WO2019219015A1 - 对蚜虫高效致死的RNAi靶标基因及其应用 - Google Patents

对蚜虫高效致死的RNAi靶标基因及其应用 Download PDF

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WO2019219015A1
WO2019219015A1 PCT/CN2019/086896 CN2019086896W WO2019219015A1 WO 2019219015 A1 WO2019219015 A1 WO 2019219015A1 CN 2019086896 W CN2019086896 W CN 2019086896W WO 2019219015 A1 WO2019219015 A1 WO 2019219015A1
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gene
seq
dsrna
insect
sequence
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PCT/CN2019/086896
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English (en)
French (fr)
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苗雪霞
李海超
关若冰
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中国科学院上海生命科学研究院
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Priority to BR112020023369-2A priority Critical patent/BR112020023369A2/pt
Priority to CA3100442A priority patent/CA3100442A1/en
Priority to JP2020564432A priority patent/JP2021523726A/ja
Priority to KR1020207035963A priority patent/KR20210010530A/ko
Publication of WO2019219015A1 publication Critical patent/WO2019219015A1/zh
Priority to US17/097,574 priority patent/US20210139902A1/en

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    • 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
    • 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
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
    • A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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    • 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
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    • 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
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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 pertains to the field of biotechnology and agricultural applications.
  • the present invention relates to an RNAi target gene that is highly lethal to aphids and uses thereof.
  • Aphids are an important worldwide pest, belonging to the Hemiptera Hemiptera, Aphidoidea, which is currently known to be more than 4,700 species. It is small in size and fast in reproduction. It is an important agricultural and horticultural pest.
  • chemical agents are still the main ones, but because of their rapid reproduction speed and strong concealment, their control effects are poor, and it is necessary to use a large amount of pesticides to inhibit their reproduction, which inevitably causes the resistance of aphids.
  • RNAi has been widely used as a tool for gene function research, especially in animals and plants where genetic manipulation tools are imperfect.
  • dsRNA must enter the cell after feeding into the insect to activate the RNAi mechanism.
  • Insect intestinal wall cells can prevent most dsRNA from entering other tissues, which is a key factor affecting the efficiency of RNAi. It is also the biggest obstacle in the application of dsRNA oral delivery method.
  • a dsRNA construct the construct of the dsRNA is double stranded, and the positive or negative strand thereof comprises the structure of formula I:
  • Seq is positive for the nucleotide sequence of a related gene or fragment of insect nymph and/or adult stage regulation
  • Seq reverses to a nucleotide sequence that is substantially complementary to the Seq forward ;
  • X is a spacer sequence located between the forward and Seq Seq the reverse, and the spacer sequence Seq Seq forward and reverse are not complementary,
  • insect nymph and/or adult stage regulation-related genes are selected from the group consisting of a DS7 gene, a DS9 gene, a DS15 gene, a DS25 gene, a DS27 gene, a DS45 gene, or a combination thereof.
  • the dsRNA is at least 21 nt in length.
  • the dsRNA has a length of 21 nt to 1350 nt, preferably 506 nt to 1093 nt, for the DS7 gene.
  • the dsRNA has a length of 21 nt to 909 nt, preferably 54 nt to 631 nt, for the DS9 gene.
  • the dsRNA is 21 nt to 2148 nt in length, preferably 516 nt to 1029 nt, for the DS15 gene.
  • the dsRNA is 21 nt to 1233 nt in length, preferably 58 nt to 674 nt.
  • the dsRNA has a length of 21 nt to 1 152 nt, preferably 219 nt to 748 nt, for the DS27 gene.
  • the dsRNA is 21 nt to 909 nt in length, preferably 42 nt to 637 nt, for the DS45 gene.
  • the homology to the dsRNA is at least 80%, preferably from 85% to 100%.
  • the Seq forward and Seq inverse lengths are at least 50 bp.
  • the dsRNA construct can form a dsRNA of formula II,
  • Seq 'Forward Forward sequence corresponds to Seq RNA sequences or fragments of sequences
  • Seq' reverse is a sequence that is substantially complementary to the Seq' forward ;
  • the dsRNA is a dsRNA without loop.
  • the dsRNA is amplified from the sequence set forth in SEQ ID NO.: 9-10.
  • the dsRNA is amplified from the sequence set forth in SEQ ID NO.: 11-12.
  • the dsRNA is amplified from the sequence set forth in SEQ ID NO.: 13-14.
  • the dsRNA is amplified from the sequence set forth in SEQ ID NO.: 15-16.
  • the dsRNA is amplified from the sequence set forth in SEQ ID NO.: 17-18.
  • the dsRNA is amplified from the sequence set forth in SEQ ID NO.: 19-20.
  • a second aspect of the invention provides a dsRNA of formula II,
  • Seq 'positive regulatory RNA sequences or fragments of sequences a nucleotide sequence-related genes or fragments of corresponding insect nymphs and / or adult stage;
  • Seq' reverse is a sequence that is substantially complementary to the Seq' forward ;
  • X' is none; or is a spacer sequence located between the Seq' forward and Seq' inversions , and the spacer sequence is not complementary to the Seq' forward and Seq' inversion ;
  • insect nymph and/or adult stage regulation-related genes are selected from the group consisting of a DS7 gene, a DS9 gene, a DS15 gene, a DS25 gene, a DS27 gene, a DS45 gene, or a combination thereof;
  • the Seq forward and Seq inverse lengths are at least 50 bp.
  • the interval sequence X' has a length of 0 to 300 bp.
  • the nymphal and/or adult stage regulation-related genes are derived from aphis.
  • sequence of the DS7 gene is set forth in SEQ ID NO.: 1 or 24.
  • sequence of the DS9 gene is set forth in SEQ ID NO.: 2 or 25.
  • sequence of the DS15 gene is set forth in SEQ ID NO.: 3 or 26.
  • sequence of the DS25 gene is set forth in SEQ ID NO.: 4 or 27.
  • sequence of the DS27 gene is set forth in SEQ ID NO.: 5 or 28.
  • sequence of the DS45 gene is set forth in SEQ ID NO.: 6 or 29.
  • the insect is a herbivorous insect, preferably a homoptera insect, most preferably a genus aphid.
  • the insect is selected from the group consisting of: peach aphid, soybean meal, or a combination thereof.
  • a third aspect of the invention provides an expression vector comprising the dsRNA construct of the first aspect of the invention.
  • a fourth aspect of the present invention provides a host cell comprising the expression vector of the third aspect of the present invention or a DNA in which a dsRNA construct corresponding to the first aspect of the present invention is integrated in a chromosome sequence.
  • the host cell is a plant cell, preferably a green leaf plant cell.
  • the plant comprises a cruciferous plant such as a vegetable or soybean.
  • a fifth aspect of the invention provides a composition comprising the dsRNA construct of the first aspect of the invention and/or the dsRNA of the second aspect of the invention, and insect feeding acceptable Carrier.
  • the insect-fed acceptable carrier comprises water.
  • the composition is a composition for inducing or causing death of a genus nymph and/or adult stage.
  • the dsRNA has the following sequence:
  • dsRNA1 having the sequence corresponding to SEQ ID NO.: 1 or 24;
  • dsRNA2 having the sequence corresponding to SEQ ID NO.: 2 or 25;
  • dsRNA3 having the sequence corresponding to SEQ ID NO.: 3 or 26;
  • dsRNA4 having the sequence corresponding to SEQ ID NO.: 4 or 27;
  • dsRNA5 having the sequence corresponding to SEQ ID NO.: 5 or 28;
  • dsRNA6 has a sequence corresponding to SEQ ID NO.: 6 or 29.
  • the DS7 gene, the DS9 gene, the DS15 gene, the DS25 gene, the DS27 gene, and/or the DS45 gene are derived from an insect, preferably from a Homoptera, preferably from Aphis.
  • the dsRNA1 content in the pharmaceutical composition is from 1 to 500 ng/ ⁇ l, preferably from 5 to 300 ng/ ⁇ l, more preferably from 50 to 150 ng/ ⁇ l.
  • the dsRNA2 content in the pharmaceutical composition is from 1 to 500 ng/ ⁇ l, preferably from 5 to 300 ng/ ⁇ l, more preferably from 50 to 150 ng/ ⁇ l.
  • the dsRNA3 content in the pharmaceutical composition is from 1 to 500 ng/ ⁇ l, preferably from 5 to 300 ng/ ⁇ l, more preferably from 50 to 150 ng/ ⁇ l.
  • the dsRNA4 content in the pharmaceutical composition is from 1 to 500 ng/ ⁇ l, preferably from 5 to 300 ng/ ⁇ l, more preferably from 50 to 150 ng/ ⁇ l.
  • the dsRNA5 content in the pharmaceutical composition is from 1 to 500 ng/ ⁇ l, preferably from 5 to 300 ng/ ⁇ l, more preferably from 50 to 150 ng/ ⁇ l.
  • the dsRNA6 content in the pharmaceutical composition is from 1 to 500 ng/ ⁇ l, preferably from 5 to 300 ng/ ⁇ l, more preferably from 50 to 150 ng/ ⁇ l.
  • a sixth aspect of the invention provides the dsRNA construct of the first aspect of the invention, or the dsRNA of the second aspect of the invention, or the host cell of the fourth aspect of the invention, or the fifth aspect of the invention.
  • a seventh aspect of the present invention provides a method for killing insects, comprising the steps of: interfering molecules that regulate expression of related genes by interfering with insect nymphs and/or adult stages, or vectors, cells, plant tissues or insects containing said interfering molecules Feeding or spraying insects with control agents;
  • the Kunn and/or adult stage control related genes are selected from the group consisting of a DS7 gene, a DS9 gene, a DS15 gene, a DS25 gene, a DS27 gene, a DS45 gene, or a combination thereof.
  • the killing insect comprises:
  • the interfering molecule is selected from the group consisting of a dsRNA, an antisense nucleic acid, a small interfering RNA, and a microscopy that inhibit or silence a target with a nymph and/or adult stage-regulated gene or a fragment thereof or a transcript thereof.
  • RNA Ribonucleic acid
  • insect nymph and/or adult stage regulation-related genes are derived from aphids.
  • the insect is a herbivorous insect, preferably from a Hemiptera insect, most preferably from the genus Aphid.
  • the method comprises the steps of: using the dsRNA construct of the first aspect of the invention, or the dsRNA of the second aspect of the invention, or the host cell of the fourth aspect of the invention, or The composition of the fifth aspect of the invention feeds or sprays insects.
  • An eighth aspect of the present invention provides a method of preparing the dsRNA of the second aspect of the present invention, comprising the steps of:
  • Seq is positive for the nucleotide sequence of a related gene or fragment of insect nymph and/or adult stage regulation
  • Seq reverses to a nucleotide sequence that is substantially complementary to the Seq forward ;
  • X is a spacer sequence located between the forward and Seq Seq the reverse, and the spacer sequence Seq Seq forward and reverse are not complementary,
  • insect nymph and/or adult stage regulation-related genes are selected from the group consisting of a DS7 gene, a DS9 gene, a DS15 gene, a DS25 gene, a DS27 gene, a DS45 gene, or a combination thereof;
  • step (ii) transferring the construct of step (i) into a host cell, thereby expressing in the host cell a dsRNA of formula II,
  • Seq 'Forward Forward sequence corresponds to Seq RNA sequences or fragments of sequences
  • Seq' reverse is a sequence that is substantially complementary to the Seq' forward ;
  • a ninth aspect of the invention provides a method of preparing an insect control agent comprising the steps of: the dsRNA construct of the first aspect of the invention, or the dsRNA of the second aspect of the invention, or the fourth aspect of the invention.
  • the host cell, or the composition of the fifth aspect of the invention is sprayed onto the surface of the plant to produce an insect control agent.
  • the plant is selected from the group consisting of soybean, radish, peach, tobacco, or a combination thereof.
  • a tenth aspect of the invention provides a method for improving plant resistance to insects, comprising:
  • the recombinant DNA construct comprises DNA encoding an RNA having substantially the same or substantially complementary to at least 21 or more contiguous nucleotides of the target gene a sequence, wherein the target gene is an insect nymph and/or adult stage regulation-related gene, selected from the group consisting of a DS7 gene, a DS9 gene, a DS15 gene, a DS25 gene, a DS27 gene, a DS45 gene, or a combination thereof.
  • the target gene is selected from the group consisting of:
  • the target gene is as shown in any one of SEQ ID NO.: 1-6, 24-29.
  • the homology to the RNA is at least 80%, preferably from 85% to 100%, more preferably from 95 to 100%.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 1350 nt, preferably 506 nt to 1093 nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 909 nt, preferably, 54 nt to 631 nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21nt-2148nt, preferably 516nt-1029nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 1233 nt, preferably, 58 nt to 674 nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 1 152 nt, preferably 219 nt to 748 nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 909 nt, preferably, 42 nt to 637 nt contiguous nucleotides of the target gene.
  • the RNA is a dsRNA comprising at least one RNA strand.
  • the RNA strand comprises at least 90%, preferably 95-100% homologous to a sequence selected from any one selected from the group consisting of SEQ ID NO.: 1-6, 24-29. Sexual sequence.
  • the recombinant DNA construct comprises a promoter, preferably a heterologous promoter.
  • the promoter is selected from the group consisting of a constitutive promoter, a space-specific promoter, a time-specific promoter, a development-specific promoter, an inducible promoter, or a combination thereof.
  • the promoter is a promoter that is functional in plants.
  • the promoter is selected from the group consisting of a pol II promoter, a pol III promoter, a pol IV promoter, a pol V promoter, or a combination thereof.
  • the recombinant DNA construct further comprises one or more additional elements selected from the group consisting of an enhancer, a small RNA recognition site, an aptamer or ribozyme, a terminator, for expression coding.
  • additional elements selected from the group consisting of an enhancer, a small RNA recognition site, an aptamer or ribozyme, a terminator, for expression coding.
  • Additional and additional expression cassettes of the sequence eg, expressing a transgene, such as a pesticidal protein or a selectable marker
  • non-coding sequences eg, expressing additional inhibitory elements
  • the plant further expresses one or more insecticide proteins selected from the group consisting of patatin, phytohemagglutinin, plant carcass, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus licheniformis insecticidal protein, Bacillus sphaericus insecticidal protein.
  • one or more insecticide proteins selected from the group consisting of patatin, phytohemagglutinin, plant carcass, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus licheniformis insecticidal protein, Bacillus sphaericus insecticidal protein.
  • the plant comprises an angiosperm and a gymnosperm.
  • the gymnosperm plant is selected from the group consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cedaraceae, Cypress, and three-pointed Cedaraceae, Taxaceae, Ephedra, Matroaceae, Monocytogenes, Centennial Orchidaceae, or a combination thereof.
  • the plant comprises a monocot and a dicot.
  • the plant comprises a herbaceous plant and a woody plant.
  • the herbaceous plant is selected from the group consisting of Solanaceae, Gramineae, Leguminous, or a combination thereof.
  • the woody plant is selected from the group consisting of kiwifruit, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of: cruciferous plants, gramineous plants, legumes, Solanaceae, kiwifruit, Malvaceae, Paeonia, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of Arabidopsis thaliana, rice, Chinese cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, radish, or combinations thereof.
  • An eleventh aspect of the present invention provides a method of preparing a transgenic plant cell, comprising the steps of:
  • RNA DNA encoding DNA
  • the RNA a sequence having substantially the same or substantially complementary to at least 21 or more contiguous nucleotides of said target gene, wherein said target gene is an insect nymph and/or adult stage regulating related gene, selected from the group consisting of: DS7 Gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene, or a combination thereof.
  • the target gene is selected from the group consisting of:
  • the homology to the RNA is at least 80%, preferably from 85% to 100%, more preferably from 95 to 100%.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 1350 nt, preferably 506 nt to 1093 nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 909 nt, preferably, 54 nt to 631 nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21nt-2148nt, preferably 516nt-1029nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 1233 nt, preferably, 58 nt to 674 nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 1 152 nt, preferably 219 nt to 748 nt contiguous nucleotides of the target gene.
  • the RNA has a sequence that is substantially identical or substantially complementary to 21 nt to 909 nt, preferably, 42 nt to 637 nt contiguous nucleotides of the target gene.
  • the transfection is performed using an Agrobacterium transformation method or a gene gun bombardment method.
  • a twelfth aspect of the present invention provides a method of preparing a transgenic plant, comprising the steps of:
  • the transgenic plant cell prepared by the method of the eleventh aspect of the invention is regenerated into a plant body, thereby obtaining the transgenic plant.
  • a thirteenth aspect of the invention provides a transgenic plant cell, which is prepared by the method of the eleventh aspect of the invention.
  • a fourteenth aspect of the invention provides a transgenic plant prepared by the method of the twelfth aspect of the invention.
  • Figure 1 shows the control effect of the target gene on aphids.
  • Figure 2 shows the results of detection of relative expression levels of target genes.
  • Figure 3 shows the control effect of three target genes of Myzus persicae in the field.
  • Figure 4 shows the results of statistical analysis of the control effect and the population loss rate of the peach aphid field.
  • the present inventors conducted extensive and intensive research to screen for a nymphal nymph and/or adult stage-related gene fragment, and unexpectedly found that the DS7 gene, SEQ ID NO., as shown in SEQ ID NO.: 1 or 24: The DS9 gene shown in 2 or 25, the DS15 gene shown in SEQ ID NO.: 3 or 26, the DS25 gene shown in SEQ ID NO.: 4 or 27, and the DS27 gene shown in SEQ ID NO.: 5 or 28.
  • the present invention can also construct plants capable of improving resistance to insects, and the method of the present invention can also effectively kill aphids, the control effect of aphids is ⁇ 80%, and the rate of reduction of insects is ⁇ 70%. On the basis of this, the inventors completed the present invention.
  • crop refers to various plants grown on agriculture. Including food crops, cash crops (oil crops, vegetable crops, flowers, grasses, trees), industrial raw materials crops, forage crops, medicinal crops, etc., and plants that can be grown in large or large areas for profit or rations (eg Cereals, vegetables, cotton, linen, etc.).
  • rice, corn, beans, potatoes, barley, broad beans, and wheat are the main crops; oil crops are mainly oilseed, vine, mustard, peanut, flax, hemp, sunflower, etc.; There are radishes, cabbage, celery, leeks, garlic, onions, carrots, melons, lotus dishes, Jerusalem artichokes, kidney beans, medlar, lettuce, yellow flowers, peppers, cucumbers, tomatoes, parsley, etc.; fruits are pears, green plums, apples, peaches , apricot, walnut, plum, cherry, strawberry, sand fruit, red dates and other varieties; wild fruits are sour pear, wild apricot, peach, jujube, mountain cherry, sea buckthorn, etc.; feed crops such as corn, green manure, Chinese milk vetch, etc. Medicinal crops such as ginseng, angelica, honeysuckle, mint, mugwort, etc.
  • RNA interference RNA interference
  • RNA interference means that some small double-stranded RNA can efficiently and specifically block the expression of specific genes in the body, promote mRNA degradation, and induce cells to exhibit specific gene deletions. Phenotype, which is also known as RNA intervention or RNA interference. RNA interference is a highly specific mechanism of gene silencing at the mRNA level.
  • small interfering RNA refers to a short-segment double-stranded RNA molecule that is capable of degrading specific mRNAs with mRNAs of homologous complementary sequences. This process is the RNA interference pathway (RNA). Interference pathway).
  • RNA interference In the present invention, the basic principle of the RNA interference is: using plants as a medium to make insects can interfere with the expression of genes (such as DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene). Interfering with RNA (siRNA), thereby inhibiting the growth of insects.
  • genes such as DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene.
  • siRNA Interfering with RNA (siRNA), thereby inhibiting the growth of insects.
  • the principle is as follows: the herbivorous feeding or the interfering substance is sprayed on the aphid, so that the RNAi enters the worm, interferes with the target gene RNA, inhibits the expression of the target gene, thereby disturbing the normal growth and development of the insect. Causes the death of aphids.
  • an intron sequence is ligated to the complementary gene sequence at both ends, and after introduction into the cell, a "neck-loop" structure can be produced, and the "neck” portion can be processed into an insect body.
  • Small RNA of about 21-25 nt, this small RNA can effectively inhibit the expression of the target gene.
  • double-stranded RNA formed by transcription complementation in Table 1 with T7 primers, respectively, can be directly used to inhibit expression of a gene of interest.
  • insect gene refers to a gene associated with insect nymph and/or adult stage regulation.
  • the insect gene is DS7 gene, DS9 gene, DS15 gene, DS25.
  • Gene, DS27 gene, and/or DS45 gene the low or no expression of the gene will lead to abnormalities in the growth, development, metabolism, reproduction and other processes of insects, and even lead to the death of insects.
  • a preferred fragment of the insect gene of the present invention has a length of at least 21 bp, such as 30 bp, 50 bp, 60 bp, 80 bp, 100 bp, 200 bp, 500 bp, 1000 bp or the entire length of the gene.
  • the gene may be a full-length gene or a gene fragment when used in the present invention, preferably, a fragment directed against the DS7 gene is set forth in SEQ ID NO: 24; a fragment directed against the DS9 gene is set forth in SEQ ID NO: 25; A fragment directed against the DS15 gene is set forth in SEQ ID NO.: 26; a fragment directed against the DS25 gene is set forth in SEQ ID NO.: 27; a fragment directed against the DS27 gene is set forth in SEQ ID NO.: 28; a fragment directed against the DS45 gene As shown in SEQ ID NO.: 29, these fragments have similar degrees of similarity to these genes of 85% to 100%, respectively, all of which produce the same insecticidal effect.
  • the present invention also provides a dsRNA directed against the DS50 gene, the sequence of which is set forth in SEQ ID NO:23.
  • the DS50 gene has a poor control effect, and the highest is only about 23%.
  • the present invention provides interfering RNA for insect nymph and/or adult stage regulation-related genes, which insects can ingest by interfering with RNAi plants or expressing dsRNA constructs or dsRNA, or by directly interfering RNAi to interfering RNAi Insect surface.
  • the dsRNA construct of the present invention is represented by Formula I, and the dsRNA is represented by Formula II, and the length of the spacer sequence X employed is not particularly limited as long as it forms a construct with the forward sequence and the reverse sequence and is After introduction into the body, the dsRNA represented by Formula II can be formed.
  • the spacer sequence of the present invention has a length of 80 to 300 bp; more preferably 100 to 250 bp.
  • the construct for expressing the insect gene dsRNA is introduced into a host cell, which may be a plant cell, tissue or organ, and the construct can express an insect gene in the plant.
  • dsRNA, dsRNA is processed into siRNA.
  • the length of the siRNA is about 21-25 nt.
  • the construct is located on an expression vector.
  • the expression vector usually further contains a promoter, 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 kalatinmycin, 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 delivering the expression vector to a plant cell.
  • the host is Agrobacterium.
  • insects exemplified in the examples of the present invention are aphids.
  • the present invention is not particularly limited to insects suitable for use in the present invention, and the insect may be any herbivorous insect that feeds on plants, for example, it may be a Hemiptera insect.
  • the present invention is not particularly limited to plants suitable for use in the present invention, and is preferably a plant to which aphids are eaten, such as soybeans, radishes, peach trees, tobacco, and the like.
  • DS7 gene tubulin alpha chain-like
  • tubulin alpha chain are used interchangeably and are a broadly distributed class of globular proteins that are the basic structural units of intracellular microtubules. It plays an important role in cell movement and division and is expressed in the nymphal stage.
  • some of the glutamic acid residues at the C-terminus of the protein are polyglutamylated, resulting in a polyglutamic acid chain on the ⁇ -carboxyl group.
  • Polyglutamylation plays a key role in microtubule cleavage of spastin (SPAST).
  • SPAST preferentially recognizes and acts on microtubules modified with a short polyglutamic acid tail: the cleavage activity of SPAST increases as the number of glutamate per tubulin increases from 1 to 8, but decreases beyond the glutamylation Threshold.
  • Some of the glutamic acid residues at the C-terminus are monoglycosylated, but not polyglycerolated.
  • Monoglycosylation is primarily limited to tubulin (cilia and flagella) incorporated into the axis. Both polyvalerylation and monoglycosylation can coexist on the same protein on adjacent residues, and lowering the level of glycosylation can increase polyvalerylation and interact.
  • an interfering RNA fragment directed against the DS7 gene is screened based on the RNAi technology using the DS7 gene as a target, and preferably, the sequence of the DS7 gene fragment is as shown in SEQ ID NO: 1 or 24.
  • DS9 gene As used herein, the terms "DS9 gene”, “ADP/ATP translocase 3-like”, “ADP/ATP carrier protein (AAC)” are used interchangeably and are responsible for the transport of phosphorylated synthetic ATP into the cytoplasm as a cell.
  • AAC ADP/ATP carrier protein
  • the protein is a transporter that allows intracellular exchange of adenosine diphosphate (ADP) and mitochondrial adenosine triphosphate across the (ATP) mitochondrial inner membrane.
  • ADP adenosine diphosphate
  • ATP oxidative phosphorylation
  • an RNA fragment directed against the DS9 gene is screened based on the RNAi technology with the DS9 gene as a target, preferably the sequence of the DS9 gene fragment is represented by SEQ ID NO: 2 or 25:
  • DS15 gene As used herein, the terms "DS15 gene”, “heat shock protein 83-like”, and “heat shock protein 83” are used interchangeably and are intracellular molecular chaperone proteins that have an important role in protein interactions, such as assistance. Fold and assist in building the right protein concept. Expressed in the nymphal stage.
  • heat shock protein is a family of proteins produced by cells in response to exposure to stress conditions. They are first related to heat shock, but are now known to be in other stresses, including exposure to cold, and healing in wounds or tissue remodeling. Many members of this group perform chaperone function by stabilizing new proteins to ensure proper folding or by helping to fold proteins that are damaged by cellular stress. Increase is the regulation of transcription. Significant upregulation of heat shock proteins is a key component of the heat shock response and is primarily induced by heat shock factor (HSF).
  • HSF heat shock factor
  • an RNA fragment directed against the DS15 gene is screened based on the RNAi technology using the DS15 gene as a target, preferably the sequence of the DS15 gene fragment is represented by SEQ ID NO: 3 or 26.
  • DS25 gene eukaryotic initiation factor 4A-like
  • eukaryotic initiation factor complex 4A eukaryotic initiation factor complex 4A
  • the eukaryotic initiation factor complex forms a ternary complex with GTP and the initiator Met-tRNA, which is regulated by guanine nucleotide exchange and phosphorylation, and is a major regulatory element of the gene expression bottleneck.
  • Many initiation factors must promote synergy between ribosomes and mRNA before translation proceeds to the extension phase and ensure that the 5'UTR of the mRNA is sufficiently deficient in secondary structure.
  • Group 4 eukaryotic initiation factors promote this binding; they are of significance in the normal regulation of translation and in the transformation and progression of cancer cells.
  • an RNA fragment directed against the DS25 gene is screened based on the RNAi technology using the DS25 gene as a target, preferably the sequence of the DS25 gene fragment is represented by SEQ ID NO: 4 or 27.
  • DS27 DS27
  • troponin T-like isoform 3 a type 3 of a cell
  • troponin is attached to the proprotein myosin and is located in a groove between the actin filaments in the muscle tissue.
  • tropomyosin blocks the attachment site of the myosin cross bridge, thereby preventing contraction.
  • the calcium channels open in the sarcoplasmic membrane and release calcium into the sarcoplasm. Some of these calcium attach to troponin, causing it to change shape, exposing the binding site of myosin (active site) on actin filaments. The binding of myosin to actin causes cross-bridge formation and begins to contract muscles.
  • Troponin activation Troponin C (red) binds to Ca2 + , a stable activation state in which troponin I (yellow) no longer binds to actin.
  • Troponin T (blue) immobilizes the complex on tropomyosin.
  • Troponin is present in skeletal muscle and myocardium, but specific versions of troponin differ in different types of muscle. The main difference is that the TnC subunit of troponin has four calcium ion binding sites in skeletal muscle, while there are only three in the myocardium. The viewpoint regarding the actual content of calcium bound to troponin differs depending on the expert and source.
  • an RNA fragment directed against the DS27 gene is screened based on the RNAi technology using the DS27 gene as a target, preferably the sequence of the DS27 gene fragment is represented by SEQ ID NO: 5 or 28.
  • DS45 gene As used herein, the terms "DS45 gene”, “Y-box protein Ct-p40-like”, and “Y-box binding protein Ct-p40-like” are used interchangeably to affect cell differentiation, cytoskeletal formation, and deletion inhibition. Intracellular and extracellular signaling pathways, involved in DNA damage repair and transcription, are expressed in nymphal stages.
  • an RNA fragment directed against the DS45 gene is screened based on the RNAi technology using the DS45 gene as a target, preferably the sequence of the DS45 gene fragment is represented by SEQ ID NO: 6 or 29.
  • the invention provides a dsRNA construct, the construct of the dsRNA is double stranded, and the positive or negative strand thereof comprises the structure of formula I:
  • Seq is positive for the nucleotide sequence of a related gene or fragment of insect nymph and/or adult stage regulation
  • Seq reverses to a nucleotide sequence that is substantially complementary to the Seq forward ;
  • X is a spacer sequence located between the forward and Seq Seq the reverse, and the spacer sequence Seq Seq forward and reverse are not complementary,
  • insect nymph and/or adult stage regulation-related genes are selected from the group consisting of a DS7 gene, a DS9 gene, a DS15 gene, a DS25 gene, a DS27 gene, a DS45 gene, or a combination thereof.
  • the Seq forward and Seq inverse lengths are at least 50 bp.
  • the dsRNA construct is ingested by an insect (such as aphid) to form a dsRNA of formula II,
  • Seq 'Forward Forward sequence corresponds to Seq RNA sequences or fragments of sequences
  • Seq' reverse is a sequence that is substantially complementary to the Seq' forward ;
  • the invention also provides the use of the dsRNA construct for: (1) improving the control effect of aphids; and/or (2) increasing the rate of decrease in the population; and/or (3) reducing nymphs and/or adults To regulate the expression level of related genes; (4) reduce the number of insect populations; and / or (5) reduce the rate of plant damage; and / or (6) reduce the degree of crop damage and improve the quality of crop products.
  • the invention also provides a dsRNA as shown in formula II,
  • Seq 'Forward Forward sequence corresponds to Seq RNA sequences or fragments of sequences
  • Seq' reverse is a sequence that is substantially complementary to the Seq' forward ;
  • the spacer sequence X has a length of 0 to 300 bp, preferably 100 bp.
  • the insect nymph and/or adult stage regulation-related gene is derived from aphids; the sequence of the DS7 gene is shown in SEQ ID NO.: 1; the sequence of the DS9 gene is shown in SEQ ID NO.: 2; The sequence of the DS15 gene is shown in SEQ ID NO.: 3; the sequence of the DS25 gene is shown in SEQ ID NO.: 4; the sequence of the DS27 gene is shown in SEQ ID NO.: 5; The sequence of the gene is shown in SEQ ID NO.: 6.
  • the insect is a herbivorous insect, preferably from a Hemiptera insect, most preferably from the genus Aphid.
  • the invention also provides the use of the dsRNA, which is used to: (1) increase the control effect of aphids; and/or (2) increase the rate of decrease in the population; and/or (3) reduce the regulation of nymphs and/or adult stages.
  • the amount of expression of the relevant gene and/or (4) reducing the number of insect populations; and/or (5) reducing the rate of plant damage.
  • the present invention also provides a composition, and the present inventors have developed an RNAi fragment targeting a target gene based on RNAi technology for the problem of efficiently killing aphids, and improving the control effect of aphids by feeding or directly spraying insects by insects. And the rate of decline of the mouth, so that RNAi can inhibit the expression of genes, and ultimately achieve the purpose of effectively killing aphids.
  • the method of the invention is efficient, convenient, fast, accurate and pollution-free.
  • the composition includes a dsRNA construct and/or dsRNA, and an insectically acceptable effective amount of the carrier.
  • the composition is a composition for inducing or causing death of a locust nymph and/or adult stage.
  • the dsRNA has the following sequence:
  • dsRNA1 having the sequence corresponding to SEQ ID NO.: 1 or 24;
  • dsRNA2 has a sequence corresponding to SEQ ID NO.: 2 or 25.
  • dsRNA3 having the sequence corresponding to SEQ ID NO.: 3 or 26;
  • dsRNA4 has a sequence corresponding to SEQ ID NO.: 4 or 27.
  • dsRNA5 having the sequence corresponding to SEQ ID NO.: 5 or 28;
  • dsRNA6 has a sequence corresponding to SEQ ID NO.: 6 or 29.
  • composition selected from the group consisting of:
  • the composition is an aqueous solution, typically having a pH of from about 5 to about 8, preferably a pH of from about 6 to about 8.
  • the term "effective amount” or “effective amount” refers to an amount that can produce a function or activity to the insect feeding and that is acceptable to the insect.
  • the amount of dsRNA1 is from about 1 to 500 ng/ ⁇ l, preferably from 5 to 300 ng/ ⁇ l, more preferably from 50 to 150 ng/ ⁇ l; and the amount of dsRNA2 is from about 1 to 500 ng/ ⁇ l, preferably, 5 -300 ng/ ⁇ l, more preferably 50-150 ng/ ⁇ l;
  • the content of dsRNA3 is about 1-500 ng/ ⁇ l, preferably 5-300 ng/ ⁇ l, more preferably 50-150 ng/ ⁇ l;
  • the content of dsRNA4 is About 1-500 ng/ ⁇ l, preferably 5-300 ng/ ⁇ l, more preferably 50-150 ng/ ⁇ l;
  • the content of dsRNA5 is about 1-500 ng/ ⁇ l, preferably 5-300 ng/ ⁇ l, more preferably Ground, 50-150 ng/
  • an "insect-feeding acceptable" ingredient is one that is suitable for the insect without excessive adverse side effects (such as toxicity, irritation, and allergies), i.e., a material having a reasonable benefit/risk ratio.
  • carrier includes various excipients and diluents. Such carriers include, but are not limited to, water, saline, buffer, dextrose, glycerol, ethanol, and combinations thereof.
  • composition of the present invention can be directly sprayed, fed, or prepared in the form of an injection, for example, by a conventional method using water, physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the composition is made under sterile or RNase free conditions.
  • the dsRNA designed according to the specific target gene of the invention can effectively kill aphids, improve the control effect of aphids ( ⁇ 80%) and the rate of decrease of insects ( ⁇ 70%);
  • the obtained dsRNA can be directly applied to aphid killing, and is convenient to use;
  • Myzus persicae is cultivated and tested on radish seedlings grown in indoor greenhouses or plastic greenhouses.
  • Aphid glycine is cultivated and tested on soybean seedlings grown in indoor greenhouses or plastic greenhouses.
  • the culture chamber temperature is 25 ⁇ 1°C
  • the relative humidity is 40-60%
  • the photoperiod is 12h: 12h.
  • a certain number of aphids were inoculated to the target plants for counting, and a certain concentration of dsRNA was sprayed and counted again at 1, 3, and 5 days, and each gene was tested 10 times. According to the counting result, the control effect of the target gene is determined.
  • the first method the control effect, is calculated as follows:
  • Control effect (%) (1-CK0 ⁇ PT1/CK1 ⁇ PT0) ⁇ 100
  • PT0 number of insects before treatment in the treatment area
  • PT1 number of insects after treatment in the treatment area
  • CK0 number of insects before application in the control area
  • CK1 number of insects after application in the control area.
  • the second method the population loss rate, is calculated as follows:
  • Infestation rate (%) [(number of insects before application - number of insects after application) / number of insects before application] ⁇ 100
  • RNAi Kit (Ambion) performs the synthesis of dsRNA and performs experimental procedures according to the instructions.
  • the T7 promoter sequence was added to the 5' end of the primer of the amplification template to facilitate the synthesis of the subsequent dsRNA.
  • pPigbac A3 EGFP was used as a template for the synthesis control dsEGFP, and dsEGFP was used as a negative control in the subsequent experiments.
  • Primers for the synthesis of dsRNAs are given in Annex S3. During the synthesis, template DNA and single-stranded RNA were removed using DNase and RNase, respectively.
  • the reagent (Invitrogen) is used to extract total RNA, and the steps are strictly in accordance with the operation manual. Take 1 ⁇ g of total RNA and use the kit ReverTra qPCR RT Master Mix with gDNA Remover (TOYOBO) for the synthesis of the first strand of cDNA.
  • the kit used in the RT-qPCR reaction is Premix Ex Taq TM II (Takara), primers are detailed in Annex S3. Three replicates of each gene sample were tested, and the expression level of the 18S rRNA was used for homogenization. Data analysis was performed with reference to 2 - ⁇ CT Method (Livak & Schmittgen, 2001). The corresponding values are obtained by calculating the mean and standard error. To eliminate individual differences, the samples from each experimental group were pools of surviving larvae formed after 2 treatments, and each experimental group was subjected to three biological replicates.
  • RNA interference technology In order to screen effective aphid target genes based on RNA interference technology, Myzus persicae and Aphid glycine were sequenced by transcriptome (the sampling and sequencing methods of the two aphids were the same). After extracting total RNA from aphids at different developmental stages, the same amount of RNA was mixed to form the total RNA of the aphid during development, and sent to Shenzhen Huada Gene Technology Service Co., Ltd. for transcriptome sequencing using the Illumina Hiseq2000 platform. After removing the linker from the sequencing result, assemble it with the denove program and then comment on the function of Unigene.
  • target gene fragments were selected from these functionally annotated Unigenes for amplification and synthesis of dsRNA.
  • the primers for amplifying and synthesizing the six target genes, the exogenous control gene GFP, and the endogenous comparative gene DS50 derived from soybean meal by the present invention are shown in Table 1.
  • the DNA sequences of the six gene fragments are shown in Table 2, wherein ds7, ds9, and ds15 are directed against aphids, especially against peach aphids, ds25, ds27, and ds45 against aphids, especially soybean meal.
  • Aphids are parthenogenetic insects that are born to be 1 year old newborn aphids. From the age of 1 year old newborn to the period of 5 to 7 days of calving (affected by ambient temperature), the aphids on a plant have obvious generational alternations, that is, different generations and various sizes (different ages). The worms coexist at the same time. Therefore, there will be mites of various ages (such as 2th-4th, and possibly adult worms) when they are tested on the plants. Thus, after various tests, the mites will soon begin to multiply and produce In the first generation, the number of mites on the plants being tested was still increasing after counting before spraying. This judgment of the control effect of aphid insecticides is a big disturbance. Therefore, there is a more strict or relatively accurate calculation method for the control effect of aphids, that is, the rate of reduction of insects (see the general method and materials section for the calculation formula). .
  • the statistical results of the present invention showed that after the dsRNA of the three peach aphid target genes were sprayed, the population loss rate of the peach aphid population at 1 day, 3 days and 5 days after treatment was as shown in Table 4, and the insect population decreased on the 5th day after spraying. The rate has reached more than 70%.
  • the dsRNA concentration for spraying was 300 ng/ ⁇ l.
  • the synthesized dsRNA was dissolved in water and each plant was sprayed with 300 ⁇ l.
  • Imidacloprid concentration was 10,000 times (Germany Bayer Emmet 70% imidacloprid 3 g, water-dispersible granules), 300 ⁇ l per plant.
  • Test method spray treatment, 4 replicates per treatment.
  • composition for efficiently killing aphids the composition being an aqueous solution comprising components:
  • the dsRNA concentration of the DS7 gene fragment shown in SEQ ID NO.: 1 or 24 is 100 ng / ⁇ l;
  • the dsRNA concentration for the sequence of the DS9 gene fragment as shown in SEQ ID NO.: 2 or 25 is 100 ng/ ⁇ l.
  • the dsRNA concentration for the DS15 gene fragment shown as SEQ ID NO.: 3 or 26 is 100 ng/ ⁇ l.
  • the dsRNA concentration for the DS25 gene fragment shown as SEQ ID NO.: 4 or 27 is 100 ng/ ⁇ l.
  • the dsRNA concentration for the DS27 gene fragment shown as SEQ ID NO.: 5 or 28 is 100 ng/ ⁇ l.
  • the dsRNA concentration for the DS45 gene fragment shown as SEQ ID NO.: 6 or 29 is 100 ng/ ⁇ l.
  • the method is the same as in Examples 1 and 2, except that the target gene is DS50, and the primers used are:
  • the amplified product was 578 bp in length.
  • the DS50 gene is a fatty acid synthase-like sequence as shown in SEQ ID NO.: 23, encoding the FASN gene.
  • Fatty acid synthase is a multi-enzyme protein that catalyzes the synthesis of fatty acids. It is not a single enzyme, but an entire enzyme system consisting of two identical 272 kDa multifunctional polypeptides, in which the substrate is delivered from one functional domain to the next, the main function of which is catalyzed by acetyl-CoA and C in the presence of NADPH. Diacyl-CoA synthesizes palmitate.
  • sequence of the DS50 gene fragment is represented by SEQ ID NO: 23:

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Abstract

提供了对蚜虫致死的RNAi靶标基因及其应用。具体地，提供了6个基于RNA干扰技术导致蚜虫若虫和/或成虫期死亡的基因片段，通过将含有dsRNA的组合物喷洒到植物上饲喂蚜虫或直接喷洒到蚜虫表面，可以导致蚜虫死亡。

Description

对蚜虫高效致死的RNAi靶标基因及其应用 技术领域
本发明属于生物技术和农业应用领域，具体地，本发明涉及对蚜虫高效致死的RNAi靶标基因及其应用。
背景技术
蚜虫是一种重要的世界性害虫，属半翅目Hemiptera，蚜总科Aphidoidea，目前已知约4700多种，体型较小，繁殖速度快，是重要的农业和园艺害虫，对于蚜虫的防治，目前依然以化学药剂为主，但是由于其繁殖速度快，隐蔽性强，其防治效果较差，需要大量使用农药抑制其繁殖，不可避免地造成了蚜虫的抗药性。
RNAi作为一种基因功能研究的工具被大量应用，尤其是在遗传操作工具不完善的动植物中被广泛应用。然而，目前在昆虫中，dsRNA通过取食进入昆虫体内后，必须进入细胞内才能激活RNAi机制。昆虫肠壁细胞可以阻止大部分dsRNA进入其他组织，是影响RNAi效率的一个关键因素，也是目前dsRNA经口传递方法在应用中存在的最大障碍。
由于不同种类昆虫对dsRNA摄取机制不同，导致其对dsRNA的响应和靶基因沉默效率存在差异，因此，不同种类昆虫的致死作用差异较大。
因此，本领域迫切需要开发一种对蚜虫高效致死的RNAi靶标基因。
发明内容
本发明的目的在于提供一种对蚜虫高效致死的RNAi靶标基因。
在本发明的第一方面，提供了一种dsRNA构建物，所述dsRNA的构建物为双链，并且其正链或负链含有式I所示的结构：
Seq 正向-X-Seq 反向    式I
式中，
Seq 正向为昆虫若虫和/或成虫期调控相关基因或片段的核苷酸序列；
Seq 反向为与Seq 正向基本上互补的核苷酸序列；
X为位于Seq 正向和Seq 反向之间的间隔序列，并且所述间隔序列与Seq 正向和Seq 反向不互补，
其中，所述的昆虫若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
在另一优选例中，所述dsRNA的长度为至少21nt.
在另一优选例中，对DS7基因来说，所述dsRNA的长度为21nt－1350nt，较佳地，506nt－1093nt。
在另一优选例中，对DS9基因来说，所述dsRNA的长度为21nt－909nt，较佳地，54nt－631nt。
在另一优选例中，对DS15基因来说，所述dsRNA的长度为21nt－2148nt，较佳地，516nt－1029nt。
在另一优选例中，对DS25基因来说，所述dsRNA的长度为21nt－1233nt，较佳地，58nt－674nt。
在另一优选例中，对DS27基因来说，所述dsRNA的长度为21nt－1152nt，较佳地，219nt－748nt。
在另一优选例中，对DS45基因来说，所述dsRNA的长度为21nt－909nt，较佳地，42nt－637nt。
在另一优选例中，与所述dsRNA的同源性至少为80％，较佳地，为85％-100％。
在另一优选例中，Seq 正向、Seq 反向的长度为至少50bp。
在另一优选例中，所述dsRNA构建物可形成式II所示的dsRNA，
Figure PCTCN2019086896-appb-000001
式中，
Seq’ 正向为Seq 正向序列对应的RNA序列或序列片段；
Seq’ 反向为与Seq’ 正向基本上互补的序列；
X’为无；或为位于Seq’ 正向和Seq’ 反向之间的间隔序列，并且所述间隔序列与Seq’ 正向和Seq’ 反向不互补，
||表示在Seq 正向和Seq 反向之间形成的氢键。
在另一优选例中，所述dsRNA为不带loop的dsRNA。
在另一优选例中，所述dsRNA由SEQ ID NO.:9-10所示的序列扩增而得。
在另一优选例中，所述dsRNA由SEQ ID NO.:11-12所示的序列扩增而得。
在另一优选例中，所述dsRNA由SEQ ID NO.:13-14所示的序列扩增而得。
在另一优选例中，所述dsRNA由SEQ ID NO.:15-16所示的序列扩增而得。
在另一优选例中，所述dsRNA由SEQ ID NO.:17-18所示的序列扩增而得。
在另一优选例中，所述dsRNA由SEQ ID NO.:19-20所示的序列扩增而得。
本发明第二方面提供了一种式II所示的dsRNA，
Figure PCTCN2019086896-appb-000002
式中，
Seq’ 正向为昆虫若虫和/或成虫期调控相关基因或片段的核苷酸序列对应的RNA序列或序列片段；
Seq’ 反向为与Seq’ 正向基本上互补的序列；
X’为无；或为位于Seq’ 正向和Seq’ 反向之间的间隔序列，并且所述间隔序列与Seq’ 正向和Seq’ 反向不互补；
其中，所述的昆虫若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合；
||表示在Seq 正向和Seq 反向之间形成的氢键。
在另一优选例中，Seq 正向、Seq 反向的长度为至少50bp。
在另一优选例中，所述的间隔序列X’的长度为0-300bp。
在另一优选例中，所述的若虫和/或成虫期调控相关基因来源于蚜虫属。
在另一优选例中，所述DS7基因的序列如SEQ ID NO.:1或24所示。
在另一优选例中，所述DS9基因的序列如SEQ ID NO.:2或25所示。
在另一优选例中，所述DS15基因的序列如SEQ ID NO.:3或26所示。
在另一优选例中，所述DS25基因的序列如SEQ ID NO.:4或27所示。
在另一优选例中，所述DS27基因的序列如SEQ ID NO.:5或28所示。
在另一优选例中，所述DS45基因的序列如SEQ ID NO.:6或29所示。
在另一优选例中，所述昆虫为植食性昆虫，较佳地为同翅目昆虫，最佳地为蚜虫属。
在另一优选例中，所述昆虫选自下组：桃蚜、大豆蚜、或其组合。
本发明第三方面提供了一种表达载体，所述表达载体含有本发明第一方面中 所述的dsRNA构建物。
本发明第四方面提供了一种宿主细胞，所述宿主细胞中含有本发明第三方面中所述的表达载体或者染色体中整合有对应于本发明第一方面中所述的dsRNA构建物的DNA序列。
在另一优选例中，所述的宿主细胞为植物细胞，较佳地为绿叶植物细胞。
在另一优选例中，所述植物包括十字花科植物(如蔬菜或大豆)。
本发明第五方面提供了一种组合物，所述组合物包括本发明第一方面中所述的dsRNA构建物和/或本发明第二方面中所述的dsRNA，以及昆虫喂食上可接受的载体。
在另一优选例中，所述的昆虫喂食上可接受的载体包括水。
在另一优选例中，所述的组合物是用于诱导或导致蚜虫属若虫和/或成虫期死亡的组合物。
在另一优选例中，所述的dsRNA具有以下序列：
dsRNA1：具有对应于SEQ ID NO.:1或24所示的序列；
dsRNA2：具有对应于SEQ ID NO.:2或25所示的序列；
dsRNA3：具有对应于SEQ ID NO.:3或26所示的序列；
dsRNA4：具有对应于SEQ ID NO.:4或27所示的序列；
dsRNA5：具有对应于SEQ ID NO.:5或28所示的序列；
dsRNA6：具有对应于SEQ ID NO.:6或29所示的序列。
在另一优选例中，DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、和/或DS45基因来自昆虫，较佳地来自同翅目昆虫，最佳地来自蚜虫属。
在另一优选例中，所述的药物组合物中，dsRNA1含量为1-500ng/μl，较佳地为5-300ng/μl，更佳地，50-150ng/μl。
在另一优选例中，所述的药物组合物中，dsRNA2含量为1-500ng/μl，较佳地为5-300ng/μl，更佳地，50-150ng/μl。
在另一优选例中，所述的药物组合物中，dsRNA3含量为1-500ng/μl，较佳地为5-300ng/μl，更佳地，50-150ng/μl。
在另一优选例中，所述的药物组合物中，dsRNA4含量为1-500ng/μl，较佳地为5-300ng/μl，更佳地，50-150ng/μl。
在另一优选例中，所述的药物组合物中，dsRNA5含量为1-500ng/μl，较佳地为5-300ng/μl，更佳地，50-150ng/μl。
在另一优选例中，所述的药物组合物中，dsRNA6含量为1-500ng/μl，较佳地为5-300ng/μl，更佳地，50-150ng/μl。
本发明第六方面提供了本发明第一方面所述的dsRNA构建物，或本发明第二方面所述的dsRNA，或本发明第四方面所述的宿主细胞、或本发明第五方面所述组合物的用途，所述用途选自下组：
(1)提高蚜虫的防治效果；和/或
(2)提高虫口减退率；和/或
(3)降低若虫和/或成虫期调控相关基因的表达量；和/或
(4)降低虫口基数；和/或
(5)减少植物受害率；和/或
(6)降低作物受害程度，提高农作物产品质量。
本发明第七方面提供了一种杀灭昆虫的方法，包括步骤：用干扰昆虫若虫和/或成虫期调控相关基因表达的干扰分子，或含所述干扰分子的载体、细胞、植物组织或昆虫防治试剂饲喂或喷洒昆虫；
较佳地，所述的昆若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
在另一优选例中，所述杀灭昆虫包括：
(1)提高蚜虫的防治效果；和/或
(2)提高虫口减退率；和/或
(3)降低若虫和/或成虫期调控相关基因的表达量；和/或
(4)降低虫口基数；和/或
(5)减少植物受害率；和/或
(6)降低作物受害程度，提高农作物产品质量。
在另一优选例中，所述的干扰分子选自：以昆虫若虫和/或成虫期调控相关基因或其片段或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA。
在另一优选例中，所述的昆虫若虫和/或成虫期调控相关基因来源于蚜虫属。
在另一优选例中，所述昆虫为植食性昆虫，较佳地来自半翅目昆虫，最佳地来自蚜虫属。
在另一优选例中，所述方法包括步骤：用本发明第一方面所述的dsRNA构建物，或本发明第二方面所述的dsRNA、或本发明第四方面所述的宿主细胞、或本发明第五方面所述组合物饲喂或喷洒昆虫。
本发明第八方面提供了一种制备本发明第二方面所述dsRNA的方法，包括步骤：
(i)制备表达dsRNA的构建物，所述构建物为双链，并且其正链或负链含有式I所示的结构：
Seq 正向-X-Seq 反向  式I
式中，
Seq 正向为昆虫若虫和/或成虫期调控相关基因或片段的核苷酸序列；
Seq 反向为与Seq 正向基本上互补的核苷酸序列；
X为位于Seq 正向和Seq 反向之间的间隔序列，并且所述间隔序列与Seq 正向和Seq 反向不互补，
其中，所述的昆虫若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合；
(ii)将步骤(i)所述的构建物转入宿主细胞，从而在宿主细胞中表达形成式II所示的dsRNA，
Figure PCTCN2019086896-appb-000003
式中，
Seq’ 正向为Seq 正向序列对应的RNA序列或序列片段；
Seq’ 反向为与Seq’ 正向基本上互补的序列；
X’为无；或为位于Seq’ 正向和Seq’ 反向之间的间隔序列，并且所述间隔序列与Seq’ 正向和Seq’ 反向不互补，
||表示在Seq 正向和Seq 反向之间形成的氢键。
本发明第九方面提供了一种制备昆虫防治试剂的方法，包括步骤：将本发明第一方面所述的dsRNA构建物，或本发明第二方面所述的dsRNA，或本发明 第四方面所述的宿主细胞，或本发明第五方面所述的组合物喷洒于植物表面，从而制得昆虫防治试剂。
在另一优选例中，所述植物选自下组：大豆，萝卜，桃树、烟草、或其组合。
本发明第十方面提供了一种提高植物对昆虫抗性的方法，包括：
在植物中表达重组DNA构建体，其中所述重组DNA构建体包含编码RNA的DNA，所述RNA具有与所述靶基因的至少21个或更多个连续核苷酸基本上相同或基本互补的序列，其中所述靶基因为昆虫若虫和/或成虫期调控相关基因，选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
在另一优选例中，所述靶基因选自下组：
(i)序列如SEQ ID NO.:1－6、24-29中的任一所示的多核苷酸；
(ii)核苷酸序列与SEQ ID NO.:1－6、24-29中的任一所示序列的同源性≥80％，较佳地，85％-90％，更佳地，为95％、96％、97％、98％、99％或100％)的多核苷酸；
(iii)在SEQ ID NO.:1－6、24-29中的任一所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(iv)与(i)-(iii)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述靶基因如SEQ ID NO.:1-6、24-29中的任一所示。
在另一优选例中，与所述RNA的同源性至少为80％，较佳地，为85％-100％，更佳地，95-100％。
在另一优选例中，对DS7基因来说，所述RNA具有与所述靶基因的21nt－1350nt，较佳地，506nt－1093nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS9基因来说，所述RNA具有与所述靶基因的21nt－909nt，较佳地，54nt－631nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS15基因来说，所述RNA具有与所述靶基因的21nt－2148nt，较佳地，516nt－1029nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS25基因来说，所述RNA具有与所述靶基因的21nt－1233nt，较佳地，58nt－674nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS27基因来说，所述RNA具有与所述靶基因的21nt－ 1152nt，较佳地，219nt－748nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS45基因来说，所述RNA具有与所述靶基因的21nt－909nt，较佳地，42nt－637nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，所述RNA是包含至少一条RNA链的dsRNA。
在另一优选例中，所述RNA链包括与选自SEQ ID NO.:1-6、24-29中的任一所示的序列具有至少90％，较佳地，95-100％同源性的序列。
在另一优选例中，所述重组DNA构建体包含启动子，较佳地，异源启动子。
在另一优选例中，所述启动子选自下组：组成型启动子、空间特异性启动子、时间特异性启动子、发育特异性启动子、诱导型启动子、或其组合。
在另一优选例中，所述启动子是在植物中有功能的启动子。
在另一优选例中，所述启动子选自下组：pol II启动子、pol III启动子、pol IV启动子、pol V启动子、或其组合。
在另一优选例中，所述重组DNA构建体还包括选自下组的一种或多种其他元件：增强子、小RNA识别位点、适体或核酶、终止子、用于表达编码序列的额外和另外的表达盒(例如，表达转基因，例如杀虫蛋白或可选择的标记物)、非编码序列(例如，表达另外的抑制元件)、或其组合。
在另一优选例中，所述植物中还表达选自下组的一种或多种杀虫剂蛋白：patatin、植物凝集素、植物甾体、苏云金芽孢杆菌杀虫蛋白、Xenorhabdus杀虫蛋白、Photorhabdus杀虫蛋白、芽孢杆菌晚疫杀虫蛋白、球形芽孢杆菌杀虫蛋白。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：茄科、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述植物选自下组：十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、水稻、白菜、大豆、番茄、玉米、烟草、小麦、高粱、萝卜、或其组合。
本发明第十一方面提供了一种制备转基因植物细胞的方法，包括步骤：
(i)将重组DNA构建体导入或转染植物细胞，使得所述植物细胞含有所述构建物从而制得所述转基因植物细胞，其中所述重组DNA构建体包含编码RNA的DNA，所述RNA具有与所述靶基因的至少21个或更多个连续核苷酸基本上相同或基本互补的序列，其中所述靶基因为昆虫若虫和/或成虫期调控相关基因，选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
在另一优选例中，所述靶基因选自下组：
(i)序列如SEQ ID NO.:1－6、24-29中的任一所示的多核苷酸；
(ii)核苷酸序列与SEQ ID NO.:1－6、24-29中的任一所示序列的同源性≥80％，较佳地，85％-90％，更佳地，为95％、96％、97％、98％、99％或100％)的多核苷酸；(请复核)
(iii)在SEQ ID NO.:1－6、24-29中的任一所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(iv)与(i)-(iii)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，与所述RNA的同源性至少为80％，较佳地，为85％-100％，更佳地，95-100％。
在另一优选例中，对DS7基因来说，所述RNA具有与所述靶基因的21nt－1350nt，较佳地，506nt－1093nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS9基因来说，所述RNA具有与所述靶基因的21nt－909nt，较佳地，54nt－631nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS15基因来说，所述RNA具有与所述靶基因的21nt－2148nt，较佳地，516nt－1029nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS25基因来说，所述RNA具有与所述靶基因的21nt－1233nt，较佳地，58nt－674nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS27基因来说，所述RNA具有与所述靶基因的21nt－ 1152nt，较佳地，219nt－748nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，对DS45基因来说，所述RNA具有与所述靶基因的21nt－909nt，较佳地，42nt－637nt个连续核苷酸基本上相同或基本互补的序列。
在另一优选例中，所述的转染采用农杆菌转化法或基因枪轰击法。
本发明第十二方面提供了一种制备转基因植物的方法，包括步骤：
将本发明第十一方面所述方法制备的所述转基因植物细胞再生为植物体，从而获得所述转基因植物。
本发明第十三方面提供了一种转基因植物细胞，所述的植物细胞是用本发明第十一方面所述的方法制备的。
本发明第十四方面提供了一种转基因植物，所述的植物是用本发明第十二方面所述的方法制备的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了靶标基因对蚜虫的防治效果。
图2显示了靶标基因的相对表达水平检测结果。
图3显示了桃蚜三个靶标基因在田间的防治效果。
图4显示了桃蚜田间防治效果和虫口减退率的统计分析结果。
具体实施方式
本发明人经过广泛而深入的研究，对蚜虫若虫和/或成虫期调控相关基因片段进行筛选，意外地发现，针对如SEQ ID NO.:1或24所示的DS7基因、SEQ ID NO.:2或25所示的DS9基因、SEQ ID NO.:3或26所示的DS15基因、SEQ ID NO.:4或27所示的DS25基因、SEQ ID NO.:5或28所示的DS27基因、SEQ ID NO.:6或29所示的DS45基因，合成干扰RNA(dsRNA)，所述dsRNA藉由植食性昆虫(如蚜虫属)取食或直接喷洒到植食性昆虫表面，从而干扰靶基因，抑制靶基因的表达，最终杀灭蚜虫。本发明还可构建能够提高对昆虫抗性的植物，并且本发 明的方法还可有效杀灭蚜虫，蚜虫的防治效果≥80％，虫口减退率≥70％。在此基础上，本发明人完成了本发明。
术语
如本文所用，所述“农作物”指农业上栽培的各种植物。包括粮食作物﹑经济作物(油料作物、蔬菜作物、花、草、树木)、工业原料作物、饲料作物，药材作物等，并且能大批长成或大面积收获，供盈利或口粮用的植物(例如谷物、蔬菜、棉花、亚麻等)。
其中，粮食作物以水稻、玉米、豆类、薯类、青稞、蚕豆、小麦为主要作物；油料作物以油籽、蔓青、大芥、花生、胡麻、大麻、向日葵等为主；蔬菜作物主要有萝卜、白菜、芹菜、韭菜、蒜、葱、胡萝卜、菜瓜、莲花菜、菊芋、刀豆、芫荽、莴笋、黄花、辣椒、黄瓜、西红柿、香菜等；果类有梨、青梅、苹果、桃、杏、核桃、李子、樱桃、草莓、沙果、红枣等品种；野生果类有酸梨、野杏、毛桃、山枣、山樱桃、沙棘等；饲料作物如玉米、绿肥、紫云英等；药用作物有人参、当归、金银花、薄荷、艾蒿等。
RNA干扰(RNAi)
如本文所用，术语“RNA干扰(RNA interference，RNAi)”是指：一些小的双链RNA可以高效、特异地阻断体内特定基因的表达，促使mRNA降解，诱使细胞表现出特定基因缺失的表型，其也称为RNA干预或者RNA干涉。RNA干扰是高度特异的在mRNA水平上的基因沉默机制。
如本文所用，术语“小干扰RNA(small interfering RNA，siRNA)”是指一种短片段双链RNA分子，能够以同源互补序列的mRNA为靶目标降解特定的mRNA，这个过程就是RNA干扰途径(RNA interference pathway)。
在本发明中，所述RNA干扰的基本原理是：以植物作为媒介，使昆虫取食可干扰其基因(如DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因)表达的小干扰RNA(siRNA)，从而抑制昆虫的生长。
具体地，所述的原理为：藉由蚜虫植食性取食或将干扰物质喷洒蚜虫，使得RNAi进入虫体，对靶基因的RNA进行干扰，抑制靶基因的表达，从而干扰 昆虫正常的生长发育，导致蚜虫死亡。
作为一种优选的方式，利用一个内含子序列，两端连接上互补的基因序列，导入细胞后，能产生“颈-环”结构，并且“颈”状部分能够在昆虫体内被加工成约21-25nt左右的小RNA，这种小RNA能特别有效的抑制目的基因的表达。
作为另一种优选的方式，利用表1中分别带有T7引物进行扩增，通过转录互补作用形成的双链RNA，这种双链RNA可直接用于抑制目的基因的表达。
昆虫基因
如本文所用，术语“昆虫基因”一词是指与昆虫若虫和/或成虫期调控相关基因，在本发明的一个优选例中，所述的昆虫基因为DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、和/或DS45基因，所述基因的低表达或不表达将导致昆虫的生长、发育、代谢、繁殖等过程产生异常，甚至导致昆虫的死亡。
作为本发明的优选方式，本发明优选的昆虫基因的片段的长度至少为21bp，比如可以是30bp、50bp、60bp、80bp、100bp、200bp、500bp、1000bp或基因的全长。所述基因在用于本发明时，可以是全长基因或基因片段，优选地，针对DS7基因的片段如SEQ ID NO：24所示；针对DS9基因的片段如SEQ ID NO：25所示；针对DS15基因的片段如SEQ ID NO.:26所示；针对DS25基因的片段如SEQ ID NO.:27所示；针对DS27基因的片段如SEQ ID NO.:28所示；针对DS45基因的片段如SEQ ID NO.:29所示，这些片段与这些基因的相似度分别为85％-100％，均为可产生相同的杀虫效果。
本发明还提供了针对DS50基因的dsRNA，所述DS50基因的序列如SEQ ID NO：23所示。与DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、和/或DS45基因相比，DS50基因的防治效果不佳，最高只达约23％。
本发明提供针对昆虫若虫和/或成虫期调控相关基因的干扰RNA，昆虫可以通过口服喷洒RNAi的植物或表达dsRNA构建物或dsRNA，来摄入所述的干扰RNAi，或将干扰RNAi直接喷洒到昆虫表面。
本发明所示的dsRNA构建物如式I所示，dsRNA如式II所示，所采用的间隔序列X的长度没有特别的限制，只要在其与正向序列和反向序列形成构建物且被导入到体内后，能够形成式II所示的dsRNA即可。作为本发明的优选方式，本发 明所述的间隔序列的长度为80-300bp；更佳地为100-250bp。
在本发明的一个优选例中，将所述的表达昆虫基因dsRNA的构建物导入到宿主细胞中，所述宿主细胞可以为植物细胞、组织或器官，所述构建物在植物体内可以表达昆虫基因dsRNA，dsRNA被加工成siRNA。一般地，siRNA的长度约在21-25nt左右。
通常，所述的构建物位于表达载体上。所述的表达载体通常还含有启动子、复制起点和/或标记基因等。本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如卡拉霉素、庆大霉素、潮霉素、氨苄青霉素抗性。
包含上述的适当基因序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主。在本发明的方法中，所述的宿主可以是任何适合于携带所述表达载体并能够将所述表达载体传递给植物细胞的宿主。优选的，所述的宿主为农杆菌。
尽管在本发明的实例中所举例的昆虫为蚜虫。然而应理解，本发明对于适用于本发明的昆虫没有特别的限制，所述昆虫可以是任何一种能以植物为食的植食性昆虫，比如其可以是半翅目昆虫。
本发明对于适用于本发明的植物没有特别的限制，较佳地蚜虫食用的植物，例如大豆，萝卜，桃树，烟草等。
DS7基因
如本文所用，术语“DS7基因”、“tubulin alpha chain-like”、“微管蛋白α链”可互换使用，均是广泛分布的一类球状蛋白质，是细胞内微管的基本结构单位，在细胞的运动和分裂中都发挥着重要作用，在若虫期表达。
在本发明中，该蛋白的C-末端的一些谷氨酸残基被聚谷氨酰化，导致γ-羧基上的聚谷氨酸链。多聚谷氨酰化在spastin(SPAST)的微管切割中起关键作用。SPAST优先识别并作用于用短聚谷氨酸尾巴修饰的微管：SPAST的切断活性随着每微管蛋白的谷氨酸盐数量从1增加到8而增加，但降低超过该谷氨酰 化阈值。
C-末端的一些谷氨酸残基是单糖基化的，但不是聚甘油化的。单甘氨酸化主要限于掺入轴丝的微管蛋白(纤毛和鞭毛)。多聚戊酰化和单甘氨酸化都可以在相邻残基上的相同蛋白质上共存，并且降低甘氨酰化水平可以增加多聚戊酰基化，并且相互作用。
在本发明的一个实施方式中，基于RNAi技术，以DS7基因作为靶标，筛选了针对DS7基因的干扰RNA片段，优选地，所述DS7基因片段的序列如SEQ ID NO：1或24所示：
Figure PCTCN2019086896-appb-000004
Figure PCTCN2019086896-appb-000005
Figure PCTCN2019086896-appb-000006
DS9基因
如本文所用，术语“DS9基因”、“ADP/ATP translocase 3-like”、“ADP/ATP载体蛋白(AAC)”可互换使用，负责将磷酸化合成的ATP转运到细胞质中，作为细胞的主要能力供给，为热力学反应提供动力，在若虫期表达。
在本发明中，该蛋白是一种转运蛋白，使细胞内的交换二磷酸腺苷(ADP)和线粒体三磷酸腺苷横跨(ATP)线粒体内膜。游离ADP从细胞质转运至线粒体基质，而由氧化磷酸化产生的ATP则从线粒体基质转运至细胞质，从而为细胞提供主要能量。
在本发明的一个实施方式中，基于RNAi技术，以DS9基因作为靶标，筛选了针对DS9基因的RNA片段，优选地，所述DS9基因片段的序列SEQ ID NO：2或25所示：
Figure PCTCN2019086896-appb-000007
Figure PCTCN2019086896-appb-000008
Figure PCTCN2019086896-appb-000009
DS15基因
如本文所用，术语“DS15基因”、“heat shock protein 83-like”、“热休克蛋白83”可互换使用，为细胞内的分子伴侣蛋白，在蛋白质相互作用中具有重要的作用，例如协助折叠和协助建立合适的蛋白质构想。在若虫期表达。
在本发明中，热休克蛋白(HSP)是细胞响应暴露于压力条件下而产生的蛋白质家族。它们首先相关于热休克，但现在已知在其它应力，包括暴露于寒冷，并在伤口愈合或组织重塑。该组的许多成员通过稳定新蛋白质以确保正确的折叠或通过帮助折叠被细胞应激损伤的蛋白质来执行伴侣分子功能。增加是转录的调控。热休克蛋白的显着上调是热休克反应的关键部分，主要由热休克因子(HSF)诱导。
在本发明的一个实施方式中，基于RNAi技术，以DS15基因作为靶标，筛选了针对DS15基因的RNA片段，优选地，所述DS15基因片段的序列SEQ ID NO：3或26所示：
Figure PCTCN2019086896-appb-000010
Figure PCTCN2019086896-appb-000011
Figure PCTCN2019086896-appb-000012
Figure PCTCN2019086896-appb-000013
DS25基因
如本文所用，术语“DS25基因”、“eukaryotic initiation factor 4A-like”、“真核起始因子复合物4A型”可互换使用，是解旋双链RNA的解旋酶，也是核糖体亚基结合所必须的功能蛋白，在若虫期表达。
在本发明中，真核起始因子复合物与GTP和引发剂Met-tRNA形成三元复合物，该过程受鸟嘌呤核苷酸交换和磷酸化调节，并且是基因表达瓶颈的主要调控元件。在翻译进行到延伸阶段之前，许多起始因子必须促进核糖体和mRNA的协同作用，并确保mRNA的5'UTR充分缺乏二级结构。第4组真核 生物起始因子促进了这种结合；在翻译的正常调节以及癌细胞的转化和进展中具有意义。
在本发明的一个实施方式中，基于RNAi技术，以DS25基因作为靶标，筛选了针对DS25基因的RNA片段，优选地，所述DS25基因片段的序列SEQ ID NO：4或27所示：
Figure PCTCN2019086896-appb-000014
Figure PCTCN2019086896-appb-000015
Figure PCTCN2019086896-appb-000016
DS27基因
如本文所用，术语“DS27”、“troponin T-like isoform 3”、“肌钙蛋白3型”可互换使用，介导Ca离子通道，调节昆虫横纹肌的收缩调节功能。在若虫、成虫期表达。
在本发明中，肌钙蛋白附着于蛋白原肌球蛋白并位于肌肉组织中肌动蛋白丝之间的凹槽内。在松弛的肌肉中，原肌球蛋白阻断肌球蛋白交叉桥的附着位点，从而防止收缩。当通过动作电位刺激肌肉细胞收缩时，钙通道在肌浆膜中开放并将钙释放到肌浆中。其中一些钙附着于肌钙蛋白，导致它改变形状，暴露肌动蛋白丝上的肌球蛋白(活性部位)的结合位点。肌球蛋白与肌动蛋白的结合引起交叉桥形成，并开始收缩肌肉。
肌钙蛋白激活。肌钙蛋白C(红色)结合Ca2 +，稳定激活状态，其中肌钙蛋白I(黄色)不再与肌动蛋白结合。肌钙蛋白T(蓝色)将复合物固定在原肌球蛋白上。
肌钙蛋白存在于骨骼肌和心肌中，但肌钙蛋白的特定版本在不同类型的肌肉中有所不同。主要区别在于肌钙蛋白的TnC亚基在骨骼肌中具有四个钙离子结合位点，而在心肌中仅有三个。关于与肌钙蛋白结合的钙的实际含量的观点因专家和来源而不同。
在本发明的一个实施方式中，基于RNAi技术，以DS27基因作为靶标，筛选了针对DS27基因的RNA片段，优选地，所述DS27基因片段的序列SEQ ID NO：5或28所示：
Figure PCTCN2019086896-appb-000017
Figure PCTCN2019086896-appb-000018
Figure PCTCN2019086896-appb-000019
DS45基因
如本文所用，术语“DS45基因”、“Y-box protein Ct-p40-like”、“Y盒结合蛋白Ct-p40-like”可互换使用，影响细胞分化，细胞骨架的形成，缺失会抑制细胞内外的信号传导途径，参与dna损伤修复和转录，在若虫期表达。
在本发明的一个实施方式中，基于RNAi技术，以DS45基因作为靶标，筛选了针对DS45基因的RNA片段，优选地，所述DS45基因片段的序列SEQ ID NO：6或29所示：
Figure PCTCN2019086896-appb-000020
Figure PCTCN2019086896-appb-000021
Figure PCTCN2019086896-appb-000022
dsRNA构建物及其应用
本发明提供了一种dsRNA构建物，所述dsRNA的构建物为双链，并且其正 链或负链含有式I所示的结构：
Seq 正向-X-Seq 反向    式I
式中，
Seq 正向为昆虫若虫和/或成虫期调控相关基因或片段的核苷酸序列；
Seq 反向为与Seq 正向基本上互补的核苷酸序列；
X为位于Seq 正向和Seq 反向之间的间隔序列，并且所述间隔序列与Seq 正向和Seq 反向不互补，
其中，所述的昆虫若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
在本发明的一个优选例中，Seq 正向、Seq 反向的长度为至少50bp。
在本发明的一个优选例中，所述dsRNA构建物被昆虫(如蚜虫)摄食后，形成式II所示的dsRNA，
Figure PCTCN2019086896-appb-000023
式中，
Seq’ 正向为Seq 正向序列对应的RNA序列或序列片段；
Seq’ 反向为与Seq’ 正向基本上互补的序列；
X’为无；或为位于Seq’ 正向和Seq’ 反向之间的间隔序列，并且所述间隔序列与Seq’ 正向和Seq’ 反向不互补，
||表示在Seq 正向和Seq 反向之间形成的氢键。
本发明还提供了所述dsRNA构建物的用途，它被用于：(1)提高蚜虫的防治效果；和/或(2)提高虫口减退率；和/或(3)降低若虫和/或成虫期调控相关基因的表达量；(4)降低虫口基数；和/或(5)减少植物受害率；和/或(6)降低作物受害程度，提高农作物产品质量。
dsRNA及其应用
本发明还提供了一种如式II所示的dsRNA，
Figure PCTCN2019086896-appb-000024
式中，
Seq’ 正向为Seq 正向序列对应的RNA序列或序列片段；
Seq’ 反向为与Seq’ 正向基本上互补的序列；
X’为无；或为位于Seq’ 正向和Seq’ 反向之间的间隔序列，并且所述间隔序列与Seq’ 正向和Seq’ 反向不互补，
||表示在Seq 正向和Seq 反向之间形成的氢键。
在另一优选例中，所述的间隔序列X的长度为0-300bp，较佳地为100bp。
所述的昆虫若虫和/或成虫期调控相关基因来源于蚜虫；所述DS7基因的序列如SEQ ID NO.:1所示；所述DS9基因的序列如SEQ ID NO.:2所示；所述DS15基因的序列如SEQ ID NO.:3所示；所述DS25基因的序列如SEQ ID NO.:4所示；所述DS27基因的序列如SEQ ID NO.:5所示；所述DS45基因的序列如SEQ ID NO.:6所示。
在另一优选例中，所述昆虫为植食性昆虫，较佳地来自半翅目昆虫，最佳地来自蚜虫属。
本发明还提供了所述dsRNA的用途，它被用于：(1)提高蚜虫的防治效果；和/或(2)提高虫口减退率；和/或(3)降低若虫和/或成虫期调控相关基因的表达量；和/或(4)降低虫口基数；和/或(5)减少植物被危害率。
组合物及其应用
本发明还提供了一种组合物，本发明人针对高效杀灭蚜虫的难题，基于RNAi技术开发了针对靶标基因的RNAi片段，并通过藉由昆虫饲喂或直接喷洒昆虫，提高蚜虫的防治效果和冲口减退率，使RNAi起到抑制基因表达的效果，最终达到高效杀灭蚜虫的目的。本发明方法高效、方便、快捷、准确且无公害。
所述组合物包括dsRNA构建物和/或dsRNA，以及昆虫喂食上可接受的有效量的载体。在另一优选例中，所述的组合物是用于诱导或导致蚜虫若虫和/或成虫期死亡的组合物。
在另一优选例中，所述的dsRNA具有以下序列：
dsRNA1：具有对应于SEQ ID NO.:1或24所示的序列；
dsRNA2：具有对应于SEQ ID NO.:2或25所示的序列。
dsRNA3：具有对应于SEQ ID NO.:3或26所示的序列；
dsRNA4：具有对应于SEQ ID NO.:4或27所示的序列。
dsRNA5：具有对应于SEQ ID NO.:5或28所示的序列；
dsRNA6：具有对应于SEQ ID NO.:6或29所示的序列。
本发明还提供了所述组合物的用途，所述用途选自下组：
(1)提高蚜虫的防治效果；和/或(2)提高虫口减退率；和/或(3)降低若虫和/或成虫期调控相关基因的表达量；(4)降低虫口基数；和/或(5)减少植物受害率；和/或(6)降低作物受害程度，提高农作物产品质量。
在本发明的一个优选例中，组合物为水溶液，pH通常约为5-8，较佳地，pH约为6-8。
如本文所用，术语“有效量”或“有效剂量”是指可对所述昆虫喂食产生功能或活性的且可被所述昆虫所接受的量。优选地，dsRNA1的含量为约1-500ng/μl，较佳地，5-300ng/μl，更佳地，50-150ng/μl；dsRNA2的含量为约1-500ng/μl，较佳地，5-300ng/μl，更佳地，50-150ng/μl；dsRNA3的含量为约1-500ng/μl，较佳地，5-300ng/μl，更佳地，50-150ng/μl；dsRNA4的含量为约1-500ng/μl，较佳地，5-300ng/μl，更佳地，50-150ng/μl；dsRNA5的含量为约1-500ng/μl，较佳地，5-300ng/μl，更佳地，50-150ng/μl；dsRNA6的含量为约1-500ng/μl，较佳地，5-300ng/μl，更佳地，50-150ng/μl。优选有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过饲喂试验或喷洒实验)。
如本文所用，“昆虫喂食上可接受的”的成分是适用于所述昆虫而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。
如本文所用，术语“载体”包括各种赋形剂和稀释剂。这类载体包括(但并不限于)：水、盐水、缓冲液、葡萄糖、甘油、乙醇、及其组合。
本发明的组合物可以直接喷洒、喂食，或被制成针剂形式，例如用水、生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的组合物宜在无菌或无RNA酶的条件下制造。
本发明的主要优点包括：
1)本发明针对特定的靶基因设计的dsRNA可高效杀灭蚜虫，提高蚜虫的防治效果(≥80％)和虫口减退率(≥70％)；
2)获得的dsRNA可直接应用于蚜虫杀灭，使用方便；
3)生产成本低，稳定性好，适用于大规模生产；
4)环境兼容性好，绿色无公害，对人畜安全。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非有特别说明，否则实施例中所用的材料和试剂均为市售产品。
通用方法和材料
1.蚜虫饲养及生物测试
桃蚜(Myzus persicae)在室内温室或塑料大棚养殖的萝卜苗上培养繁殖和测试，大豆蚜(Aphid glycine)在室内温室或塑料大棚养殖的大豆苗上培养繁殖和测试。培养室温度25±1℃，相对湿度为40-60％，光周期12h:12h。
测试前，将一定数量的蚜虫接种到目标植物上计数，喷洒一定浓度的dsRNA后分别在在1天、3天和5天再进行计数，每个基因的测试均重复10次。根据计数结果，确定靶标基因的防治效果。
2.防治效果的统计方法
本研究采用两种统计方法评估靶标dsRNA的对蚜虫的作用效果。
第一种方法，防治效果，计算方法如下：
防治效果(％)＝(1-CK0×PT1/CK1×PT0)×100
其中：PT0：处理区施药前虫数；PT1：处理区施药后虫数；
CK0：对照区施药前虫数；CK1：对照区施药后虫数。
第二种方法，虫口减退率，计算方法如下：
虫口减退率(％)＝[(施药前虫数-施药后虫数)/施药前虫数]×100
3.RNA提取及质量检测
总RNA的提取使用
Figure PCTCN2019086896-appb-000025
Reagent(Invitrogen)，并根据说明书进行操作：
1)在1mL TRIzol中加入研磨充分的玉米螟样品50-100mg，混匀，室温静置5 min。2)加入200μL氯仿，震荡混匀，室温静置3min。3)12 000rpm(4℃)离心15min，转移上层水相到另一新的离心管中，加入500μL预冷的异丙醇，震荡混匀，室温静置10min。4)12 000rpm(4℃)离心15min，小心吸取上清。5)用500μL预冷的75％乙醇洗涤，涡旋仪轻混10sec。6)12 000rpm(4℃)离心2min，小心吸取上清后室温干燥5min，加入适量DEPC灭菌水溶解，即得到总RNA样品。分光光度计下检测吸光度，1％琼脂凝胶电泳检测总RNA质量，-80℃保存待用。
4.dsRNA合成
使用试剂盒MEGA
Figure PCTCN2019086896-appb-000026
RNAi Kit(Ambion)进行dsRNA的合成，并根据说明书进行实验操作。在扩增模板的引物5’端加入T7 promoter序列，便于后续dsRNA的合成。利用pPigbac A3 EGFP作为合成对照组dsEGFP的模板，在后续试验中dsEGFP作为阴性对照参与试验组的处理。合成dsRNAs的引物见附件S3。在合成过程中，template DNA以及single-stranded RNA分别用DNase和RNase来进行去除。
5.基因表达量的检测(q-RT-PCR)
使用
Figure PCTCN2019086896-appb-000027
reagent(Invitrogen)进行总RNA的提取，步骤严格按照操作手册进行。取1μg总RNA，利用试剂盒ReverTra
Figure PCTCN2019086896-appb-000028
qPCR RT Master Mix with gDNA Remover(TOYOBO)进行cDNA第一链的合成。RT-qPCR反应所用的试剂盒为
Figure PCTCN2019086896-appb-000029
Premix Ex Taq TMII(Takara)，引物详见附件S3。对每个基因样品进行3次重复检测，其表达水平分析选用18S rRNA的表达量进行均一化处理。数据分析参照2 -ΔΔCT Method(Livak&Schmittgen,2001)。通过计算均值与标准误得到相应数值。为了消除个体差异，每个实验组的样品为2头处理后存活幼虫形成的样品池，每个实验组进行三次生物学重复。
实施例1靶标基因序列及dsRNA的合成
为了筛选有效的基于RNA干扰技术的蚜虫靶标基因，对桃蚜(Myzus persicae)和大豆蚜(Aphid glycine)分别进行了转录组测序(两种蚜虫的取样及测序分析方法相同)。将不同发育时期的蚜虫分别抽提总RNA后，取等量 的RNA混匀，组成蚜虫全发育期的总RNA，送深圳华大基因科技服务有限公司，利用Illumina Hiseq2000平台进行转录组测序。对测序结果去掉接头后用denove程序进行组装，然后对Unigene进行功能注释。本研究是从这些有功能注释的Unigene中选取目标基因片段进行扩增，并合成dsRNA。通过大量筛选，本发明扩增合成6个目的基因、外源对照基因GFP以及来自大豆蚜的内源对比基因DS50的引物如表1所示。6个基因片段的DNA序列见表2，其中ds7，ds9，ds15针对蚜虫，尤其针对桃蚜，ds25、ds27、ds45针对蚜虫，尤其针对大豆蚜。
表1.扩增并合成靶标基因dsRNA的引物序列
Figure PCTCN2019086896-appb-000030
表2. 6个靶标基因的序列片段
Figure PCTCN2019086896-appb-000031
Figure PCTCN2019086896-appb-000032
Figure PCTCN2019086896-appb-000033
Figure PCTCN2019086896-appb-000034
Figure PCTCN2019086896-appb-000035
Figure PCTCN2019086896-appb-000036
实施例2靶标基因dsRNA对蚜虫的防治效果
将一定数量的桃蚜或大豆蚜分别接种到萝卜苗或大豆苗上，首先分别记录接种到每株植物上的蚜虫数量，将合成好的dsRNA溶解到2％的吐温-80中，6个靶标基因的dsRNA浓度如表3所示。然后将1ml的dsRNA分别喷洒到接种有蚜虫的植株上，并于次日进行计数作为dsRNA处理后第一天的统计结果，然后每隔一日统计一次，共统计3次，分别记录为处理后第1天，第3天和第5天的结果。用2％的吐温-80和dsGFP作对照。统计结果表明，与只喷洒2％吐-80对照相比，3个桃蚜的靶标基因和3个大豆蚜的靶标基因对两种蚜虫的防治效果均超过80％(图1，A，B)。
表3.靶标基因dsRNA的喷洒浓度
桃蚜基因名称 浓度(ng/μl) 大豆蚜基因名称 浓度(ng/μl)
dsGFP 295 dsGFP 265
ds7 233 ds25 282
ds9 241 ds27 257
ds15 279 ds45 242
实施例3靶标基因的对蚜虫的虫口减退率统计
蚜虫属孤雌胎生昆虫，生下来就是1龄的新生蚜虫。从1龄新生蚜到其可以产仔的周期约5-7天(受环境温度影响)，一棵植物上的蚜虫存在明显的世代交替现象，也就是不同世代和各种大小(不同龄期)的虫子同时存在，因此，向测试植株接虫时会有各个龄期的蚜虫(如2th-4th，可能还有成虫)，这样，在各种测试处理之后，蚜虫很快就开始繁殖，并产生下一代，造成被测试植株上的蚜虫数量在喷药前计数后，还在不断增加。这对蚜虫类杀虫剂的防治效果判断是个很大的干扰，因此，就有了一个更加严格或相对准确的蚜虫防治效果计算方法，也就是虫口减退率(计算公式见通用方法和材料部分)。
本发明的统计结果表明，3个桃蚜靶标基因的dsRNA喷洒后，桃蚜种群在处理后1天，3天和5天的虫口减退率见表4所示，喷洒后第5天的虫口减退率均达到了70％以上。
3个大豆蚜靶标基因的dsRNA喷洒后，大豆蚜种群在处理后1天，3天和5天的虫口减退率见表5所示，喷洒后第5天的虫口减退率，除了ds45为67.61％，其他两个靶标基因均在70％以上。
表4.桃蚜靶标基因dsRNA喷洒后的虫口减退率
处理 1d 3d 5d
CK -23.55±19.99 -43.45±41.67 -111.98±80.5
dsGFP 10.99±33.56 -1.61±52.77 -58.05±74.94
ds7 27.73±16.59 61.37±17.58 74.95±9.11
ds9 29.18±21.97 63.65±11.32 70.35±14.69
ds15 38.18±22.79 62.66±19.69 71.17±13.78
表5.大豆蚜靶标基因dsRNA喷洒后的虫口减退率
处理 1d 3d 5d
CK 5.03±13.96 -50.29±29.93 -135.23±62.03
dsGFP -0.75±18.49 -28.57±19.66 -59.91±29.55
ds25 24.02±18.82 67.91±22.1 73.85±16.11
ds27 33.64±23.56 69.28±14.36 78.82±10.62
ds45 24.59±22.59 61.6±21.45 67.61±25.64
实施例4桃蚜靶标与吡虫啉的防治效果比较
实验方法：
1. 12-15d的萝卜苗，接虫100头，稳定1d后，第二天进行dsRNA喷洒处理。
2.喷洒用的dsRNA浓度为300ng/μl。合成好的dsRNA溶于水中，每株喷洒300μl。
3.吡虫啉浓度为10000倍液(德国拜耳艾美乐70％吡虫啉3g，水分散粒剂),每株喷300μl。
4.试验方法：喷雾处理，每处理4个重复。
结果如表6、表7及图3、图4所示。
表6.桃蚜田间测试：虫口减退率
Figure PCTCN2019086896-appb-000037
表7.桃蚜田间测试：防治效果
Figure PCTCN2019086896-appb-000038
Figure PCTCN2019086896-appb-000039
结果显示，针对桃蚜的三个基因在第三天开始均呈现了明显的致死效果，在第五天时虫口减退率均在70％以上，与吡虫啉相比，虫口减退率差异不明显，但是与对照dsGFP相比，虫口减退率呈现显著差异(表6)；同时，对防治效果的统计分析表明，这三个靶标基因的防治效果均达到了90％以上，且在第三天即可呈现较好的防治效果(表7)。因此，本结果表明，这三个靶标基因对桃蚜的致死效果较强，可以作为防治桃蚜的靶标基因进行害虫防治。
实施例5靶标基因的表达量检测
为了证明喷洒这些靶标基因的dsRNA对蚜虫种群的控制作用是由于抑制了目标基因的表达，分别收集6个靶标基因dsRNA处理后1天，3天和5天的蚜虫，用定量PCR(q-RT-PCR)检测目标基因是否被抑制。3个桃蚜靶标基因的检测结果见图2A，除了ds9基因处理1天后目标基因被诱导上调外，所有基因在处理3天和5天后均被显著下调，说明蚜虫的死亡与基因的表达量密切相关。
3个大豆蚜靶标基因的检测结果见图2B，除了ds25基因处理1天后目标基因的表达与对照没有显著差异外，所有基因在处理3天和5天后均被显著下调，说明蚜虫的死亡与基因的表达量密切相关。
实施例6
组合物的制备
本实施例提供了一种用于高效杀灭蚜虫的组合物，该组合物为水溶液，包括组分：
1.针对序列如SEQ ID NO.:1或24所示DS7基因片段的dsRNA浓度为100ng/μl；
2.针对序列如SEQ ID NO.:2或25所示DS9基因片段的dsRNA浓度为100ng/μl。
3.针对序列如SEQ ID NO.:3或26所示DS15基因片段的dsRNA浓度为100ng/μl。
4.针对序列如SEQ ID NO.:4或27所示DS25基因片段的dsRNA浓度为100ng/μl。
5.针对序列如SEQ ID NO.:5或28所示DS27基因片段的dsRNA浓度为100ng/μl。
6.针对序列如SEQ ID NO.:6或29所示DS45基因片段的dsRNA浓度为100ng/μl。
对比例1
方法同实施例1和2，区别在于，靶标基因为DS50，所用引物为：
primer F：
Figure PCTCN2019086896-appb-000040
primer R:
Figure PCTCN2019086896-appb-000041
扩增产物长度578bp。
DS50基因为脂肪酸合酶基因(fatty acid synthase-like)序列如SEQ ID NO.:23所示，编码FASN基因。脂肪酸合酶是一种催化脂肪酸合成的多酶蛋白。它不是单一的酶，而是由两个相同的272kDa多功能多肽组成的整个酶系统，其中底物从一个功能域递交到下一个，其主要功能是在NADPH存在下催化由乙酰辅酶A和丙二酰辅酶A合成棕榈酸酯。
结果显示，用本发明的方法针对DS50基因所设计的dsRNA对蚜虫的防治效果很差，最高只有约23％。
所述DS50基因片段的序列SEQ ID NO：23所示：
Figure PCTCN2019086896-appb-000042
Figure PCTCN2019086896-appb-000043
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 一种dsRNA构建物，其特征在于，所述dsRNA的构建物为双链，并且其正链或负链含有式I所示的结构：
    Seq 正向-X-Seq 反向  式I
    式中，
    Seq 正向为昆虫若虫和/或成虫期调控相关基因或片段的核苷酸序列；
    Seq 反向为与Seq 正向基本上互补的核苷酸序列；
    X为位于Seq 正向和Seq 反向之间的间隔序列，并且所述间隔序列与Seq 正向和Seq 反向不互补，
    其中，所述的昆虫若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
  2. 一种式II所示的dsRNA，
    Figure PCTCN2019086896-appb-100001
    式中，
    Seq’ 正向为昆虫若虫和/或成虫期调控相关基因或片段的核苷酸序列对应的RNA序列或序列片段；
    Seq’ 反向为与Seq’ 正向基本上互补的序列；
    X’为无；或为位于Seq’ 正向和Seq’ 反向之间的间隔序列，并且所述间隔序列与Seq’ 正向和Seq’ 反向不互补；
    其中，所述的昆虫若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合；
    ||表示在Seq 正向和Seq 反向之间形成的氢键。
  3. 如权利要求1所述的dsRNA构建物，其特征在于，所述昆虫为植食性昆虫，较佳地为同翅目昆虫，最佳地为蚜虫属。
  4. 一种表达载体，其特征在于，所述表达载体含有权利要求1中所述的dsRNA构建物。
  5. 一种宿主细胞，其特征在于，所述宿主细胞中含有权利要求4中所述的表达载体或者染色体中整合有对应于权利要求1中所述的dsRNA构建物的DNA序 列。
  6. 一种组合物，其特征在于，所述组合物包括权利要求1中所述的dsRNA构建物和/或权利要求2中所述的dsRNA，以及昆虫喂食上可接受的载体。
  7. 权利要求1所述的dsRNA构建物，或权利要求2所述的dsRNA，或权利要求5所述的宿主细胞、或权利要求6所述组合物的用途，其特征在于，所述用途选自下组：
    (1)提高蚜虫的防治效果；和/或
    (2)提高虫口减退率；和/或
    (3)降低若虫和/或成虫期调控相关基因的表达量；和/或
    (4)降低虫口基数；和/或
    (5)减少植物受害率；和/或
    (6)降低作物受害程度，提高农作物产品质量。
  8. 一种杀灭昆虫的方法，其特征在于，包括步骤：用干扰昆虫若虫和/或成虫期调控相关基因表达的干扰分子，或含所述干扰分子的载体、细胞、植物组织或昆虫防治试剂饲喂或喷洒昆虫；
    较佳地，所述的昆若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
  9. 一种制备权利要求2所述dsRNA的方法，其特征在于，包括步骤：
    (i)制备表达dsRNA的构建物，所述构建物为双链，并且其正链或负链含有式I所示的结构：
    Seq 正向-X-Seq 反向 式I
    式中，
    Seq 正向为昆虫若虫和/或成虫期调控相关基因或片段的核苷酸序列；
    Seq 反向为与Seq 正向基本上互补的核苷酸序列；
    X为位于Seq 正向和Seq 反向之间的间隔序列，并且所述间隔序列与Seq 正向和Seq 反向不互补，
    其中，所述的昆虫若虫和/或成虫期调控相关基因选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合；
    (ii)将步骤(i)所述的构建物转入宿主细胞，从而在宿主细胞中表达形成式II所示的dsRNA，
    Figure PCTCN2019086896-appb-100002
    式中，
    Seq’ 正向为Seq 正向序列对应的RNA序列或序列片段；
    Seq’ 反向为与Seq’ 正向基本上互补的序列；
    X’为无；或为位于Seq’ 正向和Seq’ 反向之间的间隔序列，并且所述间隔序列与Seq’ 正向和Seq’ 反向不互补，
    ||表示在Seq 正向和Seq 反向之间形成的氢键。
  10. 一种制备昆虫防治试剂的方法，其特征在于，包括步骤：将权利要求1所述的dsRNA构建物，或权利要求2所述的dsRNA，或权利要求5所述的宿主细胞，或权利要求6所述的组合物喷洒于植物表面，从而制得昆虫防治试剂。
  11. 一种提高植物对昆虫抗性的方法，其特征在于，包括：
    在植物中表达重组DNA构建体，其中所述重组DNA构建体包含编码RNA的DNA，所述RNA具有与所述靶基因的至少21个或更多个连续核苷酸基本上相同或基本互补的序列，其中所述靶基因为昆虫若虫和/或成虫期调控相关基因，选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
  12. 一种制备转基因植物细胞的方法，其特征在于，包括步骤：
    (i)将重组DNA构建体导入或转染植物细胞，使得所述植物细胞含有所述构建物从而制得所述转基因植物细胞，其中所述重组DNA构建体包含编码RNA的DNA，所述RNA具有与所述靶基因的至少21个或更多个连续核苷酸基本上相同或基本互补的序列，其中所述靶基因为昆虫若虫和/或成虫期调控相关基因，选自下组：DS7基因、DS9基因、DS15基因、DS25基因、DS27基因、DS45基因、或其组合。
  13. 一种制备转基因植物的方法，其特征在于，包括步骤：
    将权利要求12所述方法制备的所述转基因植物细胞再生为植物体，从而获得所述转基因植物。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118165090A (zh) * 2024-03-11 2024-06-11 青岛农业大学 一种苹果黄蚜Cmb4b基因及其dsRNA在防治苹果黄蚜中的应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113100235B (zh) * 2020-01-09 2023-09-12 上海植生优谷生物技术有限公司 一种提高dsRNA杀虫效果的配方
CN112175962B (zh) * 2020-09-30 2022-01-04 中国农业科学院植物保护研究所 荻草谷网蚜基因zx-5及其在害虫防控中的应用
CN113373146B (zh) * 2021-06-08 2022-04-08 湖北大学 一种防治桃蚜的质体转基因植物的制备方法及其在防治桃蚜中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005049841A1 (en) * 2003-11-17 2005-06-02 Commonwealth Scientific And Industrial Research Organisation Insect resistance using inhibition of gene expression
CN105339385A (zh) * 2013-06-03 2016-02-17 弗劳恩霍夫应用研究促进协会 新的害虫防治方法
CN105755003A (zh) * 2016-04-05 2016-07-13 中国农业科学院植物保护研究所 基于麦长管蚜Sitobion avenae 嗅觉受体SaveOrco 基因设计的siRNA 及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT1068311E (pt) * 1998-04-08 2011-07-20 Commw Scient Ind Res Org Processos e meios de obter fenótipos modificados
US9693555B2 (en) * 2011-03-18 2017-07-04 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences Insect-combating preparation and method based on RNAi technology
WO2018013333A1 (en) * 2016-07-12 2018-01-18 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005049841A1 (en) * 2003-11-17 2005-06-02 Commonwealth Scientific And Industrial Research Organisation Insect resistance using inhibition of gene expression
CN105339385A (zh) * 2013-06-03 2016-02-17 弗劳恩霍夫应用研究促进协会 新的害虫防治方法
CN105755003A (zh) * 2016-04-05 2016-07-13 中国农业科学院植物保护研究所 基于麦长管蚜Sitobion avenae 嗅觉受体SaveOrco 基因设计的siRNA 及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHRISTIAENS, 0.: "The challenge of RNAi-mediated control of hemipterans", CURRENT OPINION IN INSECT SCIENCE, vol. 6, 28 September 2014 (2014-09-28), pages 15 - 21, XP002780190, DOI: 10.1016/j.cois.2014.09.012 *
UPADHYAY, S.K. ET AL.: "RNA interference for the control of whiteflies (Bemisia tabaci) by oral route", J. BIOSCI., vol. 36, no. 1, 31 March 2011 (2011-03-31) - 29 April 2011 (2011-04-29), pages 153 - 161, XP019909465 *
WILL, T.: "Heat shock protein 83 plays pleiotropic roles in embryogene- sis, longevity, and fecundity of the pea aphid Acyrthosiphon pisum", DEVELOPMENT, GENES AND EVOLUTION, vol. 227, no. 1, 14 October 2016 (2016-10-14), pages 1 - 9, XP036123178, ISSN: 0949-944X *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118165090A (zh) * 2024-03-11 2024-06-11 青岛农业大学 一种苹果黄蚜Cmb4b基因及其dsRNA在防治苹果黄蚜中的应用

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