section and transfer the section to a new medium. A liquid pipette was used to remove the liquid M-medium and 40 mL of the Agrobacterium suspension was added to the PetriTM dish (500 segments plus 40 mL of Agrobacterium solution). The PetriTM dish was periodically vortexed to treat the hypocotyl segments for 30 minutes to keep the hypocotyl segments immersed in the Agrobacterium solution. At the end of the treatment period, the Agrobacterium solution was pipetted into a beaker of waste; autoclaved and discarded (complete removal of the Agrobacterium solution to prevent Agrobacterium overgrowth). The tweezers were used to transfer the treated hypocotyls back to the original flat plate containing the MSK1D1 medium covering the filter paper (be careful to ensure that the segments were not dry). The transformed hypocotyl segments and the untransformed control hypocotyl segments are returned to the PercivalTM growth chamber to reduce light intensity (covered with aluminum foil to cover the flat plate), and the treated hypocotyl segments are co-operated with Agrobacterium. The cultivation lasted for 3 days.

Healing tissue induction was performed on the selection medium : after co-culture for 3 days, the hypocotyl segments were individually transferred back to the healing tissue induction medium with scorpion, MSK1D1H1 (MS, 1 mg/L cytosin, 1 mg/L 2 , 4-D, 0.5 gm/L MES, 5 mg/L AgNO ₃ , 300 mg/L TimentinTM, 200 mg/L carbencillin, 1 mg/L HerbiaceTM, 3% sucrose, 0.7% plants Agar), and the growth regime is set at 22-26 °C. The hypocotyl segments were fixed to the medium but not deeply implanted in the medium.

Selection and shoot regeneration : After 7 days on healing tissue induction medium, the healing hypocotyl segments were moved to a selective shoot regeneration medium 1, MSB3Z1H1 (MS, 3 mg/L BAP, 1 mg/L zeatin, 0.5 Gm/L MES, 5 mg/L AgNO ₃ , 300 mg/L TimentinTM, 200 mg/L carbencillin, 1 mg/L HerbiaceTM, 3% sucrose, 0.7% plant agar). After 14 days, the hypocotyl segments of the developing shoots were transferred to regeneration medium 2 with increased selectivity, MSB3Z1H3 (MS, 3 mg/L BAP, 1 mg/L zeatin, 0.5 gm/L MES, 5 Mg/L AgNO ₃ , 300 mg/l TimentinTM, 200 mg/L carbencillin, 3 mg/L HerbiaceTM, 3% sucrose, 0.7% plant agar), and the growth regime was set at 22-26 °C.

Bud elongation : After 14 days, the hypocotyl segments of the developing shoots were transferred from regeneration medium 2 to shoot elongation medium, MSMESH5 (MS, 300 mg/L TimentinTM, 5 mg/l HerbiaceTM, 2% sucrose, 0.7 % TC agar), and the growth regime was set at 22-26 °C. The elongated buds are separated from the hypocotyl segments and moved to MSMESH5. After 14 days, the first round of the remaining shoots that had not been elongated on the shoot elongation medium was transferred to fresh shoot elongation medium, MSMESH5. At this stage, all remaining hypocotyl segments that are not budded are discarded.

Root induction : After 14 days of culture on shoot elongation medium, the isolated shoots were transferred to MSMEST medium (MS, 0.5 g/L MES, 300 mg/L TimentinTM, 2% sucrose, 0.7% TC agar) for Root induction was carried out at 22-26 °C. Any shoots that did not produce roots after the first transfer to MSMEST medium were transferred to the second or third round of MSMEST medium until the shoots developed roots. Example 15 : prp8 dsRNA in insect management

The Prp8 dsRNA transgene line is combined with other dsRNA molecules in gene transfer plants to provide redundant RNAi targeting and synergistic RNAi effects. Gene-transgenic plants that display dsRNA targeting prp8 , including, by way of example and not limitation, corn, soybean, and cotton, are useful for preventing feeding damage to coleopteran and hemipteran insects. The Prp8 dsRNA transgene also combines the insecticidal protein technology of Bacillus thuringiensis in plants to represent a new mode of action of the Insect Resistance Management gene pyramids. When combined with other dsRNA molecules that target insect pests and/or insecticidal proteins in genetically transgenic plants, a synergistic insecticidal effect is observed which also mitigates the development of resistant insect populations.

While the present disclosure may be susceptible to various modifications and alternative forms, specific specific examples are described herein by way of example. However, it should be understood that the disclosure is not intended to be limited to the particular forms disclosed. Rather, the disclosure is to cover all modifications, equivalents, and alternatives of the scope of the appended claims and their equivalents.

Figure 1 includes a description of the strategy used to generate dsRNA from a single transcription template and a single pair of primers.

Figure 2 includes a description of the strategy used to generate dsRNA from two transcriptional templates.

Claims (57)

An isolated nucleic acid molecule comprising at least one polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide comprises a nucleotide selected from the group consisting of Sequence: sequence identification number: 5; sequence identification number: complement of 5; sequence identification number: a fragment of at least 15 contiguous nucleotides of 5; sequence identification number: a fragment of at least 15 contiguous nucleotides of 5 the complement thereof; Lu a coding sequence one natural organism beetles (Meligethes), comprising the sequence identification number: 12; a dew beetles one organism complement of the native coding sequence, the coding sequence comprises the natural sequence identification number : 12; at least 15 contiguous nucleotides encoding a fragment of a native sequence of one exposed end of a living body a, the native coding sequence comprising the sequence identification number: 12; and the tail exposed to a biological one a native coding sequence A complement of a fragment of at least 15 contiguous nucleotides comprising the sequence ID: 12. The nucleic acid molecule of claim 1, wherein the nucleotide sequence is selected from the group consisting of: sequence identification number: 5, sequence identification number: 12, and the aforementioned complement. The nucleic acid molecule of claim 1, wherein the molecule is a vector. The nucleic acid molecule of claim 1, wherein the biological system is selected from the group consisting of: D. v. virgifera LeConte; D. barberi Smith and Lawrence; cucumber beetle Star eleven subspecies rootworm (D. u howardi.); Mexican corn rootworm (D. v zeae.); Brazilian corn rootworm (D. balteata LeConte); cucumber beetle eleven stars coccidiosis (D u. tenella ); D. u. undecimpunctata Mannerheim; D. speciosa Germar; and Meligethes aeneus Fabricius ( Pollen beetle (Pollen) Beetle)). A ribonucleic acid (RNA) molecule encoding a nucleic acid molecule of claim 1, wherein the RNA molecule comprises a polyribonucleotide encoded by the polynucleotide. An RNA molecule according to claim 5, wherein the molecule is a double-stranded ribonucleic acid (dsRNA) molecule. The dsRNA molecule of claim 6, wherein the molecule is contacted with a coleopteran pest to inhibit expression of an endogenous nucleic acid molecule that is specifically complementary to the polyribonucleotide. The dsRNA molecule of claim 7, wherein the molecule is contacted with the coleopteran pest to kill or inhibit growth and/or feeding of the pest. The dsRNA molecule of claim 6, comprising a first polyribonucleotide, a second polyribonucleotide, and a third polyribonucleotide, wherein the first polyribonucleotide is derived from the core The nucleotide sequence is encoded, wherein the third polyribonucleotide is linked to the first polyribonucleotide by the second polyribonucleotide, and wherein the third polyribonucleotide is substantially The reverse complement of the first polyribonucleotide, whereby the first and the third polyribonucleotide are hybridized to form a ribonucleic acid to form the dsRNA. The RNA molecule of claim 5, wherein the RNA molecule is a single-stranded ribonucleic acid molecule between about 15 and about 30 nucleotides in length. The vector of claim 3, wherein the heterologous promoter is functional in a plant cell, and wherein the vector is a plant-transformed vector. A cell comprising the nucleic acid molecule of claim 1. The cell of claim 12, wherein the cell is a prokaryotic cell. The cell of claim 12, wherein the cell is a eukaryotic cell. The cell of claim 14, wherein the cell is a plant cell. A plant comprising the plant cell of claim 15. A part of a plant according to claim 16, wherein the part of the plant comprises the plant cell. A portion of the plant of claim 17, wherein the portion of the plant is a member. A food product or product product produced by the plant of claim 16, wherein the product comprises a detectable amount of the nucleic acid molecule. A plant according to claim 16, wherein a cell of the plant comprises a double-stranded ribonucleic acid (dsRNA) molecule encoded by the polynucleotide. The plant cell of claim 15, wherein the cell is a Zea mays , a Brassica sp. or a grass cell. A plant according to claim 16, wherein the plant is a plant of Zea mays , Brassica sp. or Gramineae. The plant of claim 20, wherein the dsRNA molecule inhibits the expression of an endogenous polynucleotide when the coleopteran pest ingests a portion of the plant, the endogenous polynucleotide specifically complementary to the polyribo Nucleotide. 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. The nucleic acid molecule of claim 24, wherein the heterologous promoter is functional in a plant cell, and wherein the molecule is a plant-transformed vector. A method for controlling an insect pest population, the method comprising providing a preparation comprising a ribonucleic acid (RNA) molecule, a ribonucleic acid (RNA) molecule, which acts to inhibit the pest after contact with the insect pest a biological function, wherein the RNA specifically hybridizes to a polynucleotide selected from the group consisting of: sequence identification number: 91 and 92; sequence identification number: complement of any of 91 and 92 a fragment of at least 15 contiguous nucleotides of any one of 91 and 92; a complement of a fragment of at least 15 contiguous nucleotides of any one of 91 and 92; ; sequence identification number: 5 one transcript; and sequence identification number: 5 complement of one of the transcripts. The method of claim 26, wherein the RNA molecule is a double stranded RNA (dsRNA) molecule. The method of claim 26, wherein providing the formulation comprises contacting the insect pest with a sprayable composition comprising one of the formulations. The method of claim 26, wherein the providing the formulation comprises feeding a plant cell containing the RNA molecule to the insect pest. A method for controlling a coleopteran pest population, the method comprising: providing a preparation comprising a first and a second polyribonucleotide, which, upon contact with the coleopteran pest, acts to inhibit the a biological function in a coleopteran pest, wherein the first polyribonucleotide comprises a nucleotide sequence and the polyribose selected from the group consisting of sequence identification numbers: 91 and 92 From about 15% to about 100% of the nucleotides of the nucleotide have from about 90% to about 100% sequence identity, and wherein the first polyribonucleotide is specific to the second polyribonucleotide Sexually hybridize. A method for controlling a coleopteran pest population, the method comprising: providing a plant cell in a host plant of one of the coleopteran pests, the plant cell comprising the nucleic acid molecule of claim 1, wherein the polynucleotide is expressed To generate a ribonucleic acid (dsRNA) molecule that, when contacted with a coleopteran pest belonging to the group, acts to inhibit the expression of a targeted sequence in the coleopteran pest and causes The growth and/or survival of the coleopteran pest or pest population is reduced relative to the development of the same pest species on the same host plant species but not on one of the polynucleotides. The method of claim 31, wherein the coleopteran pest population is reduced relative to the same pest population group that invades the same host plant species but lacks a plant cell containing the nucleic acid molecule. A method for controlling infestation of a coleopteran pest in a plant, the method comprising providing a ribonucleic acid (RNA) molecule in a diet invading a coleopteran pest of the plant, the ribonucleic acid (RNA) molecule being selected from the group consisting of A polyribonucleotide of the group formed specifically hybridizes: sequence identification number: 91 and 92; sequence identification number: complement of any of 91 and 92; sequence identification number: 91 and 92 a fragment of at least 15 contiguous nucleotides; a sequence identity number: a complement of a fragment of at least 15 contiguous nucleotides of any of 91 and 92; sequence identification number: 5 one transcript; sequence Identification number: complement of one of five transcripts; sequence identification number: a fragment of at least 15 contiguous nucleotides of one transcript of 5; and at least 15 contiguous nucleosides of a transcript of sequence identification number: A complement of a fragment of an acid. The method of claim 33, wherein the diet comprises a plant cell comprising a polynucleotide transcribed to express the polyribonucleotide. The method of claim 33, wherein the RNA molecule is a double stranded RNA (dsRNA) molecule. A method for improving the yield of a crop, the method comprising: cultivating a plant in the crop, the plant comprising the nucleic acid molecule of claim 1 to allow expression of the polynucleotide. The method of claim 36, wherein the plant is a plant of Zea mays , Brassica sp. or Gramineae. The method of claim 36, wherein the expression of the polynucleotide produces an RNA molecule that clamps a targeting gene in a coleopteran pest that has contacted a portion of the plant, thereby inhibiting the coleopteran pest Development or growth and loss of yield due to infection with this coleopteran pest. A method for producing a gene transfer plant cell, the method comprising: transducing a plant cell with a plant-transformed vector according to claim 12; at a plant cell culture sufficient to allow for inclusion of one of the plurality of transformed plant cells The transformed plant cell is cultured under conditions of development; the transduced plant cell that has integrated the polynucleotide into its isoform is selected; and the ribonucleic acid (RNA) molecule encoded by the polynucleotide is screened. The transduced plant cell; and the plant cell that selects one of the RNAs. The method of claim 39, wherein the RNA molecule is a double stranded RNA molecule. A method for producing a genetically transgenic plant against a coleopteran pest, the method comprising: regenerating a gene-transgenic plant from a gene-transplanting plant cell comprising the nucleic acid molecule of claim 1, wherein the polynucleotide is The expression of one of the encoded ribonucleic acid (RNA) molecules is sufficient to modulate the expression of a target gene of the coleopteran pest when the coleopteran pest contacts the RNA molecule. A method for producing a gene transfer plant cell, the method comprising: transducing a plant cell with a vector comprising a member for providing a prp8 vector for protection of a leaf pest to a plant; The transformed plant cell is cultured under conditions comprising the development of a plant cell culture of one of the transformed plant cells; the member having selected the leaf beet pest protection for providing the prp8 vector is integrated into the plant etc. within the genome of the transfected plant cell shape; screening the transfected plant cell type which exhibit a member of a gene expression prp8 Diabrotica for inhibiting pests; and selecting a plant cell an expression for inhibiting Diabrotica This component of the prp8 gene expressed in the pest. A method for producing a genetically transgenic plant, the method comprising: regenerating a gene transfer plant from the gene transfer plant cell produced by the method of claim 42, wherein the plant cell of the plant comprises a strain for inhibiting A component of the prp8 gene expressed in the leaf pest. The method of the requested item 43, wherein the means for inhibiting the performance of a member within a Diabrotica insects prp8 gene expression, sufficient regulatory system that intrusion of the transgenic plants showed a pest Diabrotica a given target gene prp8. A plant gene expression comprising a member within a prp8 for inhibiting Diabrotica insects. A method for producing a transgenic plant cell, the method comprising: a carrier to form a transfected plant cell, the vector comprising a beetle pests prp8 protective medium for providing to a member of a plant; sufficient contains several conditions allowing development of the culture was transferred via one cell type plant cell, the shape of the transfected plant cell culture; has been selected for providing media prp8 pests Meligethes a protective member to the integrated plant To transgenic plant cells in the same genome; screening the transduced plant cells for expression of a member of a prp8 gene expression in an exposed-tailed pest; and selecting a plant cell for expression for inhibition A member of the Prp8 gene expressed in a pest. A method for producing a genetically transgenic plant, the method comprising: regenerating a gene transfer plant from the gene transfer plant cell produced by the method of claim 46, wherein the plant cell of the plant comprises a strain for inhibiting gene expression within a prp8 the pests Meligethes member. The method of claim 47, wherein the expression of the member for inhibiting the expression of a prp8 gene in an exposed-tailed pest is sufficient to control the expression of a target prp8 gene in an exposed-tailed pest infesting the gene-transplanting plant. . A plant gene expression comprising a member within a Nitidulidae prp8 for inhibiting pests. The nucleic acid of claim 1, which further comprises a polynucleotide encoding a pesticidal polypeptide derived from Bacillus thuringiensis , Alcaligenes spp. or a fake Species of Pseudomonas spp. The nucleic acid molecule of claim 50, wherein the insecticidal polypeptide is selected from the group consisting of B. thuringiensis insecticidal polypeptides: Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C. A plant cell according to claim 15, wherein the cell comprises a polynucleotide encoding a pesticidal polypeptide derived from a species of the genus Alcaligenes or a Pseudomonas species. The plant cell of claim 52, wherein the insecticidal polypeptide is selected from the group consisting of a S. cerevisiae insecticidal polypeptide consisting of Cry1B, Cry1I, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23 , Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C. A plant according to claim 16, wherein a cell of the plant comprises a polynucleotide encoding a pesticidal polypeptide derived from a strain of S. faecalis, a species of the genus Alcaligenes or a species of the genus Pseudomonas. The plant according to claim 54, wherein the insecticidal polypeptide is selected from the group consisting of Suribacterium insecticidal polypeptides consisting of Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C. The method of claim 30, wherein the plant cell comprises a polynucleotide encoding a pesticidal polypeptide derived from a strain of S. faecalis, a species of the genus Alcaligenes or a species of the genus Pseudomonas. The method of claim 56, wherein the insecticidal polypeptide is selected from the group consisting of C. sinensis insecticidal polypeptides consisting of Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C.