CN107603984B - 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. 11; or (b) a polynucleotide sequence of at least 15 contiguous nucleotides of SEQ ID NO:11, 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) the polynucleotide sequence shown in SEQ ID NO. 1; 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 1 target sequence for controlling coleoptera insect pest diabrotica for the first time, and the target sequence is 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 bimaculata by reducing or closing the expression of a target sequence in the Diabrotica bimaculata by an RNAi technology.

Background

Field crops are often the target of insect attack. During the past decades, there have been substantial advances in developing 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 accumulate in the food chain, especially in the food chain of higher 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 Cry toxins developed at present can only be used for controlling a relatively narrow range of coleopteran pests, such as corn rootworm and Colorado potato beetle, and no products capable of controlling Diabrotica spp. The diabrotica leaves are gradually the main pests in developed transgenic crops due to their high propagation speed and wide distribution area.

Antisense methods and compositions have been reported in the art, which are believed to exert their effect through the synthesis of single-stranded RNA molecules (which, in theory, hybridize in vivo to highly complementary sense-stranded RNA molecules). Antisense technology is difficult to use in many systems for three main reasons. First, antisense sequences expressed in transformed cells are unstable. Second, the instability of antisense sequences expressed in transformed cells has subsequently created difficulties in transporting the sequences to hosts, cell types or biological systems remote from the transgenic cells. Third, the difficulties encountered with instability and delivery of antisense sequences also make it difficult for attempts to: providing a dose effective to modulate the expression level of the sense nucleotide sequence of interest within a recombinant cell encoding the antisense sequence.

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 the use of RNAi technology for pest control is known in the art, a key factor in using this technology as a means of controlling insect infestation is the selection of the most appropriate target genes, i.e., those genes whose loss of function results in severe disruption of essential biological processes and/or death of the organism. Thus, the present invention provides for the control of insect infestation, particularly of plants, by down-regulation of specific target genes in the pest as a means of achieving control of insect infestation.

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 sequence in the following way: 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 objects, the present invention provides an isolated polynucleotide sequence comprising:

(a) the polynucleotide sequence shown in SEQ ID NO. 11; or

(b) 11, 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

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

(d) A complementary sequence of the polynucleotide sequence defined in (a), (b) or (c) above.

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 polynucleotide sequence is SEQ ID NO 2.

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 sequence 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 fragment within the target sequence, the target sequence 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.

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 fragment within the target sequence.

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

Still further, the different target fragments are derived from a single target sequence or from different target sequences.

The different target sequences are derived from the same coleopteran insect pest or different coleopteran insect pests.

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 interfering RNA sequence is SEQ ID NO 2.

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 pesticide that 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.

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

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 increasing resistance of a plant coleopteran insect pest, comprising: introducing a construct comprising the interfering ribonucleic acid sequence into a plant.

To achieve the above objects, the present invention also provides a method of 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.

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 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 coleopteran insect pest is Diabrotica biflora.

The present invention comprises a method of modulating or inhibiting the expression of one or more target sequences in a coleopteran insect pest, the 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 sequences, and such 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 sense and antisense orientations relative to at least one promoter, wherein the nucleotide sequences comprising the sense strand and the antisense strand are linked or ligated by a spacer sequence of at least about 5-1000 nucleotides, wherein the sense strand and the antisense strand may be of different lengths, and wherein at least one of the two coding sequences has at least 80% sequence identity, 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. Particularly useful are dsRNA sequences comprising about 19-300 nucleotides 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 (monoepta heliotropica (Motschulsky)) is coleoptera, phyllotaceae. Widely distributed in northeast, northwest, north China and other areas. Diabrotica biflora is a parasitic insect which eats various plants such as gramineae, cruciferae and leguminosae, and particularly has serious harm to corn, cotton and vegetables.

The diabrotica leaves overwinter in the form of eggs in soil, the larvae in June of the next year hatch and eat corn roots or weed roots in situ, and the larvae in the middle 7 th month eclosion are harmful to corn leaves, so that photosynthesis of plants is weakened. When the corn enters the silking period, the diabrotica leaf beetle imagoes cut off the corn filaments, so that pollination obstacle is caused, and further, the yield is reduced. From 2009 to 2016, the damage area of the Diabrotica leaf beetles to the corns rises 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.

With the continuous promotion of straw returning measures, the field humus is continuously abundant and the soil surface covering is 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 bimaculata 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. However, the corn grows high at this time, the pesticide application difficulty is increased, and the tragedy that pesticide application personnel are accidentally injured is easily caused. Based on the above problems, the whole plant prevention and control method of the Luciola biflora is the best solution for providing the whole growth period for the corn.

"controlling insects" or "controlling pests" or "controlling insect pests" as used herein 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 sequence" as described in the present invention is any sequence intended to be down-regulated in an insect. Insect infestation, such as disruption of essential biological processes in insects, is controlled by down-regulating target sequences. Thus, preferred target sequences include, but are not limited to, genes that play a key role in regulating feeding, survival, growth, development, reproduction, invasion, and infestation. When expression of the target sequence 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 transition of at least 30% of the insects 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 sequences may be expressed in all or part of the insect cell. Furthermore, these target sequences 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 spp. The terms "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 sequence, including but not limited to sense RNA, antisense RNA, short interfering RNA (sirna), micro RNA (mirna), double stranded RNA (dsrna), hairpin RNA (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 sequence expression by binding to a target fragment within the target sequence. Binding occurs due to base pairing between the interfering RNA and the complementary region of the target fragment. 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 fragment within a target sequence and that functions as an active portion of the interfering ribonucleic acid to direct down-regulation of expression of the target sequence. 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 fragment within a target sequence, or an interfering ribonucleic acid consisting thereof.

"target sequence expression" in the context of the present invention refers to the transcription and accumulation of RNA transcripts encoded by the target sequence and/or translation of mRNA into protein. The term "down-regulation" refers to any of the methods known in the art by which interfering ribonucleic acids reduce the level of primary RNA transcripts, mRNA, or proteins produced by a target sequence. 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 in the level of RNA or protein produced by a gene in an insect cell by at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% 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. Also, down-regulation may 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.

In the present invention, "inhibition of expression of a target sequence" refers to a reduction or absence (below a detectable threshold) of the level of protein and/or mRNA product of the target sequence. Specificity refers to the ability to inhibit a target sequence without effect on other genes of the cell and without effect on any gene within the cell that produces the 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 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.

In the present invention, the interfering ribonucleic acid down-regulates the expression of a gene by RNA interference or RNAi. RNAi is a sequence-specific gene regulation method typically mediated by double-stranded RNA molecules, such as short interfering RNA (siRNA). The siRNA comprises a 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 the target sequence. 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 fragment sequence within a target sequence. 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 sequence. 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 segment of the target sequence. The term "fully complementary" means that all bases of the silencing element nucleotide sequence are complementary or "matched" to the bases of the target fragment. 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 fragment. One skilled in the art will appreciate that in order to mediate down-regulation of target sequence expression, the silencing element need only be at least partially complementary to the target fragment. It is known in the art that RNA sequences with insertions, deletions, and mismatches relative to the target sequence can still be effective in RNAi. Preferably, the silencing element shares at least 80% or 85% sequence identity with the target fragment of the target sequence, 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 fragment. It is well known to those skilled in the art that the degree of complementarity shared between the silencing element and the target fragment varies depending on the target sequence to be down-regulated or the insect species whose gene expression is to be controlled.

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

The interfering ribonucleic acids of the present invention may comprise 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 fragment within a target sequence and functions to down-regulate expression of the target sequence 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 fragment within a particular target sequence. The silencing element within the interfering ribonucleic acid may also comprise or consist of different nucleotide sequences complementary to different target fragments. It should be clear that combinations of multiple copies of the same silencing element combined with silencing elements bound to different target fragments are also within the scope of the invention.

In order to achieve down-regulation of specific target sequences in coleopteran insects in the present invention, different target fragments may be derived from a single target sequence in one insect. In this case, the silencing elements can be combined in the interfering ribonucleic acid in the original order in which the target fragments are present in the target sequence, 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 fragments in the target sequence.

Alternatively, the different target fragments represent a single target sequence, but are derived from different insect species.

Alternatively, different target fragments may be derived from different target sequences. If the interfering ribonucleic acids are used to prevent and/or control pest infestation, it is preferred that the different target sequences 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 sequences 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 sequences 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 the 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 fragment or target sequence. 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 the processing of interfering ribonucleic acids in insect cells and thereby enhances the efficacy of interfering ribonucleic acids.

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 sequence 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 generated by the ordinary skilled person in the art by a manual or automatic reaction in a chemical or enzymatic way. 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 plant expressible promoter 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 a plant 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 sequences in the host. Preferably, the silenced gene does not exhibit significant complementarity with a nucleotide sequence other than the intended target fragment of the target sequence. 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 within a region of 17 contiguous nucleotides, more preferably within a region of 18 or 19 contiguous nucleotides, and most preferably within 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 sequence 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 fragment within the target sequence. Target sequences 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 vector, excipient or diluent, wherein the interfering ribonucleic acid functions to down-regulate expression of a target sequence 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 in 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 into contact with 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, the composition that is in contact with the insect 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 effect in the insect. 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 mediates 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 the following: 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 can be any material that the plant to be treated can tolerate, 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 a plant or any part of a plant at any stage of plant development, for example, the composition is applied to the aerial parts of the plant during the cultivation of the plant 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 contain at least one additional active agent. Thus, the composition may be provided in the form of a "kit of parts" comprising the interfering ribonucleic acid-containing composition in one container and one or more suitable active ingredients, such as chemical or biological pesticides, in a separate container. 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), isopropylphenyl methyl carbamate, indoxacarb, novaluron (chitin synthesis inhibitor), propargyl, 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, imidazolinones, sulfonylureas, and bromoxynil. The composition may also be provided in combination with at least one additional pesticide, which may be selected from any pesticide 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 sequence 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 present 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 simultaneously or sequentially to the area or organism to be treated.

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 sequence. The essential target sequence may be any insect gene involved in regulating essential biological processes required by an 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 pH 7.0.

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, or 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 genome. 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 1 target sequence 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 fragment for controlling coleopteran insect pest diabrotica does not affect the expression of non-target sequences in the host.

3. Avoid the generation of resistance. The invention provides 1 target sequence for controlling coleoptera insect pests, namely the diabrotica virgifera, and the resistance of the diabrotica virgifera can be avoided by replacing the target sequence in an irregular way or using the target sequence in a mixed way.

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 the schematic diagram of the recombinant expression vector DBN110048 vector of the nucleotide sequence and the method for controlling insect invasion of the invention.

FIG. 2 shows the lethality of feed snf7-X containing the dsRNAs of the rsbsnf7 target sequence and control feed CK-X when fed to Diabrotica bifida.

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 Flutica biflora target sequences

1. Extraction of total RNA of Diabrotica biflora

Using the first hatching instar of Diabrotica biflora as material, adopting conventional Trizol method to extract RNA, purifying by conventional method, treating with DNase to obtain the final product with concentration not less than 300 ng/microliter, total amount not less than 6 microgram and OD_260/2801.8-2.2 total RNA samples.

2. mRNA isolation and cDNA Synthesis

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

3. Screening out 1 target gene

The target gene rsbsnf7 of 1 Diabrotica diabrotica is screened out from a larva library, which has medium expression quantity and may participate in important metabolic pathways, and the nucleotide sequence is shown as SEQ ID NO. 11, and the amino acid sequence is shown as SEQ ID NO. 12.

Second example, construction of plant expression vectors

Two expression cassettes were synthesized in the order of p35S-R-tNos-p35S-Hpt-tNos by Nanjing King-King Biotech Ltd, were ligated to a plant expression vector by EcoRI and HindIII and named DBN110048, the vector schematic of which is shown in FIG. 1 (Kan: kanamycin gene; RB: right border; pr 35S: cauliflower mosaic virus 35S (SEQ ID NO: 3); R (SEQ ID NO: 2): reverse complementary nucleotide sequence of R (R is the target sequence of the target gene rsbsnf 7; SEQ ID NO:1) + spacer sequence + R; tNos: nopaline synthase gene terminator (SEQ ID NO: 4); Hpt: hygromycin phosphotransferase gene (SEQ ID NO: 5); LB: left border).

Transforming the recombinant expression vector DBN110048 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 DBN110048), and 42 ℃ water bath for 30 seconds; shaking at 37 deg.C for 1 hr (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, and white colonies were picked up and 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). The plasmid is extracted by an alkaline method. The extracted plasmid is sequenced and identified by PCR, and the result shows that the construction of the recombinant expression vector DBN110048 is correct.

Third example, transformation of Agrobacterium with recombinant expression vector

The correctly constructed recombinant expression vector DBN110048 is transformed into Agrobacterium LBA4404(Invitrgen, Chicago, USA, CAT: 18313-: 100. mu.L Agrobacterium LBA4404, 3. mu.L plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10 minutes, and carrying out warm water bath at 37 ℃ for 10 minutes; inoculating the transformed Agrobacterium LBA4404 in LB test tube, culturing at 28 deg.C and 200rpm for 2 hours, spreading on LB plate containing 50mg/L Rifampicin (Rifamicin) and 100mg/L Kanamycin (Kanamycin) until positive single clone grows out, selecting single clone, culturing and extracting plasmid, enzyme digestion verification after restriction enzymes EcoRI and HindIII are used to digest the recombinant expression vector DBN110048, the result shows that the structure of the recombinant expression vector DBN110048 is completely correct.

Fourth example, obtaining transgenic maize plants

Co-culturing the immature embryo of the sterile-cultured maize variety heddle 31(Z31) with the agrobacterium of the third embodiment according to a conventionally adopted agrobacterium infection method to transfer the T-DNA (comprising an R nucleotide sequence, a promoter sequence of a cauliflower mosaic virus 35S gene, an Hpt gene and a Nos terminator sequence) in the recombinant expression vector DBN110048 constructed in the second embodiment into a maize chromosome group, so as to obtain a maize plant with the transferred R nucleotide sequence; 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 the R 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)₆₆₀0.4-0.6, 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, ph5.3)) was infected to initiate inoculation. The young embryos are co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culture step). Preferably, the immature embryos are cultured on solid medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 20g/L, glucose 10g/L, Acetosyringone (AS)100mg/L, 2, 4-dichlorophenoxyacetic acid (2,4-D)1mg/L, agar 8g/L, pH5.8) after the infection step. After this co-cultivation phase, there may be an optional "recovery" step. In the 'recovery' step, at least one hexane in the recovery medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 2, 4-dichlorophenoxyacetic acid (2,4-D)1mg/L, plant gel 3g/L, pH5.8) is presentAn antibiotic known to inhibit the growth of Agrobacterium (cefamycin) was not added with 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 (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, hygromycin 50mg/L, 2, 4-dichlorophenoxyacetic acid (2,4-D)1mg/L, plant gel 3g/L, pH5.8) with a selective agent, resulting in selective growth of the transformed cells. Then, the callus is regenerated into a plant (step 5: regeneration step), and preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) to regenerate the plant.

The resistant callus obtained by screening was transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyladenine 2mg/L, hygromycin 50mg/L, plant gel 3g/L, pH5.8) and cultured and differentiated at 25 ℃. The differentiated plantlets are transferred to the MS rooting medium (MS salt is 2.15g/L, MS, vitamin, casein is 300mg/L, cane sugar is 30g/L, indole-3-acetic acid is 1mg/L, plant gel is 3g/L, pH is 5.8), cultured at 25 ℃ to be about 10cm high, and transferred to a greenhouse for culture until fructification. In the greenhouse, the culture was carried out at 28 ℃ for 16 hours and at 20 ℃ for 8 hours each day.

Fifth example, obtaining transgenic Soybean plants

Co-culturing cotyledonary node tissue of yellow 13 in the aseptically cultured soybean variety and the agrobacterium described in the third embodiment according to a conventionally adopted agrobacterium infection method to transfer the T-DNA (including an R 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 DBN110048 constructed in the second embodiment into a soybean genome, thereby obtaining a soybean plant with a transferred R nucleotide sequence; while wild-type soybean plants were used as controls.

For Agrobacterium-mediated soybeansBriefly, mature soybean seeds were germinated in soybean germination medium (B5 salt 3.1g/L, B5 vitamins, sucrose 20g/L, agar 8g/L, pH5.6), the seeds were inoculated on the germination medium, and cultured under the following conditions: the temperature is 25 +/-1 ℃; the photoperiod (light/dark) was 16/8 h. Taking the soybean aseptic seedling expanded at the fresh green cotyledonary node after germinating for 4-6 days, cutting off hypocotyl at 3-4 mm position below the cotyledonary node, longitudinally cutting off cotyledon, and removing terminal bud, side bud and seed root. Wounding at the cotyledonary node with the back of a scalpel, contacting the wounded cotyledonary node tissue with an Agrobacterium suspension capable of delivering the R1 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)₆₆₀0.5-0.8, medium (MS salts 2.15g/L, B5 vitamins, sucrose 20g/L, glucose 10g/L, Acetosyringone (AS)40mg/L, 2-morpholinoethanesulfonic acid (MES)4g/L, Zeatin (ZT)2mg/L, pH5.3) was infected 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 on 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, pH5.6) 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 (cephamycin) is present in the recovery medium (B5 salt 3.1g/L, B5 vitamins, 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, pH5.6) 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 tissue mass of cotyledonary node is cultured in solid screening medium with selective agent (B5 salt 3.1g/L, B5 vitamins, 2-morpholinoethyl1g/L sulfonic acid (MES), 30g/L sucrose, 1 mg/L6-benzyladenine (6-BAP), 8g/L agar, 150mg/L cephamycin, 100mg/L glutamic acid, 100mg/L aspartic acid, 50mg/L hygromycin, pH5.6) resulted in 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, cefamycin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, hygromycin 50mg/L, pH5.6), and cultured and differentiated at 25 ℃. The differentiated plantlets are transferred to the B5 rooting medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES)1g/L, sucrose 30g/L, agar 8g/L, cefamycin 150mg/L and indole-3-butyric acid (IBA)1mg/L), cultured to about 10cm high at 25 ℃ on rooting culture, and transferred to a greenhouse for culture to fructification. In the greenhouse, the culture was carried out at 26 ℃ for 16 hours and at 20 ℃ for 8 hours each day.

Seventh example, TaqMan validation of transgenic maize plants, transgenic Soybean plants

About 100mg of leaves of each R nucleotide sequence-transferred maize Plant was sampled, genomic DNA thereof was extracted with the DNeasy Plant Maxi Kit of Qiagen, and the copy number of the Hpt gene was detected by Taqman probe fluorescent quantitative PCR to determine the copy number of the R nucleotide sequence. 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 701, respectively taking 100mg of leaves of the corn plant with the transferred R nucleotide sequence and the wild corn plant, respectively grinding the leaves into homogenate in a mortar by using liquid nitrogen, and taking 3 samples for repetition;

step 702, 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 703, determining the genomic DNA concentration of the sample by using NanoDrop 2000(Thermo Scientific);

step 704, adjusting the genomic DNA concentration of the sample to the same concentration value, wherein the concentration value range is 80-100 ng/. mu.L;

705, 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 type 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: CAGGGTGTCACGTTGCAAGA is shown as SEQ ID NO. 6 in the sequence list;

primer 2: CCGCTCGTCTGGCTAAGATC is shown as SEQ ID NO. 7 in the sequence list;

1, probe 1: TGCCTGAAACCGAACTGCCCGCTG is shown as SEQ ID NO. 8 in the sequence list;

the PCR reaction system is as follows:

the 50 × primer/probe mixture contained 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 was stored in amber tubes 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 fact that the R nucleotide sequence is integrated into the chromosome group of the detected corn plant is further verified, and the corn plant transformed with the R nucleotide sequence obtains a single-copy transgenic corn plant.

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 fact that the R nucleotide sequence is integrated into the chromosome group of the detected soybean plant is further verified, and the soybean plant transformed with the R nucleotide sequence obtains a single-copy transgenic plant.

Eighth example, obtaining dsRNA

PCR amplification of a sense strand is carried out by taking the diabrotica virgae cDNA as a template (Primer-F: gaagagatgttaactaaaaaac is shown as SEQ ID NO:9 in a sequence table, and Primer-R: agcatttttcatagaatctaa is shown as SEQ ID NO:10 in the sequence table), so as to obtain a PCR product, wherein the amplification conditions are as follows: the PCR product is detected by agarose electrophoresis with the mass concentration of 1 percent at 95 ℃ for 3min, 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 30s and 72 ℃ for 10 min.

The obtained PCR product was ligated to the L4440 plasmid with the bidirectional T7 promoter. The ligation products were transformed into HT115(DE3) (from addge Inc.) competent cells (this strain was a deficient E.coli strain with the following sites F-, mcrA, mcrB, IN (rrnDrrne)1, rnc14: Tn10(DE3) lysogen: lavUV5 promoter-T7 polymerase); the RNase III gene was deleted under the induction of tetracycline by the influence of Tn10 transposon, HT115 was able to specifically express T7polymerase under the mediation of IPTG, and positive clones were screened by blue-white screening by recognizing the T7 primer sequence at the insertion site and folding to generate dsRNA.

The single clone was picked up and transferred to LB liquid medium containing 100. mu.g/mL ampicillin and 12.5. mu.g/mL tetracycline, and cultured at 37 ℃ for 10-14 hours.

Diluting the culture solution 100 times, adding 2 XYT medium (peptone 16g, yeast extract 10g, sodium chloride 4g, dissolved in 1L water, pH7.0) to OD₅₉₅When reaching 0.4, 0.4mM IPTG is added, and the mixture is incubated for 4 hours with shaking at 37 ℃, and then the dsRNA expression product of the obtained gene is obtained. (see Ravi S Kamath, Maruxa Martinez-Campos, PederZipper len, Anderw G Fraser, J.Ahringer, Effect of specific RNA-mediated interferrocenent-induced RNA in CamORha molecules, Genomebiol 2, 0002.1(2000))

The obtained bacteria can be directly used for drug application; recombinant plasmids can also be purified therefrom and the dsRNA isolated for administration.

Ninth example purification of dsRNA

The specific method for obtaining an expression product containing dsRNA according to the eighth embodiment for purification, purification of recombinant plasmid and isolation of dsRNA is as follows:

step 901, taking 2mL of bacterial liquid as a small plasmid sample to prove that the plasmid is effectively amplified; pouring the residual bacterial liquid into a 50mL centrifuge tube, and centrifuging at the temperature of 4 ℃ and the speed of 5500rpm for 10 min; inverting the centrifuge tube to remove the residual LB as much as possible;

step 902, adding 6.6mL of alkali lysis solution I (50mmol/L glucose, 25mmol/L Tris-HCl (pH7.6), 10mmol/L EDTA, dissolving in 1L of sterilized water, sterilizing at high temperature, adding 0.5mL of 1% RNase), and blowing or Vortex resuspending the bacteria; adding 1mL of 10mg/mL lysozyme which is freshly prepared; 13.3mL of newly prepared alkali lysis solution II (0.2N NaOH, 1% SDS), gently rotating and mixing, and standing at room temperature for 5-10 min; adding 10mL of ice-precooled alkaline lysis solution III (60 mL of 5mol/L potassium acetate, 11.5mL of glacial acetic acid and 28.5mL of water), slightly and completely reversing and uniformly mixing, and standing on ice in a centrifuge tube for 10 min;

step 903, centrifuging at 20000g at 4 ℃ for 10 min; adding the supernatant together with 0.6 times of isopropanol into a new centrifuge tube (or two tubes if the volume is too large), mixing, and standing at room temperature for 10 min; centrifuging at 12000g for 15min at room temperature;

step 904, discarding the supernatant, washing the tube wall with 70% ethanol by mass fraction, pouring off the ethanol, and inversely drying for 10-15 min; dissolving the precipitate with 3mL of water; precooling the nucleic acid solution with ice to a temperature of 0 ℃; adding 5mol/LLICl precooled by ice in the same volume (3mL), uniformly mixing, and centrifuging at 12000g for 10min at the temperature of 4 ℃;

step 905, transferring the supernatant to a new centrifuge tube, adding isopropanol with the same volume (6mL), uniformly mixing, and centrifuging at 12000g at room temperature for 10 min;

step 906, after the supernatant is poured out, inverting the centrifuge tube to drain the liquid, washing the precipitate and the tube wall with ethanol with the mass fraction of 70% at room temperature, and inverting the drained ethanol to volatilize the ethanol in the precipitate;

step 907, dissolving the nucleic acid precipitate with 500 μ L of water containing RNaseA (20 μ g/mL), transferring the solution to a 1.5mL centrifuge tube, and digesting for 30min at 37 ℃;

step 908, add an equal volume (500 μ Ι _ of) of phenol: mixing chloroform and Vortex, centrifuging at the highest speed at 4 deg.C for 2min, transferring supernatant to another centrifuge tube (if protein membrane is more, repeating the steps until protein is removed);

step 909, adding chloroform with the same volume (500 μ L) and Vortex mixing uniformly to extract phenol, centrifuging at the highest temperature of 4 ℃ for 2min, and transferring the supernatant to another centrifuge tube; add 2 volumes of absolute ethanol (1mL) (if protein membrane is more, repeat this step until protein is removed);

step 910, adding 1mL of ethanol with the mass fraction of 70%, reversing for washing for several times, centrifuging at the highest speed at 4 ℃ for 2min, absorbing supernatant, and inverting for 10-15min to volatilize ethanol;

911, dissolving the nucleic acid precipitate with 1mL of water, and adding DNase to digest DNA; specific dsRNA can be obtained.

Tenth example, identification of controlling ability of Flutica biflora by feeding dsRNA

According to the eighth embodiment, dsRNA-expressing bacterial solution was cultured at 37 ℃ and then cultured to OD₆₀₀About 2.0. To be OD₆₀₀When the concentration reached 0.4mM, 0.4mM IPTG was added and incubated at 37 ℃ with shaking to OD₆₀₀About 2.0.

dsRNA of the separated and purified target sequence r is uniformly mixed according to the proportion of 5 mu g/g feed and added into feed (feed formula reference Development of an intellectual di et for the western corn rootworm, Entomogia excelmentalis et application 105: 1-11,2002.) snf7-X, wherein the control feed CK-X is not added with dsRNA, and other conditions are completely consistent. Feeding the rude hatched larvae of the Diabrotica biflora by using the prepared feed, putting 30 rude hatched larvae with the hatching time not exceeding 24 hours into each dish, wherein the feed mixed with dsRNA is changed every two days and fed to the 6 th day, feeding the rude hatched larvae with the feed without adding dsRNA from the 7 th day until the 10 th day is finished (the feed is changed every two days), counting the insect death rate every two days from the beginning of feeding, setting 5 times of repetition for each treatment in the experiment, and counting the results as shown in figure 2.

The dsRNA feeding result shows that dsRNA of a target sequence r of a target gene rsbsnf7 expressed by escherichia coli has obvious inhibition effect on Diabrotica.

Eleventh example, identification of insecticidal Effect of transgenic maize against Diabrotica variegata

And (3) detecting the insect-resistant effect of the corn plant with the transferred R nucleotide sequence on the Diabrotica.

1101, selecting 5 DBN110048 corn transformation events (R-M-X) which are identified as positive single copies by taqman, 3 corn transformation events (NGM1-X) which are identified as negative by taqman, selecting 3 strains of each transformation event, and selecting 3 seedlings of each strain; wild type maize plants were also used as controls (CK 1); planting in a greenhouse until the three-leaf stage;

step 1102, taking the material in the step 1101, 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 1103, putting 10 primarily hatched larvae of the Diabrotica biflora with incubation time not more than 24 hours into each dish, tightly covering a cover of the culture dish, putting the culture dish into a raw test box with moisture-preserving gauze under the culture dish, and putting the raw test box into a raw test box with temperature of 24 +/-2 ℃, D/L of 24/0 and humidity of 70-80%;

step 1104, considering that the larvae of the ruditapes bifida 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 1105, counting the number of the survived diabrotica from the 2 nd day of inoculation to the end of the 16 th day from the outside of the culture dish every day; the surviving diabrotica leaves in the dish were transferred every 2 days to a dish containing fresh leaves and the results are shown in table 1.

TABLE 1 Experimental results of leaf feeding of Diabrotica oleracea in corn transformation events

The experimental results of table 1 show that: the corn plant with the transferred R nucleotide sequence has a good inhibition effect on the D.pyracantha, and the lethality (death rate/test number) to the D.pyracantha after 12 days can reach about 50%.

Twelfth example, identification of insecticidal Effect of transgenic Soybean against Flutica biflora

And (3) detecting the insect-resistant effect of the soybean plant with the transferred R nucleotide sequence on the Diabrotica.

Step 1201, selecting 5 DBN110048 soybean transformation events (R-S-X) which are identified as positive single copies by taqman, selecting 3 soybean transformation events (NGM2-X) which are identified as negative by taqman, selecting 3 strains of each transformation event, and selecting 3 seedlings of each strain; meanwhile, wild soybean plants are used as a control (CK 2); planting in a greenhouse until 3 true leaves grow;

step 1202, taking the material in the step 1201, 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 1203, placing 15 primarily hatched larvae of the Diabrotica longipes with incubation time not more than 24 hours into each dish, tightly covering a cover of the culture dish, placing the culture dish into a raw measurement box with moisture-preserving gauze under the culture dish, and placing 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 1204, considering that the larvae of the ruditapes biflorus 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 1205, counting the number of the surviving diabrotica diaboloides from the outside of the culture dish every day from the 2 nd day of inoculation until the 16 th day is finished; 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 2.

TABLE 2 Experimental results of leaf feeding of Diabrotica biflora in Soybean transformation event

The experimental results of table 2 show that: the soybean plant with the transferred R nucleotide sequence has a good inhibition effect on the Diabrotica bimaculata, and the lethality (death rate/test number) of the Diabrotica bimaculata after 12 days can reach more than 50%.

Fourteenth embodiment, composition

Formulation of an agriculturally pharmaceutically acceptable carrier for dsRNA (1L system): 50mM NaHPO₄(pH7.0), 10mM beta-mercaptoethanol, 10mM EDTA, 0.1% by mass sodium hexadecylsulfonate, 0.1% by mass polyethylene glycol octylphenyl ether, and then adding H₂O is complementary to 1L.

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

In conclusion, the invention discloses a target gene rsbsnf7 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 a target sequence r of the diabrotica, are efficient and specific, avoid the diabrotica to generate resistance, 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 Co., Ltd

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

<130>DBNBC122

<160>12

<170>SIPOSequenceListing 1.0

<210>1

<211>240

<212>DNA

<213>Monolepta hieroglyphica(Motschulsky)

<400>1

gcaaagaaaa atgcatcaaa aaataaaagg ctggcacttc aagcactcaa aaagaagaaa 60

cgtcttgaga agactcaatt gcaaatagat ggaactctta caactattga aatgcagagg 120

gaggcacttg aaggagctag tacaaataca gctgttttag attctatgaa aaatgctgca 180

gatgcattga aaaaagctca taaaaatttg gatgtggacg atgtgcatga cataatggac 240

<210>2

<211>578

<212>DNA

<213>Artificial sequence

<220>

<221>stem_loop

<222>()..()

<223> r nucleotide sequence + spacer sequence + reverse complement nucleotide sequence of r

<400>2

gtccattatg tcatgcacat cgtccacatc caaattttta tgagcttttt tcaatgcatc 60

tgcagcattt ttcatagaat ctaaaacagc tgtatttgta ctagctcctt caagtgcctc 120

cctctgcatt tcaatagttg taagagttcc atctatttgc aattgagtct tctcaagacg 180

tttcttcttt ttgagtgctt gaagtgccag ccttttattt tttgatgcat ttttctttgc 240

aagtactgcg atcgcgttaa cgctgtgatg tgaaacttga aattatttgt gttttgattg 300

tgattgtgag agtaacggtg gcggccgcct gcaggagcgc aaagaaaaat gcatcaaaaa 360

ataaaaggct ggcacttcaa gcactcaaaa agaagaaacg tcttgagaag actcaattgc 420

aaatagatgg aactcttaca actattgaaa tgcagaggga ggcacttgaa ggagctagta 480

caaatacagc tgttttagat tctatgaaaa atgctgcaga tgcattgaaa aaagctcata 540

aaaatttgga tgtggacgat gtgcatgaca taatggac 578

<210>3

<211>328

<212>DNA

<213>Cauliflower mosaic virus

<400>3

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>4

<211>253

<212>DNA

<213>Agrobacterium tumefaciens

<400>4

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>5

<211>1026

<212>DNA

<213>Salmonella enterica

<400>5

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>6

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<221>primer_bind

<222>()..()

<223> primer 1

<400>6

cagggtgtca cgttgcaaga 20

<210>7

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<221>protein_bind

<222>()..()

<223> primer 2

<400>7

ccgctcgtct ggctaagatc 20

<210>8

<211>24

<212>DNA

<213>Artificial Sequence

<220>

<221>primer_bind

<222>()..()

<223> Probe 1

<400>8

tgcctgaaac cgaactgccc gctg 24

<210>9

<211>22

<212>DNA

<213>Artificial Sequence

<220>

<221>primer_bind

<222>()..()

<223> target sequence r-F

<400>9

gaagagatgt taactaaaaa ac 22

<210>10

<211>21

<212>DNA

<213>Artificial Sequence

<220>

<221>primer_bind

<222>()..()

<223> target sequence R-R

<400>10

agcatttttc atagaatcta a 21

<210>11

<211>663

<212>DNA

<213>Monolepta hieroglyphica(Motschulsky)

<400>11

atgagttttt tcggaaaact atttggtgga aaaaaagaag atgttgctcc aacaccaggg 60

gaagcgatcc aaaagcttag agaaacggaa gagatgttaa ctaaaaaaca agatttttta 120

gagaaaaaaa tagaggaata caccttacta gcaaagaaaa atgcatcaaa aaataaaagg 180

ctggcacttc aagcactcaa aaagaagaaa cgtcttgaga agactcaatt gcaaatagat 240

ggaactctta caactattga aatgcagagg gaggcacttg aaggagctag tacaaataca 300

gctgttttag attctatgaa aaatgctgca gatgcattga aaaaagctca taaaaatttg 360

gatgtggacg atgtgcatga cataatggac gacatagctg aacaacatga cattgctaat 420

gagataacca atgcaattag caatccggtc ggattctcag atgatctaga cgacgatgaa 480

ttagaaaagg aattagagga actcgaacaa gaaggactgg aggaagattt gttaaaagtt 540

ccaggtccta ctgaactacc agctgtgcca actggagcaa tcgctaataa accagttaaa 600

cctgtagcta aaaaagtgga agatgatgat gacatgaagg aattggaagc ttgggcatca 660

taa 663

<210>12

<211>220

<212>PRT

<213>Monolepta hieroglyphica(Motschulsky)

<400>12

Met Ser Phe Phe Gly Lys Leu Phe Gly Gly Lys Lys Glu Asp Val Ala

1 5 10 15

Pro Thr Pro Gly Glu Ala Ile Gln Lys Leu Arg Glu Thr Glu Glu Met

20 25 30

Leu Thr Lys Lys Gln Asp Phe Leu Glu Lys Lys Ile Glu Glu Tyr Thr

35 40 45

Leu Leu Ala Lys Lys Asn Ala Ser Lys Asn Lys Arg Leu Ala Leu Gln

50 55 60

Ala Leu Lys Lys Lys Lys Arg Leu Glu Lys Thr Gln Leu Gln Ile Asp

65 70 75 80

Gly Thr Leu Thr Thr Ile Glu Met Gln Arg Glu Ala Leu Glu Gly Ala

8590 95

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

100 105 110

Leu Lys Lys Ala His Lys Asn Leu Asp Val Asp Asp Val His Asp Ile

115 120 125

Met Asp Asp Ile Ala Glu Gln His Asp Ile Ala Asn Glu Ile Thr Asn

130 135 140

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

145 150 155 160

Leu Glu Lys Glu Leu Glu Glu Leu Glu Gln Glu Gly Leu Glu Glu Asp

165 170 175

Leu Leu Lys Val Pro Gly Pro Thr Glu Leu Pro Ala Val Pro Thr Gly

180 185 190

Ala Ile Ala Asn Lys Pro Val Lys Pro Val Ala Lys Lys Val Glu Asp

195 200 205

Asp Asp Asp Met Lys Glu Leu Glu Ala Trp Ala Ser

210 215 220

Claims (26)

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

(a) the polynucleotide sequence shown in SEQ ID NO. 11; or

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

(c) A sequence completely complementary to the above (a) or (b) under stringent conditions.

2. An interfering polynucleotide consisting of the polynucleotide of claim 1 linked to its complement by a spacer sequence.

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

4. An expression cassette comprising the polynucleotide of claim 1 or the interfering polynucleotide of claim 2 or 3 under the control of an operably linked regulatory sequence.

5. A recombinant vector comprising the polynucleotide of claim 1 or the interfering polynucleotide of claim 2 or 3 or the expression cassette of claim 4.

6. Use of the interfering polynucleotide of claim 2 or 3 for interfering with the expression of a diabrotica target sequence or inhibiting the growth of diabrotica.

7. An interfering ribonucleic acid which, after uptake by diabrotica cuticula, functions to down-regulate the expression of at least one target sequence in said diabrotica cuticula, wherein the interfering ribonucleic acid 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 complementary to a target fragment within the target sequence, the target fragment comprising the polynucleotide of claim 1.

8. The interfering ribonucleic acid according to claim 7, wherein the interfering ribonucleic acid comprises at least two silencing elements, each silencing element comprising or consisting of a nucleotide sequence at least partially complementary to a target fragment within the target sequence.

9. The interfering ribonucleic acid according to claim 8, wherein the silencing elements each comprise or consist of a different nucleotide sequence complementary to a different target fragment.

10. The interfering ribonucleic acid according to claim 9, characterised in that the different target fragments are derived from a single target sequence or from different target sequences.

11. The interfering ribonucleic acid according to claim 10, wherein the different target sequences are derived from diabrotica or different coleopteran insect pests.

12. The interfering ribonucleic acid according to any of claims 7 to 11, characterised in that it further comprises a spacer sequence.

13. The interfering RNA of claim 12, wherein the sequence of the interfering RNA is the RNA sequence of SEQ ID NO. 2.

14. A composition for controlling diabrotica virescens infestation comprising at least one interfering ribonucleic acid sequence according to any one of claims 7 to 13 and at least one suitable carrier, excipient or diluent.

15. The composition for controlling diabrotica according to claim 14, wherein said composition comprises a host cell expressing or capable of expressing said interfering ribonucleic acid.

16. The composition for controlling diabrotica according to claim 15, wherein said host cell is a bacterial cell.

17. A composition for controlling diabrotica virescens infestation according to any one of claims 14-16 wherein the composition is a solid, liquid or gel.

18. The composition for controlling diabrotica according to claim 17, wherein said composition is an insecticidal spray.

19. The composition for controlling diabrotica according to any one of claims 14 to 16 or 18, further comprising at least one insecticide selected from the group consisting of chemical insecticides, potato tuber-specific proteins, bacillus thuringiensis insecticidal proteins, xenorhabdus insecticidal proteins, photorhabdus insecticidal proteins, bacillus laterosporous insecticidal proteins and bacillus sphaericus insecticidal proteins.

20. The composition for controlling diabrotica according to claim 17, further comprising at least one insecticide, said insecticide being a chemical insecticide, a potato tuber-specific protein, a bacillus thuringiensis insecticidal protein, a xenorhabdus insecticidal protein, a phoma glabrata insecticidal protein, a bacillus laterosporous insecticidal protein, or a bacillus sphaericus insecticidal protein.

21. Use of a composition for controlling diabrotica virescens infestation according to any one of claims 14-20 for the prevention and/or control of diabrotica virescens infestation.

22. A method of controlling diabrotica virescens infestation, comprising contacting diabrotica virescens with an effective amount of at least one interfering ribonucleic acid according to any one of claims 7 to 13.

23. A method for improving resistance of a plant Diabrotica bigelovii comprising: introducing into a plant a construct comprising the interfering ribonucleic acid of any one of claims 7 to 13.

24. A method of producing a plant controlling Diabrotica bimaculata comprising introducing into a plant the interfering polynucleotide of claim 2 or 3 or the expression cassette of claim 4 or the recombinant vector of claim 5.

25. A method for protecting a plant from damage caused by Diabrotica oleracea, comprising introducing the interfering polynucleotide of claim 2 or 3 or the expression cassette of claim 4 or the recombinant vector of claim 5 into a plant, wherein the introduced plant functions to inhibit the growth of Diabrotica oleracea after ingestion by Diabrotica.

26. The method of any one of claims 23-25, wherein the plant is soybean, wheat, barley, corn, tobacco, rice, canola, cotton, or sunflower.

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