WO2012163084A1 - Nouveau procédé pour prévenir et éliminer les organismes nuisibles fondé sur une technologie d'arni - Google Patents

Nouveau procédé pour prévenir et éliminer les organismes nuisibles fondé sur une technologie d'arni Download PDF

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Publication number
WO2012163084A1
WO2012163084A1 PCT/CN2012/000731 CN2012000731W WO2012163084A1 WO 2012163084 A1 WO2012163084 A1 WO 2012163084A1 CN 2012000731 W CN2012000731 W CN 2012000731W WO 2012163084 A1 WO2012163084 A1 WO 2012163084A1
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seq
nucleic acid
sequence
pest
gene
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Chinese (zh)
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苗雪霞
张�浩
李海超
王玉冰
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention is in the field of biotechnology and agrochemicals; more specifically, the present invention relates to a novel method for controlling pests based on RNAi technology. Background technique
  • the perennial damage area of Plutella xylostella accounts for about 10% of the total area of vegetable cultivation (2010 vegetable planting area is 33.33 million hm 2 , Plutella xylostella is 2.53 million hm 2 ; 2009 vegetable planting area is 30 million hm 2 , Plutella xylostella is 2.7 million Hm 2 ), the loss is about 20 ⁇ 30%. If the prevention is not timely or the measures are unreasonable, it may cause the harvest. At present, the control of Asian corn borer, cotton bollworm and diamondback moth is still dominated by chemical control, but it has caused great damage to the ecological environment and food security production. Many hazards of chemical control have made people have to seek better pest control methods. Although biological control can play a preventive role, it is not effective because of its slow effect, and it is not recognized by the public because of its environmental impact.
  • RNAi phenomenon has developed rapidly since its discovery in 1991. Studies have shown that through the specific gene RNAi, target genes of targeted interference species can be reached, and some physiological phenomena appear, which can achieve the purpose of studying gene function. At the same time, this phenomenon is extremely high. The specificity, that is, the interference effect of homologous genes on different species is not obvious, so it is an ideal pest control system.
  • RNAi technology has been reported for pest control.
  • Baum et al. (2007) demonstrated that v-ATPase has a lethal effect on Western corn rootworm WCR Diabrotica virgifera virgifera LeConte and can be used for field control.
  • RNAi technology is feasible as a new pest control method, but previous studies have also shown that RNAi-based pest control has Many problems need to be solved, such as: 1) how to quickly and high-throughput target genes; 2) how to apply them easily to field production; 3) resistance problems; 4) safety issues, etc. Therefore, applying RNAi technology In the field of pest control, it is also necessary to solve the above problems. First, it is necessary to obtain enough and effective target genes, so that a large number of target genes can be controlled by different combinations and different periods using different genes, which can effectively prevent pest resistance. The emergence of sex.
  • the present invention also provides insecticidal preparations and methods for obtaining insect-species-specific and inter-species broad-spectrum types using different fragments of the same gene. Summary of the invention
  • an isolated polypeptide selected from the group consisting of:
  • polypeptide is a polypeptide of the amino acid sequence of SEQ ID NO: 2.
  • an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
  • the polynucleotide is selected from the group consisting of - (1) a polynucleotide of the sequence of SEQ ID NO: 1;
  • the encoded protein has the same function as the polynucleotide encoded by the polynucleotide of (1) or (2).
  • a vector comprising the polynucleotide is provided.
  • a genetically engineered host cell preferably a non-reproductive or reproductive cell, comprising the vector, or a polynucleoside thereof integrated in the genome thereof acid.
  • polynucleotide fragment having a sequence of 10% or more (preferably 15% or more; more preferably 20%) of the polynucleotide sequence described above More preferably 25./.; more preferably 30%) sequence identity.
  • polynucleotide fragment is selected from the group consisting of:
  • a vector comprising one, two or more of said polynucleotide fragments is provided.
  • a genetically engineered host cell comprising the vector, or one or two or more of the polynucleotide fragments integrated in the genome of the genome.
  • the use of the polynucleotide or polynucleotide fragment is provided for use as a suppressor or silencing target for the preparation of an interfering molecule that specifically interferes with the expression of a pest gene or inhibits the growth of a pest
  • the pest gene is selected from the group consisting of a storage protein, a white gene; or as a target for the preparation of an insecticidal (such as lepidopteran) preparation.
  • the storage chalk gene is selected from the group consisting of Asian corn borer storage white gene or cotton bollworm storage white gene or Plutella xylostella storage white gene;
  • the lepidopteran insect is selected from the group consisting of Asian corn borer or cotton bollworm or diamondback moth;
  • the use of the polypeptide is provided for use as a target for screening or preparing insecticidal formulations.
  • the preparation is selected from, but not limited to, a nucleic acid inhibitor, a protein inhibitor, an antibody, a ligand, a hydrazine hydrolase, a protein binding molecule.
  • nucleic acid inhibitor is selected from the group consisting of
  • dsRNA antisense nucleic acid, small interfering RNA, microRNA; or
  • nucleic acid inhibitor selected from the group consisting of
  • polynucleotide or polynucleotide fragment, or a transcript thereof for inhibiting or silencing a target dsRNA, an antisense nucleic acid, a small interfering RNA, a microRNA; wherein any of the dsRNAs, antisense When nucleic acids, small interfering RNAs, and microRNAs are ingested by pests, they inhibit the growth of the harmful organisms; or
  • RNA wherein any of the dsRNA, antisense nucleic acid, small interfering RNA, and microRNA are ingested by a pest, and the pest gene is selected from the storage of the white gene; or
  • the nucleic acid inhibitor is a dsRNA produced by expression of the polynucleotide, wherein When the dsRNA is ingested by a pest, it inhibits the growth of the pest.
  • the nucleic acid inhibitor is dsRNA having the following structure:
  • Seq' E 3 ⁇ 4 is an RNA sequence corresponding to the polynucleotide of any one of claims 2 or 3 or a sequence fragment thereof, or a nucleotide sequence selected from the sequence defined in claim 2 or 3.
  • Seq' s i5 is a sequence substantially complementary to Seq
  • X ' is absent; or located Seq' ⁇ and Seq 'spacer sequence between s, the spacer sequence is Seq' and Seq ' ⁇ not complementary;
  • nucleic acid inhibitor is a construct comprising the following structure:
  • Seq E ft is a polynucleotide of any one of claims 2 or 3 or a sequence fragment thereof, or a core comprising at least a portion of a nucleotide sequence selected from the sequence defined in claim 2 or 3 Glycosidic acid sequence;
  • Seq S (3 ⁇ 4 is a sequence that is substantially complementary to Seq «;
  • X is a spacer sequence located between Seq j ⁇ and Seq ⁇ , and the spacer sequence is not complementary to Seq ia and Seq.
  • the construct may form the following structure -
  • Seq Seq s and X are as described above.
  • the construct is an expression vector (plasmid).
  • the polynucleotide or polynucleotide fragment can serve as a target for inhibiting mRNA transcription of the polynucleotide or inhibiting expression of a polypeptide encoded by the polynucleotide.
  • the polynucleotide or polynucleotide fragment can be used as a target for the preparation of insecticidal formulations.
  • a host cell e.g., a bacterium, a fungus, a yeast, a plant cell
  • the nucleic acid inhibitor such as the construct
  • nucleic acid inhibitor or the host cell described for the preparation of an insect control preparation.
  • an insect control preparation (such as a pesticide preparation or a pesticide composition) comprising: a safe and effective amount of a substance selected from the group consisting of: the nucleic acid inhibitor (e.g., 10-50 (g) /mL) or the host cell; And; a pharmaceutically acceptable carrier.
  • it further comprises at least one insecticide selected from the group consisting of chemical insecticides, potato glycoside, Bacillus thuringiensis, insecticidal white, pathogenic bacilli, white bacillus, light bacillus Insecticidal white, Bacillus licheniformis, and B. sphaericus insecticidal protein.
  • insecticides selected from the group consisting of chemical insecticides, potato glycoside, Bacillus thuringiensis, insecticidal white, pathogenic bacilli, white bacillus, light bacillus Insecticidal white, Bacillus licheniformis, and B. sphaericus insecticidal protein.
  • a method of controlling a pest that interferes with expression of a pest gene is selected, the pest gene being selected from the group consisting of a storage white gene.
  • the method comprises: feeding or/or spraying a pest of the nucleic acid inhibitor of any one of claims 7-10 or the host cell of claim 5 or 11.
  • a method of killing an insect comprising: reducing expression of the polypeptide in the insect; or reducing the polynucleotide or polynucleotide in the insect Transcription of the corresponding mRNA of the fragment.
  • the method of providing comprises: applying the nucleic acid inhibitor or the host cell to (eg, spraying) a subject to be controlled (eg, a plant, particularly an insect-infested plant; or an insect) ).
  • a subject to be controlled eg, a plant, particularly an insect-infested plant; or an insect
  • nucleic acid inhibitor or the host cell in another aspect of the invention, there is provided the feeding of the nucleic acid inhibitor or the host cell to an insect or a spray insect.
  • the method comprises: expressing in the plant an interfering molecule that specifically interferes with expression of the pest gene; the insect gene is selected from the group consisting of a storage protein gene.
  • a method of preparing an insect resistant plant comprising: introducing the nucleic acid inhibitor into a plant.
  • a plant or a seed thereof which plant is obtained by transformation of said polynucleotide.
  • the polynucleotide is expressed as a dsRNA in a plant cell.
  • the pest is selected from the group consisting of: insects, mites, fungi, yeasts, molds, bacteria, nematodes, weeds and parasites, and saprophytic plants.
  • the pest is an insect, including but not limited to: a Lepidoptera, a Coleoptera, a Hemiptera, and a Diptera.
  • the insect pest is selected from the group consisting of lepidopteran insects, preferably corn borer or cotton bollworm or diamondback moth.
  • a method of preparing an insect resistant plant comprising: introducing the nucleic acid inhibitor into a plant.
  • the method of preparing an insect resistant plant is:
  • step (1) (2) contacting a plant cell or tissue or organ with Agrobacterium in step (1) to transfer the nucleic acid inhibitor into a plant cell or tissue or organ.
  • the method for preparing an insect-resistant plant further comprises: (3) selecting a plant cell or tissue or organ into which the nucleic acid inhibitor has been introduced;
  • step (3) Regenerating the plant cell or tissue or organ in step (3).
  • the insects have a significant decrease in survival or death after eating the insect-resistant plants.
  • Other aspects of the invention will be apparent to those skilled in the art from this disclosure.
  • FIG. 1 Different domains of lethal efficiency at different times after dsRNA treatment of Asian corn borer.
  • EYFP is an enhanced yellow fluorescent white gene as a foreign gene control.
  • CK is a blank control and is a normal artificially reared Asian corn borer.
  • Ostrinia furnacalis Asian corn snail.
  • Figure 1 The lethality of the cotton bollworm's own SP gene dsRNA against cotton bollworm.
  • FIG. 13 Schematic representation of the pART27-dsRNA vector. detailed description
  • the inventors have found extensively a target gene useful for controlling lepidopteran insects, and based on the nucleic acid sequence of the target gene, a nucleic acid inhibitor or a host expressing a nucleic acid inhibitor can effectively kill lepidopteran insects. . the term
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • Those skilled in the art will be able to purify the protein using standard ruthenium purification techniques.
  • a substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
  • the "plant” is not particularly limited as long as the “plant” is easily attacked by insects such as lepidopterans, such as various crops, flower plants, or forestry plants.
  • the plant may be, for example, without limitation: a dicot, a monocot, or a gymnosperm.
  • the plants include (but are not limited to): cotton, wheat, barley, rye, rice, corn, sorghum, beets, apples, pears, plums, peaches, apricots, cherries, strawberries, raspberries, blackberries , beans, lentils, peas, soybeans, rapeseed, mustard, opium, olean, sunflower, coconut, castor oil plant, cocoa beans, peanuts, gourd, cucumber, watermelon, flax, hemp, jute, citrus, lemon, Grapefruit, spinach, chicory, asparagus, cabbage, Chinese cabbage, pakchoi, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, grape Trees, ramie, bananas, natural rubber trees and ornamental plants.
  • insect refers to an insect that includes a storage protein gene in any genome.
  • the insect may be a plant-feeding insect that feeds on a plant, for example, it may be a scutellaria, isoptera, coleoptera, diptera, hymenoptera, lepidoptera, orthoptera.
  • Insects or agricultural pests of the order Hemiptera, Helicoptera for example, the species of the genus Heliconia, the genus Heliconia, the genus Heliconia, the genus Diptera, the cotton leaf moth, the larvae, the yellow Moth, Moth, Moth, Maternaria, Moth, Moth, Moth, Moth, Moth, Moth, Moth, Moth, Coleoptera, apple-shaped moth, Coleoptera, Corn genus, Spodoptera frugiperda, Diamond, genus, genus, genus, genus, genus, genus, genus, genus, broad-winged Small moth, Moth, Moth, American white moth, Moth, Moth, Moth, Moth, Moth, Lepidoptera, Brassica, Tobacco, Moth, Toad Genus, European corn borer, ultra-small moth, small eyed moth, red bollworm, cotton bollworm
  • the "insect” is an insect that is harmful to plants
  • the patent application of the Chinese Patent Application No. 200680042821.5 and US 2009/0306189 Al, WO 2007/080127 A2, AU 2006/335978 AL US 2009/0285784 Al The application text is a reference for the invention, and the description of insects on pages 41-47 of CN101365795 can be incorporated into the description of the present invention.
  • lepidoptera refers to a gene (fragment) including any one of SEQ ID NO: 3-5, SEQ ID NO: 17, and SEQ ID NO: 20-21 in any genome. a sequence or a homologous gene thereof or a hybrid thereof a gene of lepidopteran insects, and these genes are genes in the insects necessary for the growth or survival of the insect.
  • the lepidoptera includes moths, examples include: Tineidae and Oecophoridae such as Tineola bisselliella (Common Clothes Moth), and Cocoonidae (Pyralidae) such as Pyralis farinalis (Meal Moth), Asian corn borer, and Noctuidae such as cotton bollworm, and the genus Moth, such as the diamondback moth.
  • RNA interference refers to a phenotype in which some RNAs efficiently and specifically block the expression of specific genes in the body, promote mRNA degradation, and induce cells to exhibit specific gene deletions. Called RNA intervention or intervention. RNA interference is a highly specific gene silencing mechanism at the mRNA level.
  • interfering RNA or “dsRNA” refers to an RNA molecule that is capable of degrading specific mRNAs with mRNAs of homologous complementary sequences, a process known as the RNA interference pathway.
  • substantially complementary means that the sequences of the nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (e.g., a stem-loop structure).
  • a secondary structure e.g., a stem-loop structure.
  • at least 70% of the nucleotides of the two "substantially complementary" nucleotide sequences are complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%.
  • mismatched nucleotides there may be up to 7 mismatched nucleotides between two sufficiently complementary molecules; preferably, up to 6 mismatched nucleotides; more preferably, up to 5 mismatched nucleosides Acid; further preferred, having up to 4 mismatched nucleotides, such as having 0, 1, 2, 3, 4 mismatched nucleotides.
  • a "complementary" sequence generally refers to a sequence that converts a sequence in the 5'-3' direction to its 3'-5' direction (eg, 5'ATCG 3'->GCTA), and then takes its complementary sequence (such as GCTA-5'CGAT 3,).
  • a “stem loop” structure also referred to as a “hairpin” structure, refers to a nucleic acid molecule that forms a secondary structure comprising a double-stranded region (stem), said double-stranded region Formed by two regions of the nucleic acid molecule (on the same molecule), the two regions are flanked by double-stranded portions; they also include at least one "loop" structure, including non-complementary nucleic acid molecules, ie, single-stranded regions. Even if the two regions of the nucleic acid molecule are not fully complementary, the double-stranded portion of the nucleic acid can remain in a double-stranded state.
  • insertions, deletions, substitutions, etc. may result in non-complementation of a small region or the formation of a stem-loop structure or other form of secondary structure by itself, however, the two regions may still be substantially complementary and are foreseeable Interaction occurs in the manner, forming a double-stranded region of the stem-loop structure.
  • Stem loop structures are well known to those skilled in the art, and typically after obtaining a nucleic acid having a nucleotide sequence of a primary structure, one skilled in the art will be able to determine whether the nucleic acid is capable of forming a stem-loop structure.
  • nucleic acid inhibitor refers to a class of substances having the activity of controlling lepidopteran insects obtained by preparing a target gene or a fragment thereof or a truncated form useful for controlling lepidopteran insects according to the present invention.
  • the "nucleic acid inhibitor” is, for example, some interfering molecule, including dsRNA (also known as double-stranded RNA, double-stranded ribonucleic acid or double-stranded ribonucleotide sequence), antisense nucleic acid, small interfering RNA, microRNA, etc. , or can A construct that expresses or forms the dsRNA, antisense nucleic acid, small interfering RNA, or microRNA.
  • operably linked refers to a spatial arrangement of the functionality of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is introduced by the promoter region such that the promoter region is “operably linked” to the nucleic acid sequence.
  • an RNA sequence corresponding to a polynucleotide (or DNA) sequence refers to an RNA sequence which is "AU” if the DNA sequence is "AT”.
  • containing includes “including”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; “mainly composed of", “consisting essentially of” and “consisting of” belonging to “contains” , the underlying concept of "with” or “including”. Storage of chalk and its coding gene
  • a target gene of the present invention which is a storage white gene.
  • Storage ⁇ white is one of the hexamer ⁇ white families.
  • the sp-related genes in insects generally have cylindrical structures, consisting of 7-8 polypeptide chains of about 50KD; there is a copper binding site. According to the amino acid structure, it can be divided into storage proteins rich in proline (> 4% methionine) and storage whites rich in aromatic amino acids (>15% aromatic amino), and spions rich in aromatic amino acids.
  • proline-rich sp does not contain glycosylation sites, and sequence identity of different species is between 40% and 90% (40°/. sequence identity is considered to be higher homology).
  • insects such as the corn borer OfSP gene, H. arm SP, Plutella xylostella PxSP1, PxSP2
  • insect storage protein genes from insects are key genes involved in insect development or growth, and the expression of these genes is down-regulated or Inhibition can cause problems with insect growth (eg, abnormal phlegm or eggs that cannot be hatched) or death.
  • genes can be prepared to inhibit the growth and development of corn borer or cotton bollworm or diamondback moth.
  • the above genes may also be derived from other organisms whose functions have been established in the literature, their nucleotide sequences are highly homologous to the corn borer gene or their gene functions are similar to those of the corn borer gene, and these heights are foreseen.
  • a homologous gene or a functionally similar gene can be used as a target to prepare specific interfering molecules and inhibit the growth of other organisms.
  • highly homologous or “highly homologous” as used herein, when referring to a nucleic acid sequence, means that under stringent conditions SEQ ID NO: 1, SEQ ID NO: 16 > SEQ ID NO: 18-19 A nucleotide sequence that hybridizes to any sequence or its complement.
  • such highly homologous sequences are exemplified by a control nucleotide sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 18-19, or a complement thereof
  • identity refers to the relationship at the nucleotide level between sequences.
  • a "percent identity” is determined by comparing the optimally aligned sequences (eg, two or more) in a comparison window, wherein the sequence portion of the comparison window is aligned with the reference sequence of the optimal sequence Ratios can include insertions or deletions. The reference sequence does not contain an insertion or deletion.
  • the reference window is selected from at least 10 contiguous nucleotides to about 50, about 100 or to about 150 nucleotides, preferably about 50 to 150 nucleotides.
  • the "percent identity” is then calculated by determining the number of nucleotides that are consistent between the sequences in the window and dividing the number by the number of nucleotides in the window and multiplying by 100.
  • the present inventors further studied target gene fragments which can effectively down-regulate these target genes, and finally found six target gene fragments whose nucleotide sequences are selected from SEQ ID NOs: 3-5, SEQ ID NO. : 17. The sequence shown in any one of SEQ ID NOS: 20-21.
  • Fragments or truncated forms of the target gene are also included in the present invention, as long as the fragments or truncated forms are used to prepare the nucleic acid inhibitor, the nucleic acid inhibitor also has a killing scale. The activity of the winged insect.
  • the inventors have unexpectedly discovered that these target gene fragments or their truncated bodies play an important role in insects, and that their inhibition, interference or silencing will result in a significant decrease in the survival rate of insects or direct death. Therefore, various nucleic acid inhibitors can be designed based on these target gene fragments for use in pest control.
  • the invention also provides a polynucleotide set (set) comprising the sequences set forth in SEQ ID NO: 3-5, SEQ ID NO: 17, SEQ ID NO: 20-21.
  • a nucleic acid inhibitor e.g., construct or dsRNA
  • 1-6 polynucleotides can be obtained from the polynucleotide set.
  • a nucleic acid inhibitor or a host expressing a nucleic acid inhibitor can be separately prepared based on a sequence of a plurality of polynucleotides (preferably, for example, 6), which can achieve a broader spectrum and more when applied simultaneously (or mixedly). Effectively kills lepidopteran insects.
  • Nucleic acid inhibitor e.g., construct or dsRNA
  • the nucleic acid inhibitor is a construct of some interfering molecule, eg, dsRNA, antisense nucleic acid, small interfering RNA or microRNA, or may express or form the dsRNA, antisense nucleic acid, small interfering RNA or micro RNA construct. More preferably, it is a dsRNA or a construct that can express the dsRNA.
  • a construct for expressing a dsRNA, an antisense nucleic acid, a small interfering RNA or a microRNA can be designed. Accordingly, the present invention provides an artificially constructed construct. Designing the constructs according to the genes provided by the present invention and their sequences is well known to those skilled in the art, and the construction can usually be made
  • the product contains an intron sequence (not complementary to the flanking sequences), and the complementary gene sequences are ligated at both ends, and after entering the cell, a "stem loop" structure can be produced, and the "stem” portion can form dsRNA, antisense nucleic acid.
  • small interfering RNA or microRNA this dsRNA, antisense nucleic acid, small interfering RNA or microRNA can be particularly effective in inhibiting the expression of the gene of interest.
  • the construct contains at least one structure as shown below -
  • nucleotide sequence of Seq ⁇ ⁇ 5 is selected from the sequence or sequence fragment shown in any one of claims 2 or 3, and the Seq ⁇ is a nucleotide sequence substantially complementary to Seq ⁇ ;
  • X is a spacer sequence located between Seq ⁇ and Seq ⁇ , and the spacer sequence is not complementary to Seq ⁇ and Seq s ;
  • the construct is located on an expression vector.
  • the present invention also encompasses a vector comprising the construct.
  • the expression vector typically also contains a promoter operably linked to the construct, an origin of replication and/or a marker gene, and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance .
  • Vectors comprising the appropriate gene sequences described above, as well as appropriate promoters or control sequences, can be used to transform a suitable host.
  • the host may be any host suitable for carrying the expression vector and capable of expressing a nucleic acid inhibitor.
  • the host is Escherichia coli, fungus, yeast, plant cells, animal cells, and the like.
  • Transformation of the host with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art, depending on the type of plant.
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 , if desired, the conversion can also be carried out by electroporation. Transformation of fungal and yeast cells, plant cells, animal cells is also well known to those skilled in the art.
  • a host carrying the construct or expression vector and capable of expressing a nucleic acid inhibitor can be directly applied to a subject (such as a plant) to be controlled to achieve the purpose of controlling lepidopteran insects.
  • the "stem"-like portion of the stem-loop structure described above is formed by the interaction of Seq ⁇ ⁇ and Seq ⁇ and can be processed to form a nucleic acid inhibitor.
  • the formed nucleic acid inhibitor has the following structure: Seq'iH to one - -,,,
  • Seq' is selected from the RNA sequence or sequence fragment corresponding to the sequence of any of claims 2 or 3; Seq' s is a sequence substantially complementary to Seq'.
  • X' is a spacer sequence located between Seq ⁇ and Seq, ⁇ , which is not complementary to Seq' ⁇ and Seq'sfi .
  • the ⁇ ' sequence can be excised in vitro or without excision. After the dsRNA enters the insect body, it is processed and excised by an enzyme (such as nuclease Dicer) in the insect.
  • the nucleic acid inhibitor can be directly applied to a subject (e.g., a plant) to be controlled to achieve the purpose of controlling lepidopteran insects.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptides of the invention may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., plants, bacteria, yeast, insect cells).
  • the polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also encompasses fragments, derivatives and analogs of the storage protein 2.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the storage buffer of the present invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (Hi) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example a polypeptide formed by fusion of a polyethylene glycol) or (iv) an additional amino acid sequence fused to the polypeptide sequence (eg, a pre-sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or fusion) protein).
  • conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptid
  • the term "storage protein 2" refers to a polypeptide of the sequence of SEQ ID NO: 2 or a variant thereof. These variations include (but are not limited to): several (usually 1 - 50, preferably 1 - 30, more preferably 1 - 20, optimally 1-10, still more preferably 1 -8 or 1-5) amino acid deletions, insertions and/or substitutions, and addition or deletion of one or several at the C-terminus and/or N-terminus (usually within 20, preferably within 10, More preferably, it is 5 or less amino acids.
  • the function of the white matter is generally not altered.
  • the addition or subtraction of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the white matter.
  • the term also encompasses active fragments and active derivatives of storage protein 2.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, mutagenized mutants, DNA encoded by DNA that hybridizes to storage protein 2 DNA under high or low stringency conditions. White and so on.
  • the invention also provides other polypeptides, such as fusion albumin comprising storage chalk 2 or a fragment thereof.
  • the present invention also encompasses soluble fragments of the storage protein 2.
  • the fragment has at least about 20 contiguous amino acids of the storage protein 2 sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, optimally at least about 100 consecutive amino acids.
  • the invention also provides an analog of the storage protein 2 or polypeptide.
  • the difference between these analogues and natural storage of chalk 2 can be The difference in amino acid sequence may also be a difference in the modification form that does not affect the sequence, or both.
  • These polypeptides include natural or induced genetic variants. Mutant variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to enhance their resistance to chalky hydrolysis or to optimize solubility properties.
  • "storage 22 2 conservative variant polypeptide” means having up to 20, preferably up to 10, more preferably up to 5, optimally compared to the amino acid sequence of SEQ ID NO: 2. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.
  • the survival rate of the insect can be significantly reduced, and it can be used for screening or preparing the target of the insecticidal preparation. Therefore, based on the target cockroach, the design can be designed.
  • insecticidal agents such as protein inhibitors, can act to kill insects after being applied to a subject.
  • the target albumin or a fragment thereof or a variant thereof is also included in the present invention, and as long as the fragment or variant is used to prepare the chalk inhibitor, the chalk inhibitor has a kill scale The activity of the winged insect.
  • the present invention also provides polynucleotides encoding the storage sputum 2 of the present invention or a conservative variant polypeptide thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include C DNA, genome
  • DNA or synthetic DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence which encodes a purine having the sequence of SEQ ID NO.-2 but differs from the coding region of SEQ ID NO: 1.
  • Polynucleotides encoding the mature polypeptide of SEQ ID NO: 2 include: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and Non-coding sequence.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • Shorter fragments derived from the target genes of the present invention are also included in the present invention as a basis for designing nucleic acid inhibitors. It is well understood by those skilled in the art that some shorter sequences can also be used as nucleic acid inhibitors, for example, some siRNA sequences are often only 18 bp.
  • the invention further relates to hybridization to the sequences described above and having at least 15%; preferably at least 18%, more preferably at least 25°/ between the two sequences. More preferably at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical Sex polynucleotide.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent (stringent) conditions.
  • stringent (strict) conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 °C; or (2) Addition of a denaturing agent, such as 50% 0 ') formamide, 0.1% calf serum / 0.1%. Ficoll, 42 °C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 80%, preferably at least 90%, more preferably 95% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to identify and/or isolate polynucleotides encoding storage ⁇ 2.
  • the full-length nucleotide sequence encoding the sputum 2 of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it often needs to be done Two or more PCR amplifications are performed, and then the amplified fragments are spliced together in the correct order.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • DN A sequence encoding the ruthenium (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis.
  • the DN A sequence can then be introduced into various existing DN A molecules (e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the chalk sequence of the present invention by chemical synthesis.
  • the above-described polynucleotide encoding the sputum white 2 of the present invention can be used as a target gene for preparing an insecticidal preparation.
  • the present inventors have found that the target gene or fragment thereof of the present invention plays an important role in insects, and that inhibition, interference or silencing thereof causes a significant decrease in the survival rate of insects or direct death. Therefore, based on the target gene, a plurality of insecticidal preparations can be designed, for example, a nucleic acid inhibitor can be designed, which can be used to kill insects after being applied to a subject.
  • the target gene or fragment thereof or truncated form or gene variant or fragment variant is also included in the invention, as long as the fragment or truncated form or variant is used to prepare the nucleic acid inhibitor,
  • the nucleic acid inhibitor also has the activity of killing lepidopteran insects.
  • the invention also relates to a vector comprising a polynucleotide of the invention, or a fragment or variant thereof, and a host cell genetically engineered using the vector of the invention or the storage sputum 2 coding sequence, and recombinantly produced to produce the invention A method of polypeptide.
  • polynucleotide sequence of the present invention can be utilized to express or produce recombinant storage protein 2 by conventional recombinant DNA techniques. Generally there are the following steps:
  • the storage protein 2 polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art.
  • any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Expression vector for translation control signals include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to refer to mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent ⁇ White (GFP), or kanamycin or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent ⁇ White (GFP), or kanamycin or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, used as a promoter to enhance transcription of a gene.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 . Conversion can also be carried out by electroporation if desired.
  • the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, rice immature embryo transformation method and the like.
  • Agrobacterium transformation or gene gun transformation such as leaf disc method, rice immature embryo transformation method and the like.
  • plants can be regenerated using conventional methods.
  • Insecticide Insecticide
  • Any substance which is prepared based on the target protein or its fragment or truncated form provided by the present invention and which has activity against lepidopteran can be used as an insecticidal preparation for controlling lepidopteran insects.
  • Such preparations are, for example, protein inhibitors, proteolytic enzymes, protein binding molecules, antibodies, ligands. Any substance which inhibits the activity or expression of the target protein is included in the present invention.
  • the nucleic acid inhibitor is preferably a construct of some interfering molecule, for example, dsRNA, antisense nucleic acid, small interfering RNA or microRNA, or can express or form the dsRNA, antisense nucleic acid, small interfering RNA or micro RNA construct. More preferably, it is a dsRNA or a construct that can express the dsRNA.
  • constructs for expressing dsRNA, antisense nucleic acid, small interfering RNA or microRNA can be designed. Accordingly, the present invention provides an artificially constructed construct. Designing the constructs according to the genes provided by the present invention and their sequences is well known to those skilled in the art, and the constructs can generally be made to comprise an intron sequence (not complementary to the flanking sequences;) The complementary gene sequence, after entering the cell, produces a "stem loop" structure, and the "stem” portion can form dsRNA, antisense nucleic acid, small interfering RNA or microRNA, such dsRNA, antisense nucleic acid, small interfering RNA Or microRNA can be particularly effective in inhibiting the expression of a gene of interest.
  • the nucleic acid inhibitor has the following structure:
  • Seq' is an RNA sequence corresponding to the polynucleotide sequence encoding the storage protein 2 or a fragment thereof; Seq, i5 is a sequence substantially complementary to Seq, ⁇ .
  • the nucleic acid inhibitor can be directly applied to a subject (such as a plant) to be controlled to achieve the purpose of controlling lepidopteran insects.
  • the Seq' is an RNA corresponding to the DNA selected from any one of SEQ ID NO: 3-5 SEQ ID NO: 17 SEQ ID NO: 20-21. More preferably, it is an RNA corresponding to the DNA selected from any one of SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 17, or SEQ ID NO: 20-21, the former (SEQ ID NO: 3) It can be used to develop a specific preparation for controlling Asian corn borer; the latter (SEQ ID NO: 5) is more suitable for the development of a broad-spectrum preparation for controlling lepidopteran pests.
  • the construct contains at least one of the structures shown below:
  • Seq ⁇ is a polynucleotide encoding the storage protein 2 or a fragment thereof, and Seq « is substantially complementary nucleotide sequence with Seq;
  • X is a spacer sequence located between Seq ⁇ and Seq ⁇ , and the spacer sequence is not complementary to Seq ⁇ and Seq s ;
  • the structure can be further cleaved and processed to form an interfering molecule (RNA molecule) in a double-stranded form, thereby exerting a gene silencing effect.
  • RNA molecule interfering molecule
  • the construct may be prepared in a form that forms more than one stem-loop structure, for example, may comprise two or more stem-loop structures.
  • the construct is located on an expression vector.
  • the present invention also encompasses a vector comprising the construct.
  • the expression vector typically also contains a promoter operably linked to the construct, an origin of replication and/or a marker gene, and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance .
  • a host carrying the construct or expression vector and capable of expressing a nucleic acid inhibitor can be directly administered to a need Control objects (such as plants) to achieve the purpose of controlling lepidopteran insects.
  • the nucleic acid inhibitor of the present invention can be obtained by a method of in vitro chemical synthesis in addition to cell expression using a construct. It will be understood that any method which can form the nucleic acid inhibitors of the present invention can be used in the present invention. Genetically modified plant
  • the present invention also relates to a method of controlling a pest by a plant or a method of improving the insect resistance of a plant, which comprises introducing the nucleic acid inhibitor into a plant.
  • the transgene of a plant is a technique well known to those skilled in the art, and for example, methods such as Agrobacterium transformation or gene gun transformation can be used, such as leaf disc method, immature embryo transformation method and the like.
  • methods such as Agrobacterium transformation or gene gun transformation can be used, such as leaf disc method, immature embryo transformation method and the like.
  • plants can be regenerated by conventional methods to obtain plants having insect resistance.
  • the basic principle of the method for controlling pests by plants or improving the insect resistance of plants is: using plants as a medium to make insects interfere with interfering RNA which can interfere with insect storage of white gene expression, Thereby inhibiting the growth of insects or killing insects.
  • interfering RNA refers to a substance having insect-control activity obtained by preparing or processing (such as being processed in vivo) based on the insect storage white gene or a fragment thereof (trunform form) provided as a target provided by the present invention.
  • the "interfering RNA” includes, for example, dsR A, antisense nucleoside acid, small interfering RNA, micro RNA, and the like.
  • a transgenic method is used to express a double-stranded RNA (dsR A) of an insect gene (full length or part) in a plant to form a high-abundance interfering RNA in a plant, when the insect feeds
  • This transgenic plant also ingests a large amount of interfering RNA, which can inhibit the expression of the insect gene after entering the insect, and interfere with the normal growth and development of the insect and even cause its death.
  • plants can be effectively improved in resistance to insects.
  • the application of RNA interference technology to transgenic plants and the development of new transgenic resistant plants are of great significance for the development of agriculture.
  • a method for improving the insect resistance of a plant comprises:
  • step (1) (2) contacting a plant cell or tissue or organ with Agrobacterium in step (1) to transfer said nucleic acid inhibitor to a plant cell or tissue or organ;
  • step (3) Regenerating the plant cell or tissue or organ in step (3).
  • the method can be carried out using any suitable conventional means, including reagents, temperatures, pressure conditions, and the like.
  • a medium such as Ying-Bo
  • nucleic acid inhibitors interfering molecules
  • Mao et al Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol (NATURE BIOTECHNOLOGY VOLUME 25
  • composition and method for controlling lepidopteran insects The invention also provides a formulation (e.g., a pesticidal composition) comprising a safe and effective amount of a host (cell) or a nucleic acid inhibitor (eg, a nucleic acid inhibitor) that carries the construct or expression vector and is capable of expressing a nucleic acid inhibitor : 10- 50 (Vg / ml; preferably 20-20 (Vg / ml; more preferably 30- 100 ⁇ 8 / ⁇ 1); pesticide and a pharmaceutically acceptable carrier.
  • a formulation e.g., a pesticidal composition
  • a formulation e.g., a pesticidal composition
  • a formulation e.g., a pesticidal composition
  • a formulation e.g., a pesticidal composition
  • a formulation e.g., a pesticidal composition
  • a formulation e.g., a pesticidal composition
  • a formulation e.g.
  • the term "containing” means that the various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms “consisting mainly of” and “consisting of” are included in the term “contains”.
  • the "pesticide-acceptable" component is suitable for agricultural use and does not cause excessive adverse side effects (such as toxicity, irritation, and allergic reaction) to humans or animals (other than lepidopteran insects). That is, a substance with a reasonable benefit/risk ratio.
  • the "pesticide-acceptable carrier” is for transferring a host carrying the construct or expression vector of the present invention and capable of expressing a nucleic acid inhibitor or a nucleic acid inhibitor of the present invention to a lepidopteran insect Acceptable solvents, suspending agents or excipients.
  • the pharmaceutically acceptable carrier can be a liquid or a solid, preferably a carrier which retains the host or nucleic acid inhibitor activity to a greater extent.
  • the dosage form of the preparation may be various, including but not limited to: aqueous solution, suspending agent, wettable powder, emulsifiable concentrate, emulsion, sprayable solution, aqueous dispersion, powder, granule Agent, or microcapsules. It is to be understood that as long as the host of the present invention carrying the construct or expression vector and capable of expressing the nucleic acid inhibitor or the nucleic acid inhibitor of the present invention can be delivered to the lepidopteran in keeping all or part of the activity, It is all desirable. Those which are easy to deliver are preferred, for example, the pesticidal composition is a liquid spray, or a spray.
  • the adjuvant is an auxiliary component which functions as an auxiliary regulating function, for example, it may be a surfactant, an adhesion aid or other type of auxiliary agent.
  • the active ingredient in the concentrated pesticide composition ie, the host carrying the construct or the expression vector and capable of expressing the nucleic acid inhibitor or the nucleic acid inhibitor of the present invention
  • the pesticide composition is diluted and used in the actual content of active ingredient is low, such as those containing a nucleic acid inhibitor 20-10 ( ⁇ g / m l, is administered in an amount of 50-1000 ⁇ 1 / 300-500 head, or under
  • the nucleic acid inhibitor content of the host (cell) expression carrying the construct or the expression vector and capable of expressing the nucleic acid inhibitor, a certain amount of the host (cell) is administered with reference to the nucleic acid inhibitor application amount.
  • Common components such as chemical agents, synergists, trace elements, stabilizers, binders, wetting agents, dispersants, emulsifiers, penetrants, oximes, solvents, fillers, etc.
  • the pesticide composition of the present invention It may also contain other active insecticides or microbicides.
  • suitable solid diluents include, but are not limited to, diatomaceous earth, corn hulls, tricalcium phosphate, cork powder, clays such as kaolin, bentonite or attapulgite, and water-soluble polymers.
  • the solid compositions may also contain one or more compatible wetting agents, dispersing agents, emulsifying agents or pigments which may also act as diluents in the solid state.
  • Such a solid composition may be in the form of a powder, granule or wettable powder.
  • the powder is usually obtained by grinding, and granules, tablets or bricks are obtained by granulation or tableting.
  • the liquid composition may be in the form of a solution, a suspension and an emulsion, or it may be coated in a natural or synthetic polymer. And may contain a wetting agent, a dispersing agent or an emulsifier.
  • emulsions, suspensions or solutions can be prepared with aqueous, organic or water-organic diluents to prepare water soluble polymers (and mixtures of the above diluents).
  • the diluent may contain, for example, the above-mentioned ionic or nonionic wetting agent, dispersing agent or emulsifier or a mixture thereof.
  • Desirable formulation adjuvants for use in the compositions of the present invention are also known, for example: Watkins “Insecticide Powder Thinner and Carrier Handbook” 2nd Edition, Darland Books, Caldwell N. I; HvOlphen, "Clay Colloid Chemistry” 2nd ed., J. Wiley & Sons, NY, Marsden, “Solvent Guide” 2nd Edition, Interscience, N Y. 1950; McCutcheon's, “Detergents and Emulsifiers", MC Publ. Corp., Ridgewood NJ; Sisley and Wood, “Encyclopedia of Surfactants", Chem Publ. Co. Inc., NY I 964; Schonfelt, " GrenzflachenGermanin
  • the wettable powder can be uniformly dispersed in water.
  • the wettable powder may further contain a non-environmentally hazardous substance such as a wetting agent, a dispersing agent, and a diluent.
  • the powder may be prepared by grinding the active material together with a solid material such as finely pulverized talc, kaolin, bentonite or the like, or a solid material such as diatomaceous earth.
  • the granules may be prepared by spraying the active substance particles onto the inert substance particles, or applying the active substance solution to a carrier (for example, sand, kaolin or inert substance) through a binder (for example, polyvinyl alcohol, sodium polyacrylate, or mineral oil). Particle) surface.
  • a suitable active substance can be prepared into granules as in the preparation of fertilizer granules.
  • the transport of insect control dsRNA to the surface of the plant by spray application provides another means of protecting plants.
  • bacteria engineered to produce and accumulate dsRNA can be fermented, and the fermented product is formulated as a spray product compatible with common agricultural applications.
  • the formulation may include: suitable gums and wetting agents for efficient leaf coverage, and UV protectants for protecting dsRNA from UV damage.
  • suitable gums and wetting agents for efficient leaf coverage and UV protectants for protecting dsRNA from UV damage.
  • UV protectants for protecting dsRNA from UV damage.
  • formulations for soil applications can include a granular formulation that acts as a bait for larvae of soil harmful insects (e.g., corn rootworm).
  • the inventors have unexpectedly discovered that the nucleic acid inhibitor can be invaded from the insect body into the insect by infiltration. Therefore, insect control can be achieved by applying various nucleic acid inhibitors or compositions containing various nucleic acid inhibitors to insect body surfaces.
  • the modes of application include, but are not limited to, spraying, coating, and dropping onto the surface of an insect.
  • the nucleic acid inhibitor is selected from the group consisting of: dsRNA, antisense nucleic acid, small interfering RNA, microRNA; or a construct capable of expressing or forming the dsRNA, antisense nucleic acid, small interfering RNA, microRNA.
  • the present invention also provides a method for controlling lepidopteran insects, the method comprising: inhibiting said host (cell) or said nucleic acid carrying said construct or expression vector capable of expressing a nucleic acid inhibitor
  • the substance is applied to a subject to be controlled (such as a plant, in particular a plant infested by a lepidopteran insect; or the insect itself).
  • the pesticidal composition can be formulated as a spray so that it can be directly sprayed for pest control.
  • the dsRNA can be directly sprayed for pest control, and the purified dsRNA is added to the penetrant or the sputum to spray the field for pest control.
  • the invention aims at the prevention and control of lepidopteran pests, and develops a target gene and an effective fragment (domain thereof) which can be used for field control of insects based on RNAi technology, and proves that the simple inhibition of the nucleic acid inhibitor can be suppressed by spraying.
  • the effect of gene expression ultimately leads to the death of lepidopteran pests, thereby achieving the purpose of prevention and treatment, which is convenient, rapid, accurate and pollution-free.
  • the present invention first revealed and confirmed a novel target gene which can be used as a target for controlling insects.
  • the present invention discloses for the first time that different target domains of genes can be used to synthesize specific and broad-spectrum target genes for controlling lepidopteran pests, and it is also proved that dsRNAs obtained based on these gene domains can be directly applied. Pest control.
  • the pest control method is convenient, fast, accurate, and can well solve the pest resistance and environmental pollution problems currently encountered in pest control.
  • the present invention is based on RNAi technology for the purpose of insecticidal, which is a novel high-efficiency, fixed-point control, pollution-free technology; the obtained dsRNA can be directly applied to the field for pest control.
  • RNA samples with concentration ⁇ 300 ⁇ 8 / ⁇ 1, total ⁇ 6 g, and OD260/280 of 1.8 ⁇ 2.2.
  • the polyA-bearing mRNA was isolated using magnetic beads with oligo-dT, and then the first strand of cDNA was synthesized using a random 6-mer and Invitrogen's Superscript II reverse transcriptase kit.
  • the target gene-specific primer (OfSPF: CATGGGGACACAGTTGAGCTTC (SEQ ID NO: 14); OfSPR: TGCTGTAAATCTAGCCTCAGTCA (SEQ ID NO: 15)) was used for amplification, and the obtained gene fragment was purified and ligated to (made by Takara) PMD-18
  • the transformation into ToplO strain, blue-white spot screening, positive strain sequencing, and the sequencing result of the full length sequence of Asian corn borer storage white 2 are as follows (SEQ ID NO: 1;
  • the inventors selected three domains of the storage protein 2 gene (SP-N, SP-M, SP-C, the sequence is shown in Table 2), and amplified.
  • the primers required for the three domains are shown in Table 3.
  • EYFP is an enhanced yellow fluorescent white gene as a foreign gene control.
  • the DNA sequences of the three domains SP-N, SP-M, and SP-C of the purine white 2 gene were amplified by the primers described in Table 3, respectively, as templates for the synthesis of dsRNA.
  • the target gene dsRNA was synthesized using the MEGAscript® T7 Kit (available from AMBION, product number AM 1334) to obtain dsRNA for bioassay.
  • the brief principles and steps are as follows: First, the total RNA of the Asian corn borer larvae tissue is used as a template to obtain cDNA by reverse transcription, and then the domains of different OfSP genes are amplified by the dsRNA synthesis primers listed in Table 3, and the products are purified. Then, using the purified product as a template, in vitro transcription using the Kit kit, obtaining dsRNA, removing the DNA in the product, and purifying the desired dsRNA.
  • the specific steps are as follows:
  • the in vitro transcription reaction is completed.
  • the enzymatic reaction system contains a small amount of template DNA, which needs to be digested by DNasel. Dnasel is added to the above reaction solution, mixed uniformly, and incubated for 37 min for 30 min.
  • reaction was terminated by adding lOO L and 15 L ammonium acetate;
  • dsSP-N The final dsRNAs were named as: dsSP-N, dsSP-M dsSP-C, and the results of electrophoresis identification are shown in Figure 4. It can be seen that the desired dsRNA was successfully obtained.
  • Example 3 Silencing effect of dsRNA on the genes of Asian corn borer
  • the present inventors directly sprayed dsSP-C (dissolved in ddH 2 0) on the first instar larva of Asian corn borer, and observed changes in gene expression.
  • the gene expression on days 0, 1, 3, and 5 after storage of the sputum white 2 gene (O yP) is shown in Fig. 5.
  • the inventors directly obtained the dsRNA (dsSP-N, dsSP-M, dsSP-C dissolved in dd 0) directly sprayed on the Asian corn borer.
  • the lethal concentration of LC 5 on the 7th day of dsSP-N is the standard concentration, the concentration is 41.55 g/mL
  • the concentration is 41.55 g/mL
  • the inventors directly sprayed the obtained dsRNA (dsSP-N dsSP-M, dsSP-C dissolved in dd3 ⁇ 40) onto the newly hatched larva of Asian corn borer.
  • the lethal medium concentration LC 5 o of dsSP-N on day 7 was used as the standard concentration, the concentration was 41.55 ⁇ g/mL), and the lethality of the larvae on day 1, 3, 5, and 7 after spraying was observed.
  • dsSP-N, dsSP-M and dsSP-C synthesized from the three domains of the target gene of the present invention were sprayed on Asian corn borer, respectively. After that, their lethal rates on day 7 were 74.82%, 73.13%, and 76.69%, respectively, while the blank control (CK) and the exogenous gene control (EYFP) had no significant lethal effect.
  • Example 7 The lethal rate of cotton bollworm by the dsRNA of SP gene of Helicoverpa armigera
  • the method described in the present invention is applied to the study of cotton bollworm.
  • the nucleotide sequence of the gene PxSP1 homologous to the corn borer sp gene in Helicoverpa armigera is shown in SEQ ID NO.
  • the present inventors prepared a dsRNA of a gene homologous to the corn borer sp gene (H.arm-SP) in the cotton bollworm, and the gene fragment homologous to the corn borer sp gene is as follows (SEQ ID NO: 17):
  • the synthesis of the target gene dsRNA was carried out by referring to the method provided in Example 2 using MEGAscript® T7 Kit (available from AMBION, product number AM1334).
  • the primer sequences are as follows:
  • the present inventors directly obtained a dsRNA (dsH.arm-SP, dissolved in dd3 ⁇ 40) of a gene homologous to the corn borer sp gene (d.H.arm-SP) in the cotton-instar worm, directly sprayed on the newly hatched larva of the cotton bollworm (
  • the lethal concentration LC 5 of Asian corn borer (Of) dsSP-N on day 7 was used as the standard concentration, the concentration was 41.55 wg/mL), and the first, third, fifth, and seventh days (d) larvae were observed after spraying. The fatal situation.
  • Example 8 The lethal rate of dsRNA of SP gene of Plutella xylostella to P. xylostella Using the method of the present invention, it is applied to the research of Plutella xylostella.
  • the nucleotide sequence of the gene PxSP l homologous to the corn borer sp gene in Plutella xylostella is shown in SEQ ID NO: 18; the nucleotide sequence of the gene PxSP2 homologous to the corn borer sp gene in Plutella xylostella is shown in FIG.
  • TGTACCTCAACCGCGACG A CAAACTCAG( GTA C' TTCACTGAAGACATCGACCTGAACA CC
  • Example 2 The method of Example 2 with reference to embodiments provided by MEGAscript® T7 Kit (available from AMBION company, producing f Num AMI 334) was synthesized dsRNA of target genes.
  • the primer sequences are as follows:
  • Px SP1 DSF TAATACGACTCACTATAGGGAGAACGTCGACCGCCAGATGAAGGA (SEQ ID NO: 32) Px SP1 DSR TMTACGACTCACTATAGGGAGATTGTGCAGGGTCATCCAGGGCA (SEQ ID NO: 33) Px SP2 DSF TAATACGACTCACTATAGGGAGAACGTCGACCGCCAGATGAAGGA (SEQ ID NO: 34) Px SP2 DSR TAATACGACTCACTATAGGGAGAACACCGTTGTGCAGCGCCAT (SEQ ID NO. 35)
  • the present inventors directly obtained the dsRNAs of P. xylostella SP 1 and SP2 (dsPxSP1 and dsPxSP2 dissolved in ddH 2 0, respectively) directly onto the newly hatched larvae of Plutella xylostella (the lethal concentration in the 7th day of Asian corn borer dsSP-N)
  • the LC50 was the standard concentration at a concentration of 41.55 g/mL), and the lethality of the larvae on days 1, 3, and 5 after the spraying was observed.
  • dsRNAs synthesized using the double-stranded RNA (PxSP l and PxSP2) of the two target genes of Plutella xylostella of the present invention were sprayed on the newly hatched larvae of Plutella xylostella, respectively, on the fifth day.
  • the lethal rates were 58.00% and 54.67%, respectively, and the mortality rate of Plutella xylostella was tested by dsSP of corn borer.
  • the 5-day mortality rate was 14.67%. There was no significant difference from the control.
  • Example 9 application of dsRNA
  • the Asian corn borer gene has the most homologous relationship with the cotton bollworm, but the Asian corn borer SP-C is utilized.
  • the dsRNA can achieve the same insecticidal effect in cotton bollworm, and it is expected that it will have a similar effect on other lepidopteran pests.
  • Example 10 SP-N, SP-M, SP-C truncated form and its effect
  • the present inventors designed a truncated form of dsRNA fragment based on SP-N (SEQ ID NO: 3), SP-M (SEQ ID NO: 4) and SP-C (SEQ ID NO: 5). as follows:
  • SP-N-M1 dsRNA The synthesis and purification method is as in Example 2, except that the target gene fragment is position 1-420 of SEQ ID NO: 3.
  • SP-N-M2 dsRNA The synthesis and purification method was as in Example 2, except that the target gene fragment was at positions 10-420 of SEQ ID NO: 3.
  • the SP-M-M1 dsRNA synthesis and purification method was as in Example 2 except that the target gene fragment was position 1-615 of SEQ ID NO: 4.
  • the SP-M-M2 dsRNA synthesis and purification method was as in Example 2 except that the target gene fragment was at positions 10-615 of SEQ ID NO: 4.
  • SP-C-M1 dsRNA synthesis and purification method as in Example 2, except that the target gene fragment is SEQ ID NO:
  • the SP-C M2 dsRNA; synthesis and purification method was as in Example 2, except that the target gene fragment was at position 10-603 in SEQ ID NO: 5.
  • Example 11 Construction and Transformation of Transgenic Vectors
  • the desired dsRNA vector as shown by pART27-dsRNA in ge 13, includes a 35S promoter, a reverse gene fragment (ie, Anti-sense), an intron of the PDK (pyruvate phosphokinase) gene (ie, Intron, approximately 742 bp), a forward gene fragment (ie Sense C1) and an OCS terminator, as well as the NOS promoter, ⁇ ⁇ (neomycin phosphotransferase gene) and NOS terminator.
  • a reverse gene fragment ie, Anti-sense
  • an intron of the PDK (pyruvate phosphokinase) gene ie, Intron, approximately 742 bp
  • a forward gene fragment ie Sense C1
  • OCS terminator as well as the NOS promoter, ⁇ ⁇ (neomycin phosphotransferase gene) and NOS terminator.
  • the expression vector was digested with Not I by a binary vector pKANNIBAL vector (purchased from the Chinese plasmid vector strain cell strain gene collection) containing the Sense-Intron-Antisense sequence, and then ligated into the pART27 vector which was also digested with Not I. From Chinese plasmid The vector strain cell line gene collection center was constructed and obtained.
  • the gene-specific primers dsGFP-2F-Kpn I and dsGFP_2R_Xho I; ds0fSP_C2F_Kpn I and ds0fSP_C2R-Xho I containing Kpn I and Xho I cleavage sites were treated with high-fidelity enzyme KOD, respectively, with GFP and Asian corn borer.
  • cDN A was used as a template for PCR amplification, and the corresponding dsGFP-2 and dsOf SP-C2 fragments were cloned.
  • the cloned fragment was double digested with Kpn I and Xho I and inserted into the corresponding cloning site (Kpn I /Xho I ) on the pKANNIBAL vector which already contained the forward dsGFP-1 and dsOfSP-C 1 fragments. between.
  • the constructed pKANNIBAL/dsGFP and pKANNIBAL/dsOfSP-C vectors were digested with Not I, respectively, and pART27 was digested with Not l, and the digested dsGFP and dsOfSP_C fragments were inserted into pART27 vector, respectively.
  • Recombinant expression vectors carrying the corresponding fragments of interest, respectively, are referred to as 35 S : dsGFP and 35S : : dsOfSP-C expression vectors, respectively.
  • Agrobacterium tumefaciens The transformation of Agrobacterium tumefaciens is carried out by freeze-thaw method.
  • a single colony GV3101, LBA4404 or EHA105 (all purchased from Invitrogen), 3ml LB medium (containing 50 ⁇ ⁇ / ⁇ 1 Kan kanamycin and 25 g / ml rifamycin Rif), 28. C, 220 rpm, culture to OD600 0.5 (about 6 hours). Place on ice for 30 minutes, centrifuge at 5000 g for 5 minutes at 4 °C. Resuspend in 1 mL of 0.15 M NaCl. 4. C, 5000 g was centrifuged for 5 minutes.
  • transgene of Arabidopsis thaliana, tobacco, and maize is taken as an example, and the transformation of other plants can also be referred to herein.
  • the flower bud part of the plant is soaked in the bacterial liquid for 5 seconds, placed in a plastic pot, moisturized, protected from light for 16 to 24 hours, and then grown in the greenhouse to flowering and seeding.
  • the TO generation seeds were treated with spring water for 4 days at 4 °C, treated with 20% bleach for 15 minutes, and washed with sterile water for 3 ⁇ times.
  • Agrobacterium LBA4404 containing the gene of interest was cultured overnight (cultured overnight at 28 ° C to OD 600 2.0).
  • the sterile tobacco leaves are cut to a size of about 1.0 cm 2 and soaked in Agrobacterium culture medium for 5 to 10 minutes.
  • the excess Agrobacterium culture medium was aspirated with sterile filter paper, and the soaked tobacco leaves were spread on 1/2 MS steroid medium and cultured in the dark for two days. Then, the leaves were moved to MS (containing 1 mg/L 6-BA)
  • the newly grown small shoots were transferred to MS medium without 6-BA, and fresh MS was replaced every 10 to 15 days until it was rooted and moved to the soil for planting.
  • Rif's YEP liquid medium was incubated at 28 °C to OD600 0.6, and centrifuged at 8000 rpm for 5 minutes to collect Agrobacterium.
  • the bacteria were suspended in the infested medium and acetosyringone (AS) 100 ⁇ /L and 0.05% surfactant Silwet L-77 were resuspended in the suspension before Agrobacterium transformation.
  • AS acetosyringone
  • Silwet L-77 surfactant Silwet L-77 were resuspended in the suspension before Agrobacterium transformation.
  • Pick the neat and full corn seeds soak them in 70% ethanol for 2 ⁇ 5 minutes, then soak them in 0.1% HgC12 for 10 ⁇ 5 minutes, then rinse them with sterile water for 3 ⁇ 5 times.
  • the T3 homozygous transgenic plants obtained in Example 12 were selected and screened by antibiotics.
  • RNA sample of the transgenic plants was extracted by a conventional method, and lOx electrophoresis sample solution was added to the RNA sample, mixed, placed at 65 ° C for 10 minutes, and cooled on ice. Electrophoresis was performed using 15% TBE-urea PAGE gel, ⁇ ⁇ running buffer. Pre-electrophoresis for 5 to 10 minutes before loading and rinse the lane with running buffer to remove the urea precipitated from the well. The amount of each lane was 10-20 total RNA, and the electric field strength was 20 V/cm, which ended when the bromophenol blue dye migrated to the bottom of the gel. The gel was equilibrated in l xTBE for 10 minutes.
  • Hofer Semi-Dry Transfer Units Amersham, Cat. 80-621 1-86. Transfer conditions: 40 mA (about 7-8 V), 2 to 4 hours, Hybond-N+ (Amersham, Cat. RPN303B) nylon membrane.
  • Electrophoresis gel storage solution 15% polyacrylamide (30% Acyl/bis, 19:1, Huajing, Cat. W443), 8M urea, ⁇ ⁇ ;
  • Probe labeling 25 ng of purified PCR product was used as a template labeling probe. The probe was labeled using Prime-a-Gene Labeling System (Promega, Cat. U1 100). 37 ° C water bath for 1 hour. The labeled probe was placed in boiling water for 5 minutes and immediately placed on ice for later use.
  • Pre-hybridization and hybridization were used (Clontech's ExpressHyb system): The nylon membrane was placed in a hybridization tube and wetted with 6xSSC to ensure no air bubbles between the membrane and the tube wall. Pour 6x SSC, add 5 mL of hybridization solution, and pre-hybridize at 37 °C for 60 minutes. After the pre-hybridization is completed, replace 5 mL of fresh hybridization solution, add the probe, mix, and hybridize overnight.

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Abstract

La présente invention concerne un nouveau procédé de prévention et d'élimination des organismes nuisibles fondé sur une technologie d'ARNi. L'invention concerne un gène cible et une protéine codée par celui-ci qui sont utiles pour la prévention et l'élimination des insectes lépidoptères. La préparation d'un inhibiteur d'acide nucléique ou d'un hôte exprimant l'inhibiteur d'acide nucléique fondé sur la séquence d'acides nucléiques du gène cible permet de tuer efficacement les insectes lépidoptères. La présente invention concerne également l'utilisation de différents fragments du même gène pour obtenir des préparations pesticides de type spécifique à une espèce d'insectes et de type interspécifique à spectre large et le procédé associé.
PCT/CN2012/000731 2011-05-27 2012-05-28 Nouveau procédé pour prévenir et éliminer les organismes nuisibles fondé sur une technologie d'arni Ceased WO2012163084A1 (fr)

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