CN110669760B - Nucleotide sequences and methods for controlling insect infestation - Google Patents

Abstract

The present invention relates to a nucleotide sequence and a method thereof for controlling insect infestation, wherein the isolated polynucleotide sequence comprises: (a) the polynucleotide sequence shown in SEQ ID NO. 1; or (b) a polynucleotide sequence of at least 15 or 17 or 19 or 21 contiguous nucleotides of SEQ ID NO:1, wherein coleopteran insect pest ingests a double-stranded RNA comprising at least one strand complementary to the polynucleotide sequence, inhibiting the growth of the coleopteran insect pest; or (c) any one of the polynucleotide sequences shown in SEQ ID NO. 3 to SEQ ID NO. 8; or (d) a polynucleotide sequence which hybridizes or is complementary to the polynucleotide sequence defined in (a), (b) or (c) above under stringent conditions. The invention discloses a plurality of target sequences of a target gene c40514 for controlling coleoptera insect pests diabrotica, which are efficient, specific, convenient and low in cost.

Description

Nucleotide sequences and methods for controlling insect infestation

Technical Field

The invention relates to a nucleotide sequence for controlling insect invasion and a method thereof, in particular to a method for controlling Diabrotica by reducing or closing the expression of a target sequence in the Diabrotica by an RNAi technology.

Background

Field crops are often the target of insect attack. During the past decades, there have been substantial advances in the development of more effective methods and compositions for insect infestation in crops. Chemical pesticides are relatively effective means for controlling pest infestation. However, the use of chemical pesticides has a number of disadvantages. First, chemical insecticides are non-selective and are intended to be applied to control insects that are harmful to a wide variety of crops and other plants, but because of their lack of selectivity, chemical insecticides can also cause damage to non-target organisms such as earthworms and the like. Also, chemical pesticides are generally barren in the field for a period of time after their application. Chemical pesticides persist in the environment and are often metabolized very slowly. This slow metabolism results in residues of chemical pesticides in the crop and the environment, which are accumulated in the food chain, especially in the high predators. The accumulation of these chemical pesticides results in the induction of diseases in higher-end species, such as human cancers. There is therefore a strong need for environmentally friendly methods for controlling or eradicating insect infestation in crop production, i.e. methods which are selective, environmentally friendly, biodegradable and which can be well used in pest resistance management systems.

During the past decades, substantial progress has been made in developing effective methods for controlling plant pests. Chemical insecticides, while very effective in eradicating plant pests, also act against non-target insects, while chemical insecticides persist in the environment, not only causing irreversible contamination of the environment, but also leading to the emergence of resistant insects. Microbial insecticides, particularly insecticides obtained from Bacillus thuringiensis (Bt) strains, play an important role in agricultural production as substitutes for chemical insecticides, have certain insecticidal activity on insects such as lepidoptera, diptera, coleoptera and the like, but have higher requirements on application environments, if the environments are not suitable for the growth of the microorganisms, the microbial insecticides need to be repeatedly applied in production, and some even repeated applications cannot achieve the purpose of controlling pests, so that the production cost is greatly increased. Transgenic plants with enhanced resistance to certain pests, such as genetically engineered Cry toxins produced by genetically engineering corn and cotton plants, have been widely used in agricultural production in the united states and provide farmers with an alternative to traditional pest control methods by genetically engineering one or more genes encoding Bt insecticidal proteins into plants. However, the transgenic crops containing the Cry toxin developed at present can only be used for controlling coleoptera pests with a relatively narrow range, such as corn rootworm and Colorado potato beetle, and for one of the main pests of corn at present, diabrotica bimaculata, no relevant report on the application of the Cry toxin for controlling exists. Meanwhile, the diabrotica leaves overwinter in the soil as eggs, and the larvae also move in the soil after hatching in 6 months of the next year; due to the large-scale popularization of straw returning in recent years, the difficulty of using chemical insecticide to prevent and control the Diabrotica bimaculata is increased year by year, particularly in the period from the bottom of 7 months to the beginning of 8 months, the adult Diabrotica bimaculata emerges, and at the moment, corns grow high and cannot be prevented and controlled by using the chemical insecticide.

RNA interference or RNAi is a method used to down-regulate gene expression in a sequence-specific manner in the cell or whole organism environment, which can achieve the goal of targeted interference of target gene expression through specific targeted selection and efficient mRNA suppression. Although it is known in the art to use RNAi technology for pest control, in view of the wide variety of insects, not only does this technology work very differently in different insects, but a key factor in using this technology as a measure for controlling insect infestation is also the selection of the most appropriate target genes, i.e. those genes whose loss of function results in severe disruption of the essential biological processes and/or death of the organism. Thus, the present invention provides for the down-regulation of specific target genes in pests as a means to achieve control of insect infestation, particularly of plants.

Disclosure of Invention

The invention aims to provide a nucleotide sequence for controlling insect invasion and a method thereof, namely, the RNAi technology is used for down-regulating the expression of a target gene: the ability of an insect to survive, grow, reproduce, colonize a particular environment and/or infest a host is impaired to achieve control of insect infestation and damage caused thereby.

To achieve the above object, the present invention provides an isolated polynucleotide sequence, wherein the polynucleotide is selected from the group consisting of:

(a) 1, a polynucleotide sequence shown in SEQ ID NO; or

(b) 1, wherein a coleopteran insect pest ingests a double-stranded RNA comprising at least one strand complementary to the polynucleotide sequence that inhibits growth of the coleopteran insect pest; or

(c) 1, wherein a coleopteran insect pest ingests a double-stranded RNA comprising at least one strand complementary to the polynucleotide sequence, inhibiting the growth of the coleopteran insect pest; or

(d) 1, wherein a coleopteran insect pest ingests a double-stranded RNA comprising at least one strand complementary to the polynucleotide sequence, inhibiting the growth of the coleopteran insect pest; or

(e) 1, wherein a coleopteran insect pest ingests a double-stranded RNA comprising at least one strand complementary to the polynucleotide sequence that inhibits growth of the coleopteran insect pest; or

(f) 3 to 8 of SEQ ID NO; or

(g) A polynucleotide sequence which hybridizes or is complementary to a polynucleotide sequence defined in any one of (a) to (f) above under stringent conditions.

Further, the polynucleotide sequence also includes a complementary sequence of the polynucleotide sequence.

Still further, the polynucleotide sequence further comprises a spacer sequence.

Preferably, the spacer sequence is SEQ ID NO 11.

On the basis of the technical scheme, the coleoptera insect pests are the diabrotica virgifera.

To achieve the above object, the present invention also provides an expression cassette comprising the polynucleotide sequence under the control of an operably linked regulatory sequence.

In order to achieve the above object, the present invention also provides a recombinant vector comprising the polynucleotide sequence or the expression cassette.

To achieve the above objects, the present invention also provides a use of the polynucleotide sequence for interfering with expression of a coleopteran insect pest target sequence or inhibiting growth of a coleopteran insect pest.

To achieve the above objects, the present invention also provides an interfering ribonucleic acid sequence which, upon ingestion by a coleopteran insect pest, functions to down-regulate the expression of at least one target gene in the coleopteran insect pest, wherein the interfering ribonucleic acid sequence comprises at least one silencing element, wherein the silencing element is a double-stranded RNA region comprising annealed complementary strands, one of which comprises or consists of a nucleotide sequence at least partially complementary to a target sequence within the target gene, the target gene comprising the polynucleotide sequence.

Further, the silencing element comprises or consists of a sequence of at least 15 contiguous nucleotides that is complementary to at least partially complementary to a target fragment within the target sequence.

Further, the silencing element comprises or consists of a sequence of at least 17 contiguous nucleotides that is complementary to at least partially complementary to a target fragment within the target sequence.

Further, the silencing element comprises or consists of a sequence of at least 19 contiguous nucleotides that is complementary to at least partially complementary to a target fragment within the target sequence.

Further, the silencing element comprises or consists of a sequence of at least 21 contiguous nucleotides that is complementary to, at least partially complementary to, a target fragment within the target sequence.

Optionally, the interfering ribonucleic acid sequence comprises at least two silencing elements, each of which comprises or consists of a nucleotide sequence at least partially complementary to a target sequence within the target gene.

Further, the silencing elements each comprise or consist of a different nucleotide sequence that is complementary to a different target sequence.

Still further, the different target sequences are derived from a single one of the target genes or from a different target gene than the target gene.

The target gene different from the target gene is derived from the same coleopteran insect pest or a different coleopteran insect pest.

Preferably, the coleopteran insect pest is diabrotica spp.

On the basis of the technical scheme, the interfering RNA sequence also comprises a spacer sequence.

Specifically, the spacer sequence is SEQ ID NO 11.

To achieve the above objects, the present invention also provides a composition for controlling coleopteran insect pest infestation comprising at least one of the interfering ribonucleic acid sequences and at least one suitable carrier, excipient, or diluent.

Further, the composition comprises a host cell expressing or capable of expressing the interfering ribonucleic acid sequence. In particular, the host cell is a bacterial cell.

Still further, the composition is a solid, liquid or gel. In particular, the composition is an insecticidal spray.

Optionally, the composition further comprises at least one insecticide, said insecticide is a chemical insecticide, a potato tuber-specific protein, a bacillus thuringiensis insecticidal protein, a xenorhabdus insecticidal protein, a photorhabdus insecticidal protein, a bacillus laterosporous insecticidal protein, or a bacillus sphaericus insecticidal protein.

To achieve the above objects, the present invention also provides a use of the coleoptera insect pest infestation control composition for preventing and/or controlling coleoptera insect pest infestation.

Preferably, the coleopteran insect pest is diabrotica spp.

To achieve the above objects, the present invention also provides a method of controlling coleopteran insect pest infestation comprising contacting a coleopteran insect pest with an effective amount of at least one of the interfering ribonucleic acid sequences.

To achieve the above objects, the present invention also provides a method for enhancing resistance of a plant to a coleopteran insect pest, comprising introducing into the plant the polynucleotide sequence or the expression cassette or the recombinant vector or a construct comprising the interfering ribonucleic acid sequence.

To achieve the above objects, the present invention also provides a method for producing a plant for controlling a coleopteran insect pest, comprising introducing into a plant the polynucleotide sequence or the expression cassette or the recombinant vector or a construct comprising the interfering ribonucleic acid sequence.

To achieve the above objects, the present invention also provides a method for protecting a plant from damage caused by a coleopteran insect pest, comprising introducing the polynucleotide sequence or the expression cassette or the recombinant vector or the construct comprising the interfering ribonucleic acid sequence into a plant, the introduced plant functioning to inhibit the growth of the coleopteran insect pest after being ingested by the coleopteran insect pest.

On the basis of the technical scheme, the plant is soybean, wheat, barley, corn, tobacco, rice, rape, cotton or sunflower.

The invention encompasses a method of modulating or inhibiting the expression of one or more target genes in a coleopteran insect pest, said method comprising: introducing part or all of the stabilized double-stranded RNA (such as dsRNA) or modified form thereof (e.g., small interfering RNA sequence) into a cell or extracellular environment in an invertebrate pest insect. In the insect, dsRNA or siRNA enters the cell and inhibits the expression of at least one or more target genes, and this inhibition results in a reduction in the ability of the insect to survive, grow, reproduce, and invade the host.

The invention provides a separated and purified polynucleotide sequence shown as SEQ ID NO. 1. The present invention provides stabilized double stranded RNA molecules for inhibiting expression of target sequences from these sequences and fragments in coleopteran pests. The stabilized double stranded RNA comprises at least two coding sequences arranged in a sense and antisense orientation relative to at least one promoter, wherein the nucleotide sequences comprising the sense and antisense strands are linked or ligated by a spacer sequence of at least about 5-1000 nucleotides, wherein the sense and antisense strands may be of different lengths, and wherein at least one of the two coding sequences has at least 80%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one or more of the nucleotide sequences set forth in SEQ ID No. 1.

When expressed as dsRNA and provided to a pest, the fragment can be defined as causing death, feeding inhibition, blocking or cessation of the pest. The fragment may, for example, comprise at least about 19, 21, 23, 25, 40, 60, 80, 100, 125 or more contiguous nucleotides, or about 19 to about 100 nucleotides, or more, of any one or more of SEQ ID NO. 1 or the complement thereof, such as SEQ ID NO. 3-8. Particularly useful are dsRNA sequences comprising about 19-300 nucleotides that are homologous to a target sequence of a pest. The invention also provides RNA, including dsRNA, expressed from any of the polynucleotide sequences. The sequence selected for expression of the gene inhibitor can be constructed from a single sequence from one or more target pests and used to express RNA that inhibits a single gene or gene family in one or more target pests, or the DNA sequence can be constructed as a chimera from a variety of DNA sequences.

The plants described herein may include any reproductive or propagation material of a plant and may also include plant cells, plant protoplasts, plant tissue cultures, plant calli, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, husks, stems, roots, root tips, and the like.

The diabrotica biflora (Motschulsky) is a holomorphic insect of the genus Flutica in the family of the family Leptoviridae of the order Coleoptera, the eggs, larvae and pupae of which live in the soil, and the adult insects emerge from the soil after eclosion, and are a generation of one year of insects for diapause of the eggs to live through the winter. Hatching the diapause eggs in 5 months every year, and observing larvae in field soil at the beginning of 5-7 months; pupae are visible in the field from the bottom of 6 months to the middle of 7 months; occasionally, adult insects are eclosively flying into corn, soybean and other fields to be damaged in the middle ten days of the 7 month; the emergence peak period is from the bottom of 7 months to the beginning of 8 months, the spawning can be started about 15 days after the emergence, and the spawning period lasts about 1 month.

The larvae of the diabrotica leaves mainly feed the roots of crops in the field, and the larvae are harmful and can not be reflected on the overground part; adults can be found to harm leaves in corn and soybean fields beginning in late 7 months each year; a large amount of adults are mainly harmful to the corn silks from the bottom of 7 months to the beginning of 8 months, the corn silks are bitten off, pollination is seriously affected, sharp spikes and spindle-shaped spikes are caused, and the yield of the corn is reduced; and then, the diabrotica virgifera leafbud is transferred to a soybean field to eat soybean leaves, and can also be transferred to a peripheral vegetable field to be harmful vegetables. From 2009 to 2016, the damage area of the diabrotica phyllotreta to the corn is increased from 1600 ten thousand mu times to nearly 4000 ten thousand mu times, and the occurrence area is doubled by 2 times. And the districts causing the harm are also spread from northwest and the like to northeast, northwest China and other major corn production areas.

Meanwhile, with the continuous promotion of straw returning measures, field humus is continuously abundant and soil surface coverings are increased, so that the difficulty of applying the pesticide to the soil is increased, and the prevention and control of the larvae of the Diabrotica biflora are more and more difficult. That is to say, the straw returning to the field provides natural protection for the larvae of the diabrotica virgifera, which may cause the survival rate of the larvae of the diabrotica virgifera to be greatly increased, thereby causing the population density of the diabrotica virgifera to be increased. The diabrotica adults are good flying jumping insects, and begin to be harmful corns after eclosion in the middle and last 7 months when the corns enter the silking period. At the moment, the corn grows high, the pesticide application difficulty is increased, pesticide application personnel are easily injured by mistake, and meanwhile, the non-selective insecticidal action of the corn can cause damage to crops and non-target organisms. In addition, chemical pesticides may have a cumulative effect in the human body, being mutagens or carcinogens. There is therefore a need for an accurate, environmentally friendly method for controlling the infestation of Diabrotica bimaculata by a simple and easy-to-handle method for farmers. The crop can keep a certain insect-resistant efficacy in the whole growth period by a transgenic mode, and the whole plant can be protected in the whole growth period. Based on the problems, the control of the Diabrotica bimaculata by adopting a transgenic RNAi means provides a whole growth period for the corn, and the mode of preventing and controlling the Diabrotica bimaculata of the whole plant is an optimal solution.

As used herein, "controlling insects" or "controlling pests" or "controlling insect pests" refers to any action that results in the damage caused by an insect being limited and acting on the insect, including, but not limited to, killing the insect, inhibiting the development of the insect, altering the fertility or growth of the insect in such a way that the insect provides less damage to the plant, reducing the number of progeny produced by the insect, producing fewer normal insects, producing insects that are more susceptible to predators, or preventing the insect from gnawing the plant.

A "target gene" as referred to herein is any sequence intended to be down-regulated in an insect. Insect infestation is controlled by down-regulating target genes, for example, by disrupting essential biological processes in the insect. Thus, preferred target genes include, but are not limited to, genes that play a key role in regulating feeding, survival, growth, development, reproduction, invasion, and infestation. When the expression of the target gene is down-regulated or inhibited, at least 30% of the insects are killed; or preventing/retarding/hindering/delaying/hindering growth of at least 30% of the insects, preventing reproduction of at least 30% of the insects, preventing at least 30% of the insects from shifting through the life cycle; or damage caused by an insect and/or a reduced ability of an insect to infest or infest an environment, surface and/or plant or crop species; or at least 30% of the insects stop feeding from their natural food sources (such as plants and plant products). These target genes may be expressed in all or part of the insect cell. Furthermore, these target genes may be expressed only at specific stages of the insect's life cycle, such as the adult or larval or egg stages.

In the present invention, the term "pest" is preferably an insect causing plant attack/infestation and belongs to the order coleoptera, preferably diabrotica purpurea. The terms "infestation," "infestation," and/or "attack" are generally used interchangeably throughout.

The present invention "RNA interference (RNAi)" refers to the phenomenon that some RNA can effectively and specifically block the expression of specific genes in vivo, promote the degradation of mRNA, and induce cells to show the phenotype of specific gene deletion, which is also called RNA interference or interference. RNA interference is a highly specific gene silencing mechanism at the mRNA level.

"nucleic acid" as used herein refers to a single or double-stranded polymer of deoxyribonucleic acid or ribonucleic acid bases read from the 5 'to the 3' end. Alternatively, a "nucleic acid" may also contain non-naturally occurring or altered bases that allow for proper reading by a polymerase without reducing the expression of the polypeptide encoded by the nucleic acid. "nucleotide sequence" refers to the sense and antisense strands of a nucleic acid that exists as separate single strands or in a duplex. "ribonucleic acids" (RNA) include RNAi (RNA interference), dsRNA (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (microRNA), tRNA (transfer RNA, charged with or without the corresponding acylated amino acids), and cDNA and genomic DNA and DNA-RNA hybrids. "nucleic acid fragments", "nucleic acid sequence fragments" or more generally "fragments" will be understood by those skilled in the art as: including genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operator sequences and smaller engineered nucleotide sequences that express or can be engineered to express proteins, polypeptides or peptides.

The "interfering ribonucleic acid" of the present invention encompasses any type of RNA molecule capable of down-regulating or "silencing" the expression of a target gene, including but not limited to sense RNA, antisense RNA, siRNA, miRNA, dsRNA, hairpin RNA, and the like. Methods for determining functional interfering RNA molecules are well known in the art and have been disclosed.

The interfering ribonucleic acids of the present invention effect specific down-regulation of target gene expression by binding to a target sequence within the target gene. Binding occurs due to base pairing between the interfering RNA and the complementary region of the target sequence.

The term "silencing element" refers to a portion or region of an interfering ribonucleic acid that comprises or consists of a nucleotide sequence that is complementary or at least partially complementary to a target sequence within a target gene and that functions as an active portion of the interfering ribonucleic acid to direct down-regulation of expression of the target gene. The silencing element comprises a sequence of at least 15 contiguous nucleotides, preferably at least 18 or 19 contiguous nucleotides, more preferably at least 21 contiguous nucleotides, even more preferably at least 22, 23, 24 or 25 contiguous nucleotides complementary to a target sequence within a target gene, or an interfering ribonucleic acid consisting thereof.

"target gene expression" in the present invention refers to transcription and accumulation of RNA transcripts encoded by a target gene, and/or translation of mRNA into protein.

The term "downregulate" refers to any of the methods known in the art whereby interfering ribonucleic acids reduce the level of primary RNA transcripts, mRNA, or protein produced from a target gene. By down-regulation is meant the situation whereby the level of RNA or protein produced by a gene is reduced by at least 10%, preferably at least 33%, more preferably at least 50%, even more preferably at least 80%. In particular, down-regulation refers to a reduction of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% of the level of RNA or protein produced by one gene in an insect cell as compared to a suitably controlled insect (e.g., an insect that has not been exposed to an interfering ribonucleic acid or has been exposed to a control interfering ribonucleic acid). Methods for detecting a decrease in RNA or protein levels are well known in the art and include RNA solution hybridization, northern hybridization, reverse transcription (e.g., quantitative RT-PCR analysis), microarray analysis, antibody binding, enzyme-linked immunosorbent assay (ELISA), and Western blotting. Meanwhile, down-regulation may also refer to a reduction in the level of RNA or protein, as compared to appropriate insect control, sufficient to result in a detectable change in the insect phenotype, such as cell death, growth arrest, and the like. Thus, downregulation can be measured by phenotypic analysis of insects using techniques conventional in the art.

"inhibition of expression of a target gene" in the context of the present invention refers to a reduction or absence (below a detectable threshold) of the level of protein and/or mRNA product of the target gene. Specificity refers to the ability to inhibit a target gene and thus have no effect on other genes of the cell and no effect on any gene within the cell that produces a dsRNA molecule.

As used herein, "sense" RNA refers to RNA transcripts corresponding to sequences or fragments in the form of mRNA that can be translated into protein by plant cells. "antisense" RNA as used herein refers to RNA that is complementary to all or a portion of the mRNA normally produced in a plant. The complementarity of the antisense RNA can be to any portion of the transcript of a particular gene, i.e., the 5 'non-coding sequence, the 3' non-coding sequence, an intron, or a coding sequence. In the present invention, "RNA transcript" refers to a product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfectly complementary copy of the DNA sequence, it is referred to as the primary transcript, or it may be RNA obtained from post-transcriptional processing of the primary transcript, which is referred to as mature RNA.

The interfering ribonucleic acids of the present invention down-regulate the expression of the gene by RNA interference or RNAi. RNAi is a sequence-specific gene regulation method typically mediated by double-stranded RNA molecules, such as siRNA. siRNA comprises one sense RNA strand that anneals by complementary base pairing to an antisense RNA strand. The sense strand or "guide strand" of the siRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence located within an RNA transcript of a target gene. Thus, the sense strand of the siRNA is capable of annealing to RNA transcripts by Waston-Crick-type (Waston-Crick-type) base pairing and targeting the RNA for degradation within a cellular complex known as the RNAi-induced silencing complex or RISC. In the case of the preferred interfering ribonucleic acids of the present invention, the silencing element may be a double stranded region comprising annealed complementary strands, wherein at least one strand comprises or consists of a nucleotide sequence that is complementary or at least partially complementary to a target sequence within a target gene. The double-stranded region has a length of at least about 15 to about 25 base pairs, or about 25 to about 100 base pairs, or even about 3000 base pairs.

The dsRNA molecules of the invention can serve as precursors to active siRNA molecules that direct RNA transcripts to the RISC complex for subsequent degradation. dsRNA molecules present in an organism or its cellular environment can be taken up by the organism and processed by an enzyme called DICER to give siRNA molecules. Alternatively, the dsRNA molecule may be produced in vivo, i.e., transcribed from one or more polynucleotides encoding the dsRNA present in a cell (e.g., a bacterial cell or a plant cell), and processed by DICER in a host cell or preferably in an insect cell after uptake of a longer precursor dsRNA. The dsRNA can be formed from two separate (sense and antisense) RNA strands that anneal by means of complementary base pairing. Alternatively, the dsRNA may be a single strand that is capable of refolding itself to form a hairpin RNA or stem-loop structure. In the case of an RNA, the double-stranded region or "stem" is formed from two regions or segments of the RNA that are essentially inverted repeats of each other and have sufficient complementarity to allow formation of a double-stranded region. One or more functional double-stranded silencing elements may be present in this "stem region" of the molecule. Inverted repeat regions are typically separated by a region or segment of the RNA referred to as a "loop" region. This region may comprise any nucleotide sequence that confers sufficient flexibility to allow self-pairing to occur between the flanking complementary regions of the RNA, and in general, the loop region is substantially single-stranded and serves as a spacer sequence between the inverted repeats.

The interfering ribonucleic acid of the present invention comprises at least one double-stranded region, typically a silencing element of the interfering ribonucleic acid, which comprises a sense RNA strand that anneals by complementary base pairing to an antisense RNA strand, wherein the sense strand of the dsRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence located within an RNA transcript of a target gene. The silencing element or at least one strand thereof (when the silencing element is double-stranded) may be fully complementary or partially complementary to a target sequence of the target gene. The term "fully complementary" refers to the silencing element nucleotide sequence of all bases complementary or "matching" with the target sequence of bases. The term "at least partially complementary" means that there is less than 100% match between the bases of the silencing element and the bases of the target sequence. One skilled in the art will appreciate that in order to mediate down-regulation of target gene expression, the silencing element need only be at least partially complementary to the target sequence. It is known in the art that RNA sequences with insertions, deletions, and mismatches relative to the target gene can still be effective in RNAi. Preferably, the silencing element shares at least 80% or 85% sequence identity with the target sequence of the target gene, preferably at least 90% or 95% sequence identity, or more preferably at least 97% or 98% sequence identity and even more preferably at least 99% sequence identity. Alternatively, the silencing element may comprise 1,2 or 3 mismatches over each length of 24 partially complementary nucleotides, as compared to the target sequence. It is well known to those skilled in the art that the degree of complementarity shared between the silencing element and the target sequence varies depending on the insect species whose target gene expression is to be down-regulated.

The target sequence in the present invention may be selected from any suitable region or nucleotide sequence of the target gene or its RNA transcript. For example, the target sequence may be located within the 5'UTR or 3' UTR of the target gene or RNA transcript, or within an exon or intron region of the gene.

The interfering ribonucleic acids of the present invention may comprise or consist of one or more silencing elements, wherein each silencing element comprises or consists of a nucleotide sequence that is at least partially complementary to a target sequence within a target gene and functions to down-regulate expression of the target gene upon ingestion by an insect. The term "plurality" means at least two, at least three, at least four, etc. and up to at least 10, 15, 20 or at least 30. Interfering ribonucleic acids comprise multiple copies of a single silencing element, i.e., repeats of a silencing element that bind to a particular target sequence within a particular target gene. The silencing element within the interfering ribonucleic acid may also comprise or consist of a different nucleotide sequence that is complementary to a different target sequence. It should be clear that combinations of multiple copies of the same silencing element combined with silencing elements bound to different target sequences are also within the scope of the invention.

In order to achieve down-regulation of specific target genes in coleopteran insects, different target sequences can be derived from a single target gene in an insect. In this case, the silencing elements can be combined in the interfering ribonucleic acid in the original order in which the target sequence is present in the target gene, or the silencing elements can be shuffled and randomly combined in any hierarchical order in the environment of the interfering ribonucleic acid as compared to the order of the target sequence in the target gene.

Alternatively, the different target sequences represent a single target gene, but are derived from different insect species.

Alternatively, different target sequences may be derived from different target genes. If the interfering ribonucleic acid is used to prevent and/or control pest infestation, it is preferred that the different target genes are selected from the group of genes that regulate essential biological functions of the insect, including but not limited to survival, growth, development, reproduction, and pathogenicity. The target genes may regulate the same or different biological pathways or processes.

In the present invention, the different genes targeted by different silencing elements are derived from the same insect. This approach can be used to achieve enhanced attack against a single insect. In particular, different target genes may be differentially expressed at different stages of the insect life cycle, such as mature adult, immature larval, and egg stages. Accordingly, the interfering ribonucleic acids of the present invention may be used to prevent and/or control insect infestation during more than one stage of the insect life cycle. Alternatively, different genes targeted by different silencing elements are derived from different insects, and thus, the interfering ribonucleic acids of the present invention may also be used to simultaneously prevent and/or control the infestation of more than one insect.

The silencing element of the invention may be a contiguous region of interfering ribonucleic acid or may be separated by the presence of a linker sequence. The adaptor sequence may comprise a short random nucleotide sequence that is not complementary to any target sequence or target gene. The linker sequence may be a conditional self-cleaving RNA sequence, preferably a pH-sensitive linker or a hydrophobic-sensitive linker. The linker may also comprise a nucleotide sequence equivalent to the intron sequence. The linker sequence may be in the range of 1 base pair to about 10000 base pairs in length, provided that the linker does not impair the ability of the interfering ribonucleic acid to down-regulate gene expression.

In addition to one or more silencing elements and any linker sequences, the interfering ribonucleic acids of the invention may comprise at least one additional polynucleotide sequence. The additional polynucleotide sequence is selected from (1) a sequence capable of protecting an interfering ribonucleic acid from RNA processing; (2) sequences that affect the stability of interfering ribonucleic acids; (3) A sequence that allows the binding of a protein to facilitate the uptake of interfering ribonucleic acids by insect cells; (4) sequences that facilitate large-scale production of interfering ribonucleic acids; (5) Sequences that are aptamers that bind to receptors or to molecules on the surface of insect cells to facilitate uptake; or (6) a sequence that catalyzes an interfering ribonucleic acid processing in an insect cell and thereby enhances the efficacy of the interfering ribonucleic acid.

The interfering ribonucleic acids of the present invention need to be long enough to be taken up by the cells of the insect and to down regulate the target genes of the insect. The upper limit of the length can depend on (1) the requirement that the interfering ribonucleic acid be taken up by the insect cell and (2) the requirement that the interfering ribonucleic acid be processed in the insect cell to mediate gene silencing via the RNAi pathway, and can also be tailored by the production method and formulation used to deliver the interfering ribonucleic acid into the cell. Preferably, the interfering ribonucleic acids of the invention will be between 19 and 10000 nucleotides in length, preferably between 50 and 5000 nucleotides or between 100 and 2500 nucleotides in length, more preferably between 80 and 2000 nucleotides in length.

The interfering ribonucleic acids of the present invention may comprise non-natural backbone linkages or modifications of DNA bases, non-natural bases or sugar-phosphate backbones, for example to enhance stability during storage or to enhance resistance to nuclease degradation. In addition, the interfering RNA can be manually or automatically generated by the technicians in this field by chemical or enzymatic methods. Alternatively, the interfering ribonucleic acid may be transcribed from the polynucleotide encoding it. Thus, the invention provides isolated polynucleotides encoding any of the interfering ribonucleic acids.

The polynucleotides of the invention may be inserted into DNA constructs or vectors known in the art by conventional molecular cloning techniques. The DNA construct may be a recombinant DNA vector, such as a bacterial, viral or yeast vector. The DNA construct is an expression construct and the polynucleotide is operably linked to at least one control sequence capable of driving expression of the polynucleotide sequence. The term "control sequence" refers to any nucleotide sequence capable of affecting the expression of an operably linked polynucleotide, including but not limited to promoters, enhancers, and other naturally occurring or synthetic transcriptional activation elements. The control sequence may be located at the 5 'or 3' end of the polynucleotide sequence. The term "operably linked" refers to a functional linkage between a control sequence and a polynucleotide sequence such that the control sequence drives expression of the polynucleotide. The operably linked elements may be continuous or discontinuous.

The regulatory sequence of the present invention may be a promoter, preferably, the promoter is a promoter expressible in plants, and the "promoter expressible in plants" refers to a promoter which ensures the expression of the polynucleotide linked thereto in plant cells. The promoter expressible in plants may be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, 35S promoter derived from cauliflower mosaic virus, maize ubi promoter, promoter of rice GOS2 gene, and the like. Alternatively, the promoter expressible in a plant may be a tissue-specific promoter, i.e., a promoter that directs expression of the coding sequence at a higher level in some tissues of the plant, e.g., in green tissues, than in other tissues of the plant (as can be determined by conventional RNA assays), e.g., the PEP carboxylase promoter. Alternatively, the promoter expressible in plants may be a wound-inducible promoter. A wound-induced promoter or a promoter that directs a wound-induced expression pattern means that when a plant is subjected to mechanical or insect feeding induced wounds, the expression of the polynucleotide under the control of the promoter is significantly increased compared to under normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, promoters of potato and tomato protease-inhibitory genes (pin I and pin II) and maize protease-inhibitory gene (MPI).

Alternatively, one or more transcription termination sequences may be incorporated into an expression construct of the invention. The term "transcription termination sequence" encompasses a control sequence at the end of a transcription unit that signals transcription termination, 3' processing, and polyadenylation of a primary transcript. Additional regulatory elements, including but not limited to transcriptional or translational enhancers, may be incorporated into the expression construct, for example, the dual enhancer CaMV35S promoter.

The method for producing any interfering ribonucleic acid in the invention comprises the following steps: (1) Contacting a polynucleotide encoding the interfering ribonucleic acid or a DNA construct comprising the polynucleotide with a cell-free component; (2) The polynucleotide encoding the interfering ribonucleic acid or a DNA construct comprising the polynucleotide is introduced (e.g., by transformation, transfection, or injection) into a cell.

In the present invention, the host cell comprising any one of the interfering ribonucleic acids of the present invention, any one of the polynucleotides of the present invention, or a DNA construct comprising these polynucleotides, may be a prokaryotic cell, including but not limited to gram-positive and gram-negative bacterial cells; or a eukaryotic cell, including but not limited to a yeast cell or a plant cell. Preferably, the host cell is a bacterial cell or a plant cell. The polynucleotide or DNA construct of the invention may be present or maintained as an extrachromosomal element in the host cell, or may be stably incorporated into the host cell genome.

In the present invention, where the interfering ribonucleic acid is expressed in a host cell and/or is used to prevent and/or control insect infestation of a host organism, it is preferred that the interfering ribonucleic acid does not exhibit a significant "off-target" effect, i.e., the interfering ribonucleic acid does not affect the expression of non-target genes in the host. Preferably, the silenced gene does not exhibit significant complementarity to a nucleotide sequence other than the intended target sequence of the target gene. The silencing element exhibits less than 30%, more preferably less than 20%, more preferably less than 10% and even more preferably less than 5% sequence identity with any gene of the host cell or organism. If genomic sequence data is available for the host organism, one can cross-test for identity to the silencing element using standard bioinformatic tools. There is no sequence identity between the silencing element and the gene from the host cell or organism over a region of 17 contiguous nucleotides, more preferably over a region of 18 or 19 contiguous nucleotides, and most preferably over a region of 19 or 20 or 21 contiguous nucleotides.

The composition for preventing and/or controlling insect infestation of the present invention comprises at least one interfering ribonucleic acid and optionally at least one suitable carrier, excipient or diluent, wherein the interfering ribonucleic acid, upon ingestion by an insect, functions to down-regulate the expression of a target gene in said insect. The interfering ribonucleic acid comprises or consists of at least one silencing element, and said silencing element is a double stranded RNA region comprising annealed complementary strands, wherein one strand (the sense strand) comprises a nucleotide sequence that is at least partially complementary to a target sequence within a target gene. Target genes include, but are not limited to, genes that regulate insect survival, growth, development, reproduction, and pathogenicity. Optionally, the composition comprises at least one host cell comprising at least one interfering ribonucleic acid or a DNA construct encoding the interfering ribonucleic acid and optionally at least one suitable carrier, excipient or diluent, wherein the interfering ribonucleic acid functions to down-regulate the expression of a target gene in an insect upon ingestion of the host cell by said insect.

The compositions of the present invention may take any suitable physical form for application to insects. For example, the composition may be in solid form (powder, pellet or bait), liquid form (including as an insecticidal spray) or gel form. The composition may be a coating, paste or powder which may be applied to a substrate to protect the substrate from insects. The composition may be used to protect any substrate or material susceptible to insect infestation or damage caused by insects.

The nature of the excipients and the physical form of the composition may vary depending on the nature of the substrate that it is desired to treat. For example, the composition may be a liquid that is brushed or sprayed on or printed into the material or substrate to be treated; or a coating or powder which is applied to the material or substrate to be treated.

In the present invention, the composition may be in the form of a bait. The bait is used to attract insects to contact the composition. Upon contact therewith, the composition is then internalized by the insect, e.g., through ingestion, and mediates RNAi, thereby killing the insect. The bait may comprise a food, such as a protein-based food, for example fish meal. Boric acid may also be used as a bait. The bait may depend on the species targeted. An attractant may also be used, for example, the attractant may be a pheromone, such as a male or female sex pheromone. The attractant acts to attract insects to the composition and may be targeted to a particular insect or may attract a full range of insects, increasing the chance of contact of these attracted insects with the composition of the invention, and thus serving the purpose of killing a large number of insects. The bait may be in any suitable form, such as a solid, paste, pellet or powder form.

The bait may also be carried back to the community by the insect. The bait can then serve as a food source for the other members of the community, thereby providing an effective control of a large number of insects and potentially the entire insect community. The bait may also be provided in a suitable "housing" or "trap".

In addition, compositions that come into contact with insects may remain on the epidermis of the insect. When cleaning, whether an individual insect cleaning itself or the insects clean each other, these compositions can be ingested and can thereby mediate their effects in the insects. This requires that the composition be sufficiently stable so that the interfering ribonucleic acid remains intact and capable of mediating RNAi even after exposure to external environmental conditions for a period of time (e.g., days).

The composition of the present invention may be provided in the form of a spray. Thus, the human user can directly spray the composition on the insects. The composition is then internalized by the insect, where it can mediate RNA interference, thereby controlling the insect. The spray is preferably a pressurized/atomised spray or a pump spray. These particles may be of a suitable size so that they adhere to the insects, for example to the exoskeleton, and may be absorbed therefrom.

The carrier of the composition of the present invention is an electrostatically charged powder or granule which adheres to the insects. Alternatively, the carrier of the composition may comprise magnetic particles which adhere to the insect cuticle. Optionally, the carrier of the composition comprises metal particles which are initially unmagnetized but capable of becoming magnetically polarized upon being subjected to an electric field provided by the insect body. Preferably, the composition is incorporated into a vector that increases the uptake of interfering RNA in insects. The carrier may be a lipid-based carrier, preferably comprising one or more of: oil-in-water emulsions, micelles, cholesterol, lipopolyamines, and liposomes. Other agents that facilitate uptake of the constructs of the invention are well known to those skilled in the art and include polycations, dextrans and cationic lipids, such as CS096, CS102, and the like. Optionally, the carrier of the composition is a nucleic acid condensing agent, preferably the nucleic acid condensing agent comprises spermidine or protamine sulfate or derivatives thereof.

Where the compositions of the present invention are suitable for use in preventing and/or controlling insect infestation of plants, the compositions may comprise an agriculturally suitable carrier. The carrier may be any material which is tolerated by the plant to be treated, which does not cause undue damage to the environment or other organisms therein and which allows the interfering ribonucleic acids to remain effective against insects. In particular, the compositions of the present invention may be formulated for delivery to plants in accordance with conventional agricultural practices used in the biopesticide industry. The composition may contain additional components capable of performing other functions including, but not limited to, (1) enhancing or promoting uptake of interfering ribonucleic acids by insect cells and (2) stabilizing the active components of the composition. Such additional components contained in the composition comprising the interfering ribonucleic acid can be yeast tRNA or yeast total RNA.

The composition may be formulated for direct administration or as a concentrated form of the primary composition requiring dilution prior to use. Alternatively, the composition may be supplied in the form of a kit comprising the interfering ribonucleic acid or the host cell comprising/expressing the interfering ribonucleic acid in one container and a suitable diluent or vector for the RNA or host cell in a separate container. In the use of the invention, the composition may be applied to the plant or any part of the plant at any stage of plant development, for example, the composition is applied to the aerial parts of the plant during the plant's cultivation in the field; the composition is applied to the plant seed while the plant seed is in storage or after it is planted in soil. In summary, it is important to obtain good control of insects early in the growth of the plant, as this is the period in which the plant is likely to be most severely damaged by insects.

In the present invention, the composition can be applied to the environment of the insect by various techniques including, but not limited to, spraying, atomizing, dusting, scattering, pouring, coating seeds, seed treatment, introduction into soil, and introduction into irrigation water. In treating plants susceptible to insect infestation, the composition can be delivered to the plant or part of the plant prior to the emergence of the insect (for prophylactic purposes) or after the onset of signs of insect infestation (for control purposes).

The compositions of the present invention may be formulated to comprise at least one additional active agent. Thus, the composition may be provided in the form of a "kit of parts" comprising in one container the composition comprising the interfering ribonucleic acid and in a separate container one or more suitable active ingredients, such as chemical or biological pesticides. Alternatively, the composition may be provided in the form of a mixture which is stable and used in combination with each other.

Suitable active ingredients that can act in a complementary manner on the interfering ribonucleic acids of the invention include, but are not limited to, the following: chlorpyrifos, allethrin, resmethrin, tetrabromoethyl, dimethanol-cyclopropanecarboxylic acid (generally included in the liquid composition); and hydramethylnon, abamectin, chlorpyrifos, sulfluramid, hydroprene, fipronil (GABA receptor), isopropylphenylmethyl carbamate, indoxacarb, novaluron (chitin synthesis inhibitor), imiprothrin, abamectin (glutamate-gated chloride channel), imidacloprid (acetylcholine receptor) (generally included in the bait composition). Preferably, the active ingredient is known to be an insecticide, such as hydramethylnon and abamectin, for health and environmental considerations.

The composition of the invention may be formulated to include at least one additional agronomic agent, such as a herbicide or an additional pesticide. "additional pesticide" or "second pesticide" refers to a pesticide other than the first or original interfering RNA molecule of the composition. Alternatively, the composition of the invention may be delivered in combination with at least one other agronomic agent (e.g., a herbicide or a second pesticide). The composition may be provided in combination with a herbicide selected from any herbicide known in the art, such as glyphosate, 2,4-D, imidazolinones, sulfonylureas, and bromoxynil. The composition may also be provided in combination with at least one additional insecticide, which may be selected from any insecticide known in the art and/or may comprise an interfering ribonucleic acid that, upon ingestion by an insect, functions to down-regulate expression of a target gene in said insect. The target pest is an insect and the interfering ribonucleic acid is any one selected from the interfering ribonucleic acids of the invention. The additional pesticide comprises an interfering ribonucleic acid that functions to down-regulate the expression of a known gene in any target pest. The composition of the original interfering ribonucleic acid and the second or additional pesticide may be targeted to the same or different insects. For example, the primary interfering ribonucleic acid and the second pesticide may target different insects or may target insects of different families or classes, such as fungi or nematodes or insects. It will be clear to one of ordinary skill in the art how to test the synergistic effect of interfering ribonucleic acids in combination with other agronomic agents. Preferably, the composition comprises a first interfering ribonucleic acid and one or more additional pesticides, each of which is toxic to the same insect, wherein the one or more additional pesticides is selected from the group consisting of potato tuber-specific protein, bacillus thuringiensis insecticidal protein, xenorhabdus insecticidal protein, phoma glabrata insecticidal protein, bacillus laterosporous insecticidal protein, bacillus sphaericus insecticidal protein, and lignin. The different components may be delivered to the area or organism to be treated simultaneously or sequentially.

The methods of the invention for preventing and/or controlling insect infestation comprise contacting an insect with an effective amount of at least one interfering ribonucleic acid, wherein the interfering ribonucleic acid, when ingested by the insect, functions to down-regulate expression of an essential insect target gene. The essential target gene may be any insect gene involved in regulating essential biological processes required by the insect to initiate or sustain infestation, including but not limited to survival, growth, development, reproduction, and pathogenicity.

The method of the invention for preventing and/or controlling insect infestation in a field of crop plants comprises expressing an effective amount of the interfering ribonucleic acid in said plants, and in the case of the method for controlling insect infestation, the term "effective amount" refers to the amount or concentration of the interfering ribonucleic acid required to produce a phenotypic effect on the insect such that the number of insects infesting the host organism is reduced and/or the amount of damage caused by the insect is reduced. The phenotypic effect may be death of the insect and at least 20%, 30%, 40%, preferably at least 50%, 60%, 70%, more preferably at least 80% or 90% mortality of the insect compared to a control insect is achieved using interfering RNA. Phenotypic effects may also include, but are not limited to, hampering insect growth, feeding cessation, and reducing egg production. Thus, the total number of insects which infest the host organism may be reduced by at least 20%, 30%, 40%, preferably at least 50%, 60%, 70%, more preferably at least 80% or 90% compared to the control insects. Alternatively, the damage caused by the insect may be reduced by at least 20%, 30%, 40%, preferably at least 50%, 60%, 70%, more preferably at least 80% or 90% compared to a control insect. Thus, the present invention may be used to achieve at least 20%, 30%, 40%, preferably at least 50%, 60%, 70%, more preferably at least 80% or 90% insect control.

The methods and compositions of the invention can be used to limit or eliminate coleopteran pest infestation, preferably diabrotica biflora, within or on the surface of any pest host, pest consortium, or environment in which the pest is present, by providing one or more compositions of the invention comprising dsRNA molecules in the diet of the pest. The method is particularly beneficial for preventing insects from attacking plants, pests being defined as digestive system pH of about 4.5 to about 9.5, about 5 to about 9, about 6 to about 7 and about pH7.0.

The nucleotide sequences of the present invention may comprise inverted repeats separated by "spacer sequences". The spacer sequence may be a region comprising any nucleotide sequence that promotes secondary structure formation between each repeat, if desired. The spacer sequence is part of the sense or antisense coding sequence for the mRNA. Alternatively, the spacer sequence may comprise any combination of nucleotides or homologues thereof capable of covalent attachment to a nucleic acid molecule. The spacer sequence may comprise a nucleotide sequence of at least about 10-100 nucleotides in length, alternatively at least about 100-200 nucleotides in length, at least about 200-400 nucleotides in length, or at least about 400-500 nucleotides in length.

When the interfering ribonucleic acid is "introduced" into a plant in the present invention, it is meant to occur by direct transformation methods such as Agrobacterium-mediated transformation of plant tissue, microprojectile emission bombardment, electroporation, and the like; or may be performed by crossing a plant having a heterologous nucleotide sequence with another plant such that the progeny have the nucleotide sequence incorporated into their genomes. Such breeding techniques are well known to those skilled in the art.

The invention provides a nucleotide sequence for controlling insect invasion and a method thereof, and the nucleotide sequence has the following advantages:

1. the invention discloses a plurality of target sequences of a target gene c40514 for controlling coleoptera insect pest diabrotica, and simultaneously verifies that a nucleic acid inhibitor obtained based on the target sequences can be directly used for controlling coleoptera insect pest invasion.

2. Is highly specific. The target sequence for controlling coleoptera insect pests Diabrotica highly and specifically acts on the Diabrotica and species which are close to the relativity of the Diabrotica and have consistent sequences.

3. Avoid the generation of resistance. The invention does not depend on the combination of specific dsRNA and receptor protein in the insect body, and can effectively avoid similar risks of Bt toxic protein resistance of the insect.

4. The RNAi technology used by the invention has high efficiency and specificity, can directly apply the obtained dsRNA to the field for controlling the attack of coleoptera insect pests, and has the advantages of convenience, low cost and good environmental compatibility.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is an electrophoresis chart of the expression level of a target gene c40514 of the nucleotide sequence and the method for controlling insect infestation according to the present invention;

FIG. 2 is a schematic diagram of a recombinant expression vector DBNR40514C1 vector of the nucleotide sequence and method for controlling insect infestation.

Detailed Description

The technical scheme of the nucleotide sequence and the method for controlling insect invasion of the invention is further illustrated by the specific examples.

First example, determination of Diabrotica Blumei target sequence

1. Extraction of total RNA of Diabrotica biflora

Taking the first hatching beetle of Diabrotica biflora as a material, extracting RNA by a conventional Trizol method, purifying by a conventional method, treating with DNA enzyme to obtain a protein with the concentration of more than or equal to 300 ng/mu L, the total amount of more than or equal to 6 mu g and OD _260/280 1.8-2.2 total RNA samples.

2. Isolation of mRNA and Synthesis of cDNA

PolyA-bearing mRNA was isolated using oligo-dT-bearing magnetic beads, and the first strand cDNA was synthesized using random hexamers and the Superscript II reverse transcriptase kit from Invitrogen.

3. Screening out 1 target gene

The target gene c40514 of 1 Diabrotica diabrotica is screened out from the larva library with moderate expression amount and possibly participating in important metabolic pathway, the full-length nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.

4. Selection of target sequences

6 target sequences of different positions and/or different lengths of the ORF of the target gene c40514 were selected as shown in table 1:

sequence information of the 6 target sequences in tables 1

Target sequences	Serial number
		c40514_g1-01	SEQ ID NO:3
c40514_g1-02	SEQ ID NO:4
		c40514_g1-03	SEQ ID NO:5
c40514_g1-04	SEQ ID NO:6
		c40514_g1-05	SEQ ID NO:7
c40514_g1-06	SEQ ID NO:8

Second example, obtaining dsRNA

Referring to the MEGAscript RNAi Kit specification of the thermo Fisher company, 6 pieces of dsRNA of the target sequences are synthesized, namely c40514_ g1-01 to c40514_ g1-06; the sizes of the products were checked by agarose electrophoresis at a mass concentration of 1%, and the concentrations of c40514_ g1-01 to c40514_ g1-06 were measured by NanoDrop 2000 (Thermo Scientific), respectively.

Third example, evaluation of control ability of Diabrotica Bimaculata by feeding dsRNA

The separated and purified c40514_ g1-01 to c40514_ g1-06 are respectively and uniformly mixed and added into the feed according to the proportion of 50 μ g/g and 5 μ g/g feed (the feed formula refers to the Development of an aromatic di et for the western corn rootworm, entomologia Experimentalis et application 105, 1-11, 2002.) to respectively obtain c40514_ g1-01-50 to c40514_ g1-06-50 feed and c40514_ g1-01-5 to c40514_ g1-06-5 feed, wherein the control feed is added with CK irrelevant dsRNA (SEQ ID NO: 17), and other conditions are completely consistent. The method comprises the following steps of feeding rudinidae hatched larvae of diabrotica by using a prepared feed, putting 30 rudinidae hatched larvae with the hatching time not exceeding 24 hours into each dish, replacing the feed mixed with dsRNA every two days and feeding the feed to the 14 th day, counting the insect death rate every two days from the beginning of feeding, and measuring the expression quantity of a target gene from the 0 th day, the 4 th day and the 8 th day from the beginning of feeding, wherein the specific method comprises the following steps:

301, collecting the insect bodies fed with the c40514_ g1-01-50 to c40514_ g1-06-50 feed and the c40514_ g1-01-5 to c40514_ g1-06-5 feed on the 0 th, 4 th and 8 th days respectively, and freezing and storing the insect bodies with liquid nitrogen;

step 302, respectively extracting the total RNA of the polypide by adopting a Trizol method;

step 303, carrying out reverse transcription on the total RNA of the polypide respectively by adopting a full-type gold kit to obtain cDNA;

304, carrying out PCR amplification by using Ubiquitin-C as an internal reference gene, and carrying out agarose gel electrophoresis with the mass concentration of 1% on 10uL of an amplification product after amplification.

Each treatment was replicated 5 times in the above experiment, and the statistical results are shown in fig. 1 and table 2.

The results of measuring the expression amount of the target gene in fig. 1 show that the dsRNA (50 mug/g) of the target sequence c40514_ g1-01 has a significant inhibiting effect on the expression of the target gene c40514 in the diabrotica, the expression amount of the target gene c40514 is obviously reduced at the 4 th day of feeding, and the expression of the target gene c40514 is hardly detected at the 8 th day.

The dsRNA feeding result in table 2 shows that dsRNA of target sequences c40514_ g1-01 to c40514_ g1-06 of target genes c40514 has obvious lethal effect on Diabrotica, especially no survival larva in most of the repetition of 50ug/g concentration test on the 14 th day of feeding.

TABLE 2 survival rate test results of dsRNA fed to Diabrotica biflora

Material numbering

DAI0

DAI2

DAI4

DAI6

DAI8

DAI10

DAI12

DAI14

CK-d s RNA

100％±0％

100％±0％

98％±3％

95％±4％

91％±8％

88％±9％

85％±11％

83％±11％

C40514_g1-01-50

100％±0％

100％±0％

98％±3％

93％±7％

88％±11％

67％±21％

57％±23％

38％±25％

c40514_g1-01-5

100％±0％

100％±0％

97％+3％

93％+6％

82％+9％

66％+148％

53％+17％

43％+14％

c40514_g1-02-50

100％±0％

100％±0％

97％±3％

96％±3％

88％±9％

55％±26％

31％±31％

17％±26％

c40514_g1-02-5

100％±0％

100％±0％

92％+3％

91％+5％

85％+12％

68％+6％

50％+11％

49％+11％

c40514_g1-03-50

100％±0％

100％±0％

98％+3％

91％+6％

92％+10％

69％+13％

34％+14％

14％+10％

c40514_g1-03-5

100％±0％

100％±0％

90％+3％

92％+5％

81％+8％

57％+10％

56％+11％

44％+12％

c40514_g1-04-50

100％±0％

100％±0％

100％+4％

91％+4％

91％+8％

60％+8％

31％+13％

12％+13％

c40514_g1-04-5

100％±0％

100％±0％

94％+5％

95％+5％

85％+6％

69％+8％

51％+9％

47％+10％

c40514_g1-05-50

100％±0％

100％±0％

100％+1％

92％+7％

90％+9％

68％+12％

34％+10％

27％+14％

c40514_g1-05-5

100％±0％

100％±0％

97％+2％

93％+7％

85％+12％

62％+12％

53％+10％

46％+8％

c40514_g1-06-50

100％±0％

100％±0％

94％+1％

94％+6％

90％+8％

57％+12％

30％+13％

28％+9％

c40514_g1-06-5

100％±0％

100％±0％

98％+0％

92％+8％

80％+10％

52％+8％

50％+7％

48％+11％

Fourth embodiment, interference with expression of the same Gene from different insects has unexpected technical effects

The 54kDa protein of the signal recognition particle belongs to one peptide chain in a signal recognition particle complex, and the main function of the 54kDa protein is that when the pre-secretory protein is exposed from the ribosome, the protein is rapidly bound to the signal sequence of the pre-secretory protein and transferred to the translocation-associated membrane protein. Related literature indicates that the expression of the coding gene of the 54kDa protein of the interfering signal recognition particle can generate lethal effect on various coleoptera insects, for example, julia Ulrich et al (2015) reports that RNAi interference is carried out on the coding gene of the protein in the tribolium castaneum in an injection mode (injection sequence code iB _ 00404), and the tribolium castaneum is found to be basically completely lethal in about 4 days of injection. RNAi interference of the gene encoding this protein in Drosophila by injection (Table 1) was also reported by Avet-Rochex et al (2010), with results indicating that the injection is essentially all lethal to the fly.

Based on the literature report and the high homology of the sequence, the coding gene of the protein in the diabrotica virens is screened out, and a sequence M1 at a corresponding position is selected according to the sequences injected by the tribolium castaneum and the drosophila melanogaster, and is shown as SEQ ID NO. 18; the sequence M2 of the non-corresponding position is shown as SEQ ID NO. 19. The method of feeding dsRNA (feed ratio of 50. Mu.g/g) in the third embodiment of the invention was used to identify the control ability of Diabrotica biflora. As shown in table 3, the experimental results show that: neither the sequence M1 at the corresponding position nor the sequence M2 at the non-corresponding position can produce a significant lethal effect on Diabrotica biflora, and is substantially different from the control. Similar experimental results are verified in WO 2018/026770, and the obtained transcriptome is verified by RNAi lethal genes such as nematode and fruit fly, that is, RNAi interference is performed on corresponding genes in corn rootworm according to a plurality of known lethal genes in nematode and fruit fly, and no obvious lethal effect is basically obtained. In conclusion, the technical effects of interfering with the expression of the same gene in different insects are unpredictable and are not necessarily linked to the technical effects and sequence homology of known interferences.

TABLE 3 lethality test results of dsRNA fed to Diabrotica biflora

Material numbering

DAI

4

DAI 6

DAI 8

DAI 10

DAI 12

DAI 14

CK-dsRNA

96％±6％

85％±9％

75％±16％

71％±16％

69％±13％

69％±14％

M1-dsRNA-50

98％±3％

92％±6％

89％±7％

83％±9％

69％±15％

63％±18％

M2-dsRNA-50

91％±8％

88％±10％

84％±11％

76％±13％

69％±15％

67％±17％

Fifth example construction of plant expression vectors

Two expression cassettes were synthesized in the order of p35S-RX-tNos-p35S-Hpt-tNos (X: 1-6) and linked to a plant expression vector by EcoRV and BamHI and named DBNR40514CX (X: 1-6) with DBNR40514C1 vector as schematically shown in FIG. 2 (Kan: kanamycin gene; RB: right border; pr35S: cauliflower mosaic virus 35S (SEQ ID NO: 9); R1 (SEQ ID NO: 10): g1-01 nucleotide sequence (g 1-01 is the target sequence 1, SEQ ID NO.

Transforming the recombinant expression vector DBNR40514C1 into an Escherichia coli T1 competent cell by a heat shock method, wherein the heat shock condition is as follows: 50 μ L of Escherichia coli T1 competent cells, 10 μ L of plasmid DNA (recombinant expression vector DBNR40514C 1), and 42 ℃ water bath for 30s; shaking at 37 deg.C for 1h (shaking table at 100 rpm); then, the cells were cultured on LB solid plates (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50mg/L Kanamycin (Kanamycin) at 37 ℃ for 12 hours, white colonies were picked up, and the cells were cultured overnight at 37 ℃ in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, kanamycin 50mg/L, pH adjusted to 7.5 with NaOH). Extracting the plasmid by an alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial solution with 100 μ L ice-precooled solution I (25 mM Tris-HCl, 10mM EDTA (ethylene diamine tetraacetic acid), 50mM glucose, pH 8.0); add 200. Mu.L of freshly prepared solution II (0.2M NaOH,1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix and place on ice for 3-5min; adding 150 μ L ice-cold solution III (3M potassium acetate, 5M acetic acid), mixing, and standing on ice for 5-10min; centrifuging at 4 deg.C and 12000rpm for 5min, adding 2 times volume of anhydrous ethanol into the supernatant, mixing, and standing at room temperature for 5min; centrifuging at 4 deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; the precipitate was dissolved by adding 30. Mu.L of TE (10 mM Tris-HCl,1mM EDTA, pH 8.0) containing RNase (20. Mu.g/mL); bathing at 37 deg.C for 30min to digest RNA; storing at-20 deg.C. The extracted plasmid is sequenced and identified by PCR, and the result shows that the construction of the recombinant expression vector DBNR40514C1 is correct.

The recombinant expression vector DBNR40514C2-DBNR40514C6 is respectively constructed according to the method, and the vector structure is as follows: kan: a kanamycin gene; RB: a right boundary; pr35S: cauliflower mosaic virus 35S (SEQ ID NO: 9); RX: g1-0X nucleotide sequence (g 1-0X is the target sequence X of target gene c40514, X is 2-6) + spacer sequence (SEQ ID NO: 11) + the reverse complement of g1-0X nucleotide sequence); and tNos: a terminator of the nopaline synthase gene (SEQ ID NO: 12); hpt: hygromycin phosphotransferase gene (SEQ ID NO: 13); LB: on the left border, the recombinant expression vector DBNR40514C2-DBNR40514C6 is transformed into competent E.coli T1 cell by heat shock method and its plasmid is extracted by alkali method.

Sixth example, agrobacterium transformation with recombinant expression vector

The correctly constructed recombinant expression vectors DBNR40514C1-DBNR40514C6 are respectively transformed into Agrobacterium LBA4404 (Invitron, chicago, USA, CAT: 18313-015) by a liquid nitrogen method under the following transformation conditions: 100. Mu.L Agrobacterium LBA4404, 3. Mu.L plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10min, and warm water bath at 37 deg.C for 10min; inoculating the transformed Agrobacterium LBA4404 in an LB test tube, culturing for 2h at the temperature of 28 ℃ and the rotation speed of 200rpm, coating on an LB plate containing 50mg/L Rifampicin (Rifampicin) and 100mg/L Kanamycin (Kanamycin) until a positive monoclonal is grown, picking out the monoclonal for culturing and extracting the plasmid, carrying out enzyme digestion verification after the recombinant expression vector DBNR40514C1-DBNR40514C6 is digested by restriction enzymes EcoRV and BamH I, and the result shows that the structure of the recombinant expression vector DBNR40514C1-DBNR40514C6 is completely correct.

Seventh example, obtaining transgenic maize plants

Co-culturing immature embryos of a sterile-cultured corn variety Huo 31 (Z31) and the agrobacterium of the sixth embodiment according to a conventionally adopted agrobacterium infection method to transfer T-DNA (comprising an RX nucleotide sequence, a promoter sequence of a cauliflower mosaic virus 35S gene, an Hpt gene and a Nos terminator sequence) in a recombinant expression vector DBNR40514C1-DBNR40514C6 constructed in the fifth embodiment into a corn chromosome set, so as to obtain corn plants with the RX nucleotide sequence (X is 1-6); wild type maize plants were also used as controls.

For Agrobacterium-mediated transformation of maize, briefly, immature embryos are isolated from maize and the embryos are contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of delivering an RX nucleotide sequence to at least one cell of one of the embryos (step 1: the infection step). In this step, the young embryos are preferably immersed in an Agrobacterium suspension (OD) ₆₆₀ =0.4-0.6, infect medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, ph 5.3)) to initiate inoculation. The young embryos are co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, the immature embryos are cultured on a solid medium (4.3 g/L MS salt, MS vitamins, 300mg/L casein, 20g/L sucrose, 10g/L glucose, 100mg/L Acetosyringone (AS), 1 mg/L2, 4-dichlorophenoxyacetic acid (2, 4-D), 8g/L agar, pH 5.8) after the infection step. After this co-cultivation phase, there may be an optional "recovery" step. In the "recovery" step, at least one antibiotic known to inhibit the growth of Agrobacterium (cefamycin) is present in the recovery medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, plant gel 3g/L, pH 5.8) without the addition of a selection agent for plant transformants (step 3: recovery step). Preferably, the immature embryos are cultured on solid medium with antibiotics but without a selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells. Next, the inoculated immature embryos are cultured on a medium containing a selection agent (hygromycin) and the growing transformed calli are selected (step 4: selection step). Preferably, the immature embryos are cultured on selective solid medium with selection agent (MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, hygromycin 50mg/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, phytogel 3g/L, pH 5.8) resulting in selective growth of the transformed cells. Then, the callus is regenerated into a plant (step 5: regeneration step), preferably, callus grown on a medium containing a selection agent is on a solid medium (MS differentiation medium and MS)Rooting medium) to regenerate the plant.

The resistant callus obtained by screening is transferred to the MS differentiation medium (4.3 g/L MS salt, 2mg/L MS vitamin, 300mg/L casein, 30g/L sucrose, 2 mg/L6-benzyladenine, 50mg/L hygromycin, 3g/L plant gel, pH5.8), and cultured and differentiated at 25 ℃. The differentiated plantlets are transferred to the MS rooting medium (2.15 g/L of MS salt, 300mg/L of MS vitamin, 300mg/L of casein, 30g/L of sucrose, 1mg/L of indole-3-acetic acid, 3g/L of plant gel, pH 5.8), cultured at 25 ℃ to the height of about 10cm, and transferred to a greenhouse for culture until fructification. In the greenhouse, each day at 28 ℃ for 16 hours, and then at 20 ℃ for 8 hours.

Eighth example, obtaining transgenic Soybean plants

Co-culturing cotyledonary node tissues of yellow 13 in the soybean variety subjected to sterile culture and the agrobacterium described in the sixth embodiment according to a conventionally adopted agrobacterium infection method so as to transfer T-DNA (comprising an RX nucleotide sequence, a promoter sequence of a cauliflower mosaic virus 35S gene, an Hpt gene and a Nos terminator sequence) of the recombinant expression vector DBNR40514C1-DBNR40514C6 constructed in the fifth embodiment into a soybean genome, thereby obtaining a soybean plant transferred with the RX nucleotide sequence (X is 1-6); while wild-type soybean plants were used as controls.

For Agrobacterium-mediated transformation of soybean, briefly, mature soybean seeds were germinated in soybean germination medium (B5 salt 3.1g/L, B5 vitamins, sucrose 20g/L, agar 8g/L, pH 5.6), the seeds were inoculated on germination medium and cultured under the following conditions: the temperature is 25 +/-1 ℃; the photoperiod (light/dark) was 16/8h. Taking the soybean aseptic seedling which is expanded at the fresh green cotyledonary node after germinating for 4-6 days, cutting off hypocotyl at the position 3-4mm below the cotyledonary node, longitudinally cutting cotyledon, and removing terminal bud, lateral bud and seed root. Wounding is performed at the cotyledonary node with the back of a scalpel, and wounded cotyledonary node tissue is contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of delivering the RX nucleotide sequence to the wounded cotyledonary node tissue (step 1: infection step). In this step, the cotyledonary node tissue is preferably immersed in an Agrobacterium suspension (OD) ₆₆₀ =0.5-0.8, infestation medium (MS salts 2.15g/L, B5 vitamins, sucrose 20g ^ er ^ s)L, glucose 10g/L, acetosyringone (AS) 40mg/L, 2-morpholinoethanesulfonic acid (MES) 4g/L, zeatin (ZT) 2mg/L, pH 5.3) to initiate inoculation. The cotyledonary node tissues were co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culture step). Preferably, the cotyledonary node tissue is cultured after the infection step on a solid medium (MS salts 4.3g/L, B5 vitamins, sucrose 20g/L, glucose 10g/L, 2-morpholinoethanesulfonic acid (MES) 4g/L, zeatin 2mg/L, agar 8g/L, pH 5.6). After this co-cultivation phase, there may be an optional "recovery" step. In the "recovery" step, at least one antibiotic known to inhibit the growth of Agrobacterium (cephamycin) is present in the recovery medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholinoethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 2mg/L, agar 8g/L, cephamycin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH 5.6) without the addition of a selection agent for plant transformants (step 3: recovery step). Preferably, the regenerated tissue mass of cotyledonary nodes is cultured on solid medium with antibiotics but without a selective agent to eliminate Agrobacterium and provide a recovery period for the infected cells. Next, the regenerated tissue mass of the cotyledonary node was cultured on a medium containing a selection agent (hygromycin) and the growing transformed callus was selected (step 4: selection step). Preferably, the regenerated cotyledonary node tissue mass is cultured on selective screening solid medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholinoethanesulfonic acid (MES) 1g/L, sucrose 30g/L, 6-benzyladenine (6-BAP) 1mg/L, agar 8g/L, cephamycin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, hygromycin 50mg/L, pH 5.6) to cause selective growth of the transformed cells. Then, the transformed cells are regenerated into plants (step 5: regeneration step), and preferably, the cotyledonary node-regenerated tissue pieces grown on a medium containing a selection agent are cultured on a solid medium (B5 differentiation medium and B5 rooting medium) to regenerate the plants.

The resistant tissue blocks obtained by screening are transferred to the B5 differentiation medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 1mg/L, agar 8g/L, cefuromycin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, hygromycin 50mg/L, pH5.6), and are cultured and differentiated at 25 ℃. Transferring the differentiated plantlets to the B5 rooting culture medium (3.1 g/L of B5 salt, 1g/L of B5 vitamin, 1g/L of 2-morpholinoethanesulfonic acid (MES), 30g/L of sucrose, 8g/L of agar, 150mg/L of cefuroxime axetil and 1mg/L of indole-3-butyric acid (IBA)), culturing at 25 ℃ to a height of about 10cm on the rooting culture medium, and transferring to a greenhouse for culturing to fructification. In the greenhouse, each day at 26 degrees C were cultured for 16 hours, and then at 20 degrees C were cultured for 8 hours.

Ninth example, taqMan validation of transgenic maize plants, transgenic Soybean plants

Approximately 100mg of leaves of maize plants into which RX nucleotide sequences (X: 1-6) were transferred were sampled, genomic DNAs thereof were extracted with the DNeasy Plant Maxi Kit from Qiagen, and the copy number of the Hpt gene was determined by the Taqman probe fluorescent quantitative PCR method. Meanwhile, wild corn plants are used as a control, and detection and analysis are carried out according to the method. The experiment was repeated 3 times and the average was taken.

The specific method for detecting the copy number of the Hpt gene comprises the following steps:

step 901, respectively taking 100mg of leaves of a corn plant with an RX nucleotide sequence and a wild corn plant, respectively grinding the leaves into homogenate in a mortar by using liquid nitrogen, and taking 3 samples for repetition;

step 902, extracting the genomic DNA of the sample by using DNeasy Plant Mini Kit of Qiagen, and referring to the product specification of the specific method;

step 903, measuring the genomic DNA concentration of the sample by using NanoDrop 2000 (Thermo Scientific);

step 904, adjusting the genomic DNA concentration of the sample to the same concentration value, wherein the concentration value range is 80-100 ng/. Mu.L;

step 905, identifying the copy number of the sample by adopting a Taqman probe fluorescent quantitative PCR method, taking the sample with known copy number after identification as a standard substance, taking the sample of a wild corn plant as a control, repeating each sample for 3 times, and taking the average value; the fluorescent quantitative PCR primer and the probe sequence are respectively as follows:

the following primers and probes were used to detect the Hpt nucleotide sequence:

primer 1: the caggtgcacgttgcaagaga is shown as SEQ ID NO. 14 in the sequence table;

primer 2: ccgctcgtctggctaagatc is shown as SEQ ID NO 15 in the sequence table;

1, probe 1: the tgcctgaaaccgaactgaccgccgctg is shown as SEQ ID NO 16 in the sequence table;

the PCR reaction system is as follows:

the 50 × primer/probe mixture contains 45 μ L of each primer at a concentration of 1mM, 50 μ L of probe at a concentration of 100 μ M and 860 μ L of 1 × TE buffer and is stored in a centrifuge tube at 4 ℃.

The PCR reaction conditions are as follows:

data were analyzed using SDS2.3 software (Applied Biosystems).

Through the experimental result of analyzing the copy number of the Hpt gene, the RX nucleotide sequences are respectively integrated into the chromosome group of the detected corn plants, and the corn plants transformed with the RX nucleotide sequences (X is 1-6) obtain single-copy transgenic corn plants.

The transgenic soybean plants were tested and analyzed according to the above method for verifying transgenic maize plants with TaqMan. Through the experimental result of analyzing the copy number of the Hpt gene, the RX nucleotide sequence is integrated into the chromosome group of the detected soybean plant, and the soybean plant transformed with the RX nucleotide sequence (X is 1-6) obtains a single-copy transgenic plant.

Tenth example, identification of insecticidal Effect of transgenic maize against Diabrotica

And (3) detecting the insect-resistant effect of the corn plant with the RX nucleotide sequence (X is 1-6) on the Diabrotica.

Step 1001, selecting 10 strains of each corn conversion event (RX-M) of DBNR40514C1-DBNR40514C6 identified as positive single copies by taqman and 3 strains of corn conversion events (NGM 1) identified as negative by taqman; wild type maize plants were also used as controls (CK 1); planting in a greenhouse to a trefoil stage;

step 1002, taking the materials in the step 1001, taking a third tender leaf from each seedling, cutting the third tender leaf into 1 × 2cm leaves with main veins removed, and flatly spreading the leaves into a culture dish paved with moisturizing filter paper;

step 1003, putting 10 primarily hatched larvae of the Diabrotica biflora with incubation time not more than 24h into each dish, tightly covering a cover of the culture dish, putting the culture dish into a raw measurement box with moisture-preserving gauze below, and putting the raw measurement box into a raw measurement box with temperature of 24 +/-2 ℃, D/L of 24/0 and humidity of 70-80%;

step 1004, considering that the larvae of the ruditania bimaculata which are hatched for the first time are weak and easy to have mechanical damage, keeping the culture dish as still as possible on the day of inoculation and on the 1 st day after inoculation;

step 1005, counting the number of the survived diabrotica from the 2 nd day of inoculation to the 16 th day from the outside of the culture dish every day; the surviving diabrotica virgifera leafworms in the culture dishes were transferred to the culture dishes containing fresh leaves every 2 days, and the results of the experiment are shown in table 4.

TABLE 4 Experimental results of leaf feeding Diabrotica in corn transformation events

The experimental results of table 4 show that: the corn plant transferred with the RX nucleotide sequence (X is 1-6) has a good inhibition effect on the Diabrotica bimaculata Blume, and the survival rate (survival rate = survival number/test number) of the Diabrotica bimaculata Blume on day 16 is about 40%.

Eleventh example for identifying insecticidal Effect of transgenic Soybean against Diabrotica

Detecting the insect-resistant effect of the soybean plant with the RX nucleotide sequence (X is 1-6) on the Diabrotica.

Step 1101, selecting 10 strains of each soybean transformation event (RX-S) of DBNR40514C1-DBNR40514C6 identified as positive single copies by taqman, and selecting 3 strains of soybean transformation events (NGM 2) identified as negative by taqman; meanwhile, wild soybean plants are used as a control (CK 2); planting in a greenhouse until 3 true leaves grow out;

step 1102, taking the material in the step 1101, taking a true leaf of about 2 x 2cm on each seedling, and flatly laying the true leaves into a culture dish paved with moisturizing filter paper;

step 1103, placing 15 primarily hatched larvae of the diabrotica virens with hatching time not more than 24 hours into each dish, tightly covering a cover of the culture dish, placing the culture dish into a raw test box with moisturizing gauze on the bottom, and placing the raw test box into a raw test box with the temperature of 24 +/-2 ℃, the D/L of 24/0 and the humidity of 70-80%;

1104, considering that the larvae of the diabrotica virgifera larvae which are hatched at first are weak and easy to have mechanical damage, keeping the culture dish as still as possible on the day of inoculation and on the 1 st day after inoculation;

step 1105, counting the number of the survived diabrotica from the outside of the culture dish every day from the 2 nd day of inoculation until the end of the 16 th day; the surviving D.biflorus leaf beetles in the culture dish were transferred to a culture dish containing fresh true leaves every 2 days, and the experimental results are shown in Table 5.

TABLE 5 Experimental results of leaf feeding Diabrotica in Soybean transformation event

The experimental results of table 5 show that: the soybean plant with RX nucleotide sequence (X is 1-6) has good inhibition effect on the Diabrotica biflora, and the survival rate (survival rate = survival number of heads/number of heads tested) of the Diabrotica biflora on day 16 is below 45%.

Twelfth embodiment, compositions

Formulation of an agriculturally pharmaceutically acceptable carrier for dsRNA (1L system): 50mM NaHPO ₄ (pH7.0), 10mM beta-mercaptoethanol, 10mM EDTA, 0.1% by mass sodium hexadecylsulfonate, 0.1% by mass polyethylene glycol octylphenyl ether, and then adding H ₂ O is complementary to 1L.

The above formula is a buffer solution formula, and any purified dsRNA is only required to be directly added into the buffer solution until the final concentration reaches the requirement, such as 50mg/L. Can also be made into concentrated preparation if necessary.

In conclusion, the invention discloses a target gene c40514 for controlling coleoptera insect pest diabrotica and a target sequence thereof for the first time, and obtains transgenic plants (corn and soybean) by utilizing RNAi technology, wherein the transgenic plants control the invasion of the diabrotica by introducing a dsRNA sequence formed by the target sequence, are efficient and specific, avoid similar risks of Bt poison protein resistance generated by the diabrotica, and have good environmental compatibility, convenience and low cost.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Beijing Dabei agricultural Biotechnology Limited

<120> nucleotide sequences and methods thereof for controlling insect infestation

<130> DBNBC132

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1167

<212> DNA

<213> Diabrotica (Monolepta thermophila)

<400> 1

atgccgatcg aaaacttgga agaccagggt cttgagaaaa accctgattt agaacttgcc 60

cactgtaagt tcctcttaaa tttacccgaa tatcgaaacg atagaaatgt ccactcaaaa 120

gtcactgaag ctatcaagaa agatgacatg gccccttggt atgaactcgt ttgcaaagat 180

gctggttgga aattggatgc aaatctgctt aaaaccctaa aaactaagaa cgccgaacaa 240

attaaattgt tagatgaagc aatcgaagac gcagaaaaga atttgggaga aatggaagtt 300

cgcgaagctt atttaaggaa agcggaatac tatagccgta ttggagataa agaaaatgca 360

gtaagcacat ttagacaaac atatgataag actgtctctc ttggtcatcg cttagatata 420

atttttcatt tgataagaat cggtcttttc ttcatggacc atgatctcat taccagaaat 480

atcgacaaag ctaaaagctt aatagaagaa gggggtgatt gggatagaag aaatcgttta 540

aaggtgtatc aaggtgctta ctgcatgtct gtaagagatt ttaagtctgc tgccaacttg 600

tttttagaca cagttagcac tttcacctct tacgaactta tggattacaa ggcatttgta 660

agatacacag tttatacatc tattataagc ttgcccagaa atcagctcag ggataaggta 720

gtaaaagggt ctgaaatttt ggaagtatta cattcagaac cttttgtcaa ggattatttg 780

ttttctttgt ataactgtca gtatgccgaa ttctttacaa atttagcaga agttgaaaca 840

atcctgagaa aagactactt cttaaatcca cactacagat actacgtgag agaaatgaag 900

attcaggcat acacacaact actagaatca tacagatccc ttactctaca atacatggcc 960

gaagcatttg gagttaccat ggaatacatt gatgaggaat tatccacttt cattgcaact 1020

ggaagactac actgcaaaat tgatagagtt ggtggaattg tagagaccaa tagacctgac 1080

ctgaagaatg cccaatttaa ttctgttgtt aaacaaggag atttgctatt gaatagagtc 1140

cagaagttgt cgagagttat aaacatt 1167

<210> 2

<211> 389

<212> PRT

<213> Diabrotica (monolepta heliotropica)

<400> 2

Met Pro Ile Glu Asn Leu Glu Asp Gln Gly Leu Glu Lys Asn Pro Asp

1 5 10 15

Leu Glu Leu Ala His Cys Lys Phe Leu Leu Asn Leu Pro Glu Tyr Arg

20 25 30

Asn Asp Arg Asn Val His Ser Lys Val Thr Glu Ala Ile Lys Lys Asp

35 40 45

Asp Met Ala Pro Trp Tyr Glu Leu Val Cys Lys Asp Ala Gly Trp Lys

50 55 60

Leu Asp Ala Asn Leu Leu Lys Thr Leu Lys Thr Lys Asn Ala Glu Gln

65 70 75 80

Ile Lys Leu Leu Asp Glu Ala Ile Glu Asp Ala Glu Lys Asn Leu Gly

85 90 95

Glu Met Glu Val Arg Glu Ala Tyr Leu Arg Lys Ala Glu Tyr Tyr Ser

100 105 110

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

115 120 125

Asp Lys Thr Val Ser Leu Gly His Arg Leu Asp Ile Ile Phe His Leu

130 135 140

Ile Arg Ile Gly Leu Phe Phe Met Asp His Asp Leu Ile Thr Arg Asn

145 150 155 160

Ile Asp Lys Ala Lys Ser Leu Ile Glu Glu Gly Gly Asp Trp Asp Arg

165 170 175

Arg Asn Arg Leu Lys Val Tyr Gln Gly Ala Tyr Cys Met Ser Val Arg

180 185 190

Asp Phe Lys Ser Ala Ala Asn Leu Phe Leu Asp Thr Val Ser Thr Phe

195 200 205

Thr Ser Tyr Glu Leu Met Asp Tyr Lys Ala Phe Val Arg Tyr Thr Val

210 215 220

Tyr Thr Ser Ile Ile Ser Leu Pro Arg Asn Gln Leu Arg Asp Lys Val

225 230 235 240

Val Lys Gly Ser Glu Ile Leu Glu Val Leu His Ser Glu Pro Phe Val

245 250 255

Lys Asp Tyr Leu Phe Ser Leu Tyr Asn Cys Gln Tyr Ala Glu Phe Phe

260 265 270

Thr Asn Leu Ala Glu Val Glu Thr Ile Leu Arg Lys Asp Tyr Phe Leu

275 280 285

Asn Pro His Tyr Arg Tyr Tyr Val Arg Glu Met Lys Ile Gln Ala Tyr

290 295 300

Thr Gln Leu Leu Glu Ser Tyr Arg Ser Leu Thr Leu Gln Tyr Met Ala

305 310 315 320

Glu Ala Phe Gly Val Thr Met Glu Tyr Ile Asp Glu Glu Leu Ser Thr

325 330 335

Phe Ile Ala Thr Gly Arg Leu His Cys Lys Ile Asp Arg Val Gly Gly

340 345 350

Ile Val Glu Thr Asn Arg Pro Asp Leu Lys Asn Ala Gln Phe Asn Ser

355 360 365

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

370 375 380

Arg Val Ile Asn Ile

385

<210> 3

<211> 516

<212> DNA

<213> Diabrotica (Monolepta thermophila)

<400> 3

ttaaaggtgt atcaaggtgc ttactgcatg tctgtaagag attttaagtc tgctgccaac 60

ttgtttttag acacagttag cactttcacc tcttacgaac ttatggatta caaggcattt 120

gtaagataca cagtttatac atctattata agcttgccca gaaatcagct cagggataag 180

gtagtaaaag ggtctgaaat tttggaagta ttacattcag aaccttttgt caaggattat 240

ttgttttctt tgtataactg tcagtatgcc gaattcttta caaatttagc agaagttgaa 300

acaatcctga gaaaagacta cttcttaaat ccacactaca gatactacgt gagagaaatg 360

aagattcagg catacacaca actactagaa tcatacagat cccttactct acaatacatg 420

gccgaagcat ttggagttac catggaatac attgatgagg aattatccac tttcattgca 480

actggaagac tacactgcaa aattgataga gttggt 516

<210> 4

<211> 492

<212> DNA

<213> Diabrotica (Monolepta thermophila)

<400> 4

atgccgatcg aaaacttgga agaccagggt cttgagaaaa accctgattt agaacttgcc 60

cactgtaagt tcctcttaaa tttacccgaa tatcgaaacg atagaaatgt ccactcaaaa 120

gtcactgaag ctatcaagaa agatgacatg gccccttggt atgaactcgt ttgcaaagat 180

gctggttgga aattggatgc aaatctgctt aaaaccctaa aaactaagaa cgccgaacaa 240

attaaattgt tagatgaagc aatcgaagac gcagaaaaga atttgggaga aatggaagtt 300

cgcgaagctt atttaaggaa agcggaatac tatagccgta ttggagataa agaaaatgca 360

gtaagcacat ttagacaaac atatgataag actgtctctc ttggtcatcg cttagatata 420

atttttcatt tgataagaat cggtcttttc ttcatggacc atgatctcat taccagaaat 480

atcgacaaag ct 492

<210> 5

<211> 343

<212> DNA

<213> Diabrotica (Monolepta thermophila)

<400> 5

atgccgatcg aaaacttgga agaccagggt cttgagaaaa accctgattt agaacttgcc 60

cactgtaagt tcctcttaaa tttacccgaa tatcgaaacg atagaaatgt ccactcaaaa 120

gtcactgaag ctatcaagaa agatgacatg gccccttggt atgaactcgt ttgcaaagat 180

gctggttgga aattggatgc aaatctgctt aaaaccctaa aaactaagaa cgccgaacaa 240

attaaattgt tagatgaagc aatcgaagac gcagaaaaga atttgggaga aatggaagtt 300

cgcgaagctt atttaaggaa agcggaatac tatagccgta ttg 343

<210> 6

<211> 130

<212> DNA

<213> Diabrotica (monolepta heliotropica)

<400> 6

aagcacattt agacaaacat atgataagac tgtctctctt ggtcatcgct tagatataat 60

ttttcatttg ataagaatcg gtcttttctt catggaccat gatctcatta ccagaaatat 120

cgacaaagct 130

<210> 7

<211> 193

<212> DNA

<213> Diabrotica (Monolepta thermophila)

<400> 7

agaaatcgtt taaaggtgta tcaaggtgct tactgcatgt ctgtaagaga ttttaagtct 60

gctgccaact tgtttttaga cacagttagc actttcacct cttacgaact tatggattac 120

aaggcatttg taagatacac agtttataca tctattataa gcttgcccag aaatcagctc 180

agggataagg tag 193

<210> 8

<211> 267

<212> DNA

<213> Diabrotica (Monolepta thermophila)

<400> 8

gaaccttttg tcaaggatta tttgttttct ttgtataact gtcagtatgc cgaattcttt 60

acaaatttag cagaagttga aacaatcctg agaaaagact acttcttaaa tccacactac 120

agatactacg tgagagaaat gaagattcag gcatacacac aactactaga atcatacaga 180

tcccttactc tacaatacat ggccgaagca tttggagtta ccatggaata cattgatgag 240

gaattatcca ctttcattgc aactgga 267

<210> 9

<211> 328

<212> DNA

<213> Cauliflower mosaic virus

<400> 9

ccattgccca gctatctgtc actttattgt gaagatagtg gaaaaggaag gtggctccta 60

caaatgccat cattgcgata aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg 120

tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac 180

gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg acgcacaatc 240

ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac 300

acgctgacaa gctgactcta gcagatct 328

<210> 10

<211> 1182

<212> DNA

<213> Artificial Sequence-R1 nucleotide Sequence (Artificial Sequence)

<400> 10

ttaaaggtgt atcaaggtgc ttactgcatg tctgtaagag attttaagtc tgctgccaac 60

ttgtttttag acacagttag cactttcacc tcttacgaac ttatggatta caaggcattt 120

gtaagataca cagtttatac atctattata agcttgccca gaaatcagct cagggataag 180

gtagtaaaag ggtctgaaat tttggaagta ttacattcag aaccttttgt caaggattat 240

ttgttttctt tgtataactg tcagtatgcc gaattcttta caaatttagc agaagttgaa 300

acaatcctga gaaaagacta cttcttaaat ccacactaca gatactacgt gagagaaatg 360

aagattcagg catacacaca actactagaa tcatacagat cccttactct acaatacatg 420

gccgaagcat ttggagttac catggaatac attgatgagg aattatccac tttcattgca 480

actggaagac tacactgcaa aattgataga gttggtaagt actgcgatcg cgttaacgct 540

ttatcacgat accttctacc acatatcact aacaacatca acactcatca ctctcgacga 600

catccactcg atcactactc tcacacgacc gattaactcc tcatccacgc ggccgcctgc 660

aggagcacca actctatcaa ttttgcagtg tagtcttcca gttgcaatga aagtggataa 720

ttcctcatca atgtattcca tggtaactcc aaatgcttcg gccatgtatt gtagagtaag 780

ggatctgtat gattctagta gttgtgtgta tgcctgaatc ttcatttctc tcacgtagta 840

tctgtagtgt ggatttaaga agtagtcttt tctcaggatt gtttcaactt ctgctaaatt 900

tgtaaagaat tcggcatact gacagttata caaagaaaac aaataatcct tgacaaaagg 960

ttctgaatgt aatacttcca aaatttcaga cccttttact accttatccc tgagctgatt 1020

tctgggcaag cttataatag atgtataaac tgtgtatctt acaaatgcct tgtaatccat 1080

aagttcgtaa gaggtgaaag tgctaactgt gtctaaaaac aagttggcag cagacttaaa 1140

atctcttaca gacatgcagt aagcaccttg atacaccttt aa 1182

<210> 11

<211> 150

<212> DNA

<213> Artificial Sequence-spacer Sequence (Artificial Sequence)

<400> 11

aagtactgcg atcgcgttaa cgctttatca cgataccttc taccacatat cactaacaac 60

atcaacactc atcactctcg acgacatcca ctcgatcact actctcacac gaccgattaa 120

ctcctcatcc acgcggccgc ctgcaggagc 150

<210> 12

<211> 253

<212> DNA

<213> Agrobacterium tumefaciens

<400> 12

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgttactag atc 253

<210> 13

<211> 1026

<212> DNA

<213> Salmonella enterica

<400> 13

atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60

agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120

gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180

cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240

ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300

caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat 360

gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420

atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480

cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540

ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600

tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660

atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720

tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg 780

cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840

ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900

gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960

tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020

gaatag 1026

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence-primer 1 (Artificial Sequence)

<400> 14

cagggtgtca cgttgcaaga 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence-primer 2 (Artificial Sequence)

<400> 15

ccgctcgtct ggctaagatc 20

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence-Probe (Artificial Sequence)

<400> 16

tgcctgaaac cgaactgccc gctg 24

<210> 17

<211> 334

<212> DNA

<213> Artificial Sequence-unrelated dsRNA (Artificial Sequence)

<400> 17

ggaaatcgcc actgctaaga aaaatggaca gaaaaataag agagcggcac ttcaagcact 60

caagcggaag aagcggtatg agaaacagtt gcagcagatt gatggaacat tatcaactat 120

tgaaatgcag agagaagctt tagagggtgc caacactaat acagctgttc tcacaacaat 180

gaaagatgct gcggacgccc tcaaagctgc tcacaaacac atggatgtcg atcaagttca 240

tgatatgatg gatgacattg ccgaacagca agatgtagct agagaaattt ctgatgccat 300

atccaaccca gttgcatttg gtcatgatat tgat 334

<210> 18

<211> 541

<212> DNA

<213> Diabrotica (monolepta heliotropica)

<400> 18

atttgttgat actagtggta gacataaaca agaagaatca ctatttgaag aaatgttggc 60

agtttctaat gctgtgagac cagataatat tattttcgtt atggatgcaa ctattggtca 120

agcttgtgag tctcaggcta aagctttcaa agaaaaggta gatgtaggct ctgtaattat 180

aacaaaatta gatggacatg caaaaggagg tggtgcactc agtgctgtgg cagccactaa 240

cagtcctatt atattcattg gtacaggaga acatatagat gacttagaac cttttaaaac 300

aaaacctttc attagtaaat tattaggaat gggtgatata gaaggtttaa ttgataaagt 360

aaacgaatta aagttagagg ataatgaaga attgttagaa aaaattaaac atgggcaatt 420

cacactcaga gacatgtatg aacagttcca aaatattatg aaaatgggac ctttctcaca 480

aataatggga atgatccctg gatttagcca agatttcatg tcaaaaggaa gtgaacaaga 540

a 541

<210> 19

<211> 394

<212> DNA

<213> Diabrotica (Monolepta thermophila)

<400> 19

aataatggac agtatgaatg attatgaatt agataaccga gatggtgcaa aattatttac 60

aaagcaaaat ggtagagtta ttagagttgc acaagggtct ggtgttacag aaagagaagt 120

aaaagatttg atcacgcaat acacgaagtt tgccgccgta gtaaagaaaa tgggcggcat 180

aaagggtctt tttaaaggcg gcgatatggc taaaaatgtc aatcacaacc aaatggccaa 240

acttaatcaa caaatggcca agatgatgga tcctcgagtg cttcagcaaa tgggcggcat 300

ggctggatta cagaacatga tgagacagct acaagcgggc gcggcaggag gcttgggagg 360

tttgggtaac cttatgggtg gttttggagg gaaa 394

Claims (27)

1. An isolated polynucleotide sequence, wherein said polynucleotide is selected from the group consisting of:

(a) 1, or a polynucleotide sequence shown in SEQ ID NO;

(b) 1, wherein the diabrotica pest ingests a double-stranded RNA comprising at least one strand complementary to the polynucleotide sequence, inhibiting the growth of the diabrotica pest;

(c) Any one of the polynucleotide sequences shown in SEQ ID NO. 3 to SEQ ID NO. 8; and

(d) A complete complement of a polynucleotide sequence defined in any one of (a) - (c).

2. A polynucleotide encoding an interfering ribonucleic acid, comprising:

(a) The polynucleotide sequence defined in any one of (b) - (d) of claim 1; and

(b) A spacer sequence; and

(c) A complete complement of the polynucleotide sequence of item (a);

wherein (a) and (c) are connected by (b).

3. The polynucleotide of claim 2, wherein the spacer sequence is SEQ ID NO. 11.

4. An expression cassette comprising the polynucleotide of claim 2 or 3 encoding an interfering ribonucleic acid under the control of an operably linked control sequence.

5. A recombinant vector comprising the polynucleotide of claim 2 or 3 encoding an interfering ribonucleic acid or the expression cassette of claim 4.

6. Use of the polynucleotide of claim 1 to interfere with the expression of a diabrotica pest target gene or to inhibit the growth of a diabrotica pest.

7. Use of a polynucleotide sequence encoding an interfering ribonucleic acid according to claim 2 or 3 for interfering with the expression of a diabrotica pest target gene or inhibiting the growth of a diabrotica pest.

8. An interfering ribonucleic acid sequence which, upon ingestion by a diabrotica pest, functions to down-regulate the expression of at least one target gene in said diabrotica pest, wherein the interfering ribonucleic acid sequence comprises at least one silencing element, wherein the silencing element is a double stranded RNA region comprising annealed complementary strands, one of which comprises or consists of a sequence fully complementary to a target sequence within the target gene, which target gene is a polynucleotide sequence as set forth in SEQ ID NO:1, wherein the target sequence is selected from the group consisting of:

(a) 1, wherein the diabrotica pest ingests a double-stranded RNA comprising at least one strand complementary to the polynucleotide sequence, inhibiting the growth of the diabrotica pest; and

(b) 3 to 8, respectively, or a combination thereof.

9. The interfering ribonucleic acid sequence of claim 8, wherein the interfering ribonucleic acid sequence comprises at least two silencing elements, and wherein one strand of the double stranded RNA comprised in each silencing element comprises or consists of a sequence that is fully complementary to a target sequence within the target gene.

10. The interfering ribonucleic acid sequence of claim 9, wherein one strand of the double stranded RNA comprised in each silencing element further comprises or consists of a different nucleotide sequence that is the complete complement of a different target sequence.

11. The interfering ribonucleic acid sequence of claim 10, wherein the different target sequences are derived from a single target gene or from a target gene different from the target gene.

12. The interfering ribonucleic acid sequence of claim 11, wherein the target gene that is different from the target gene is derived from the same diabrotica pest or a different coleopteran insect pest.

13. The interfering ribonucleic acid sequence according to any of claims 8 to 12, characterised in that it further comprises a spacer sequence.

14. The interfering ribonucleic acid sequence according to claim 13, wherein the spacer sequence is SEQ ID NO. 11.

15. A composition for controlling infestation of diabrotica pests comprising at least one interfering ribonucleic acid sequence according to any one of claims 8 to 14 and at least one suitable carrier, excipient or diluent.

16. The composition for controlling infestation of a diabrotica pest according to claim 15 wherein said composition comprises a host cell expressing or capable of expressing said interfering ribonucleic acid sequence.

17. A composition for controlling infestation by a diabrotica pest according to claim 16 wherein the host cell is a bacterial cell.

18. A composition for controlling infestation by diabrotica pests according to any one of claims 15 to 17 wherein the composition is a solid, liquid or gel.

19. The composition for controlling infestation of diabrotica pests according to claim 18 wherein the composition is a pesticidal spray.

20. The composition for controlling diabrotica pest infestation according to any one of claims 15-17, 19 wherein the composition further comprises at least one pesticide which is a chemical pesticide, a potato tuber specific protein, a bacillus thuringiensis insecticidal protein, a xenorhabdus insecticidal protein, a photorhabdus insecticidal protein, a bacillus laterosporous insecticidal protein or a bacillus sphaericus insecticidal protein.

21. The composition for controlling a diabrotica pest infestation according to claim 18, wherein the composition further comprises at least one pesticide that is a chemical pesticide, a tuber-specific protein, a bacillus thuringiensis insecticidal protein, a xenorhabdus insecticidal protein, a photorhabdus insecticidal protein, a bacillus laterosporous insecticidal protein, or a bacillus sphaericus insecticidal protein.

22. Use of a composition for controlling infestation by diabrotica pests according to any one of claims 15 to 21 for the prevention and/or control of infestation by diabrotica pests.

23. A method of controlling infestation of diabrotica species by a diabrotica species pest, comprising contacting the diabrotica species pest with an effective amount of at least one interfering ribonucleic acid sequence according to any one of claims 8 to 14.

24. A method for increasing the resistance of a plant to a diabrotica pest, comprising introducing into the plant a polynucleotide encoding an interfering ribonucleic acid according to claim 2 or 3, or an expression cassette according to claim 4, or a recombinant vector according to claim 5, or a construct comprising an interfering ribonucleic acid sequence according to any one of claims 8 to 14.

25. A method of producing a plant for controlling a diabrotica pest, comprising introducing into the plant a polynucleotide encoding an interfering ribonucleic acid according to claim 2 or 3, or an expression cassette according to claim 4, or a recombinant vector according to claim 5, or a construct comprising an interfering ribonucleic acid sequence according to any one of claims 8 to 14.

26. A method for protecting a plant from damage caused by a diabrotica pest, comprising introducing into the plant a polynucleotide encoding an interfering ribonucleic acid according to claim 2 or 3 or an expression cassette according to claim 4 or a recombinant vector according to claim 5 or a construct comprising an interfering ribonucleic acid sequence according to any one of claims 8 to 14, the introduced plant acting to inhibit the growth of the diabrotica pest after being ingested by the diabrotica pest.

27. The method of any one of claims 24 to 26, wherein the plant is soybean, wheat, barley, corn, tobacco, rice, canola, cotton or sunflower.

Images