RNAI TARGET GENE THAT IS HIGHLY LETHAL TO APHIDS AND USE THEREOF

Abstract

Provided are a RNAi target gene that is lethal to aphids and the use thereof. Specifically, provided are six gene fragments resulting in the death of aphid nymphs and/or death thereof in the adult stage based on RNA interference technology. The death of the aphids can be caused by spraying a dsRNA-containing composition onto plants to feed aphids or directly spraying same onto the skin of the aphids.

Claims

1. A dsRNA construct, wherein the dsRNA construct is double-stranded, and its positive or negative strand contains a structure as shown in Formula I:
Seq.sub.forward-X-Seq.sub.reverse  Formula I wherein Seq.sub.forward is a nucleotide sequence of insect nymph and/or adult stage regulation-related gene or fragment; Seq.sub.reverse is a nucleotide sequence that is basically complementary to Seq.sub.forward; X is an intervening sequence between the Seq.sub.forward and the Seq.sub.reverse, and the intervening sequence is not complementary to the Seq.sub.forward and the Seq.sub.reverse, wherein the insect nymph and/or adult stage regulation-related gene is selected from the group consisting of DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene and a combination thereof.

2. A dsRNA as shown in Formula II, ##STR00006## wherein Seq′.sub.forward is a RNA sequence or sequence fragment corresponding to a nucleotide sequence of an insect nymph and/or adult stage regulation-related gene or fragment; Seq′.sub.reverse is a sequence that is basically complementary to the Seq′.sub.forward; X′ is none; or is an intervening sequence located between Seq′.sub.forward and Seq′.sub.reverse, and the intervening sequence is not complementary to Seq′.sub.forward and Seq′.sub.reverse; wherein, the insect nymph and/or adult stage regulation-related gene is selected from the group consisting of: DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene, and a combination thereof; ∥ represents the hydrogen bond formed between Seq forward and Seq.sub.reverse.

3. The dsRNA of claim 1, the insect is a phytophagous insect, preferably a homoptera insect, most preferably Aphis.

4. An expression vector containing the dsRNA construct of claim 1.

5. A host cell that contains an expression vector containing the dsRNA construct of claim 1 or having the DNA sequence corresponding to the dsRNA construct integrated into the chromosome.

6. A composition comprising the dsRNA construct of claim 1, and an acceptable carrier for insect feeding.

7. A method of: (1) improving the control effect of aphids; (2) increasing the dropping rate of insect population; (3) decreasing the expression level of nymph and/or adult stage regulation-related gene; (4) reducing the initial number of insect population; (5) reducing plant damage rate; and/or (6) reducing crop damage degree and improving the quality of crop products, the method comprising administering the dsRNA construct of claim 1.

8. A method for killing insects, comprising the steps of: using an interference molecule that interferes with the expression of an insect nymph and/or adult stage regulation-related gene, or feeding or spraying an insect with a vector, cell, plant tissue or insect prevention and control reagent containing the interference molecule; preferably, the insect nymph and/or adult stage regulation-related gene is selected from the group consisting of DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene, and a combination thereof.

9. A method for preparing the dsRNA of claim 2, comprising the steps: (i) preparing a construct expressing dsRNA, and the construct is double-stranded, and its positive or negative strand contains a structure as shown in Formula I:
Seq.sub.forward-X-Seq.sub.reverse  Formula I wherein Seq.sub.forward is a nucleotide sequence of insect nymph and/or adult stage regulation-related gene or fragment; Seq.sub.reverse is a nucleotide sequence that is basically complementary to Seq.sub.forward; X is an intervening sequence located between the Seq.sub.forward and the Seq.sub.reverse, and the intervening sequence is not complementary to the Seq.sub.forward and the Seq.sub.reverse, wherein the insect nymph and/or adult stage regulation-related gene is selected from the group consisting of DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene and a combination thereof; (ii) transforming the construct as described in step (i) into a host cell, thereby expressing and forming a dsRNA as shown in Formula II in the host cell, ##STR00007## wherein Seq′.sub.forward is a RNA sequence or sequence fragment corresponding to the Seq.sub.forward sequence; Seq′.sub.reverse is a sequence that is basically complementary to the Seq′.sub.forward; X′ is none; or is an intervening sequence located between Seq′.sub.forward and Seq′.sub.reverse, and the intervening sequence is not complementary to Seq′.sub.forward and Seq′.sub.reverse, ∥ represents the hydrogen bond formed between Seq.sub.forward and Seq.sub.reverse.

10. A method for preparing an insect prevention and control reagent comprising the steps of: spraying the dsRNA construct of claim 1 on the surface of the plant, thereby producing the insect prevention and control agent.

11. A method for improving a plant resistance to an insect, comprising: expressing a recombinant DNA construct in a plant, wherein the recombinant DNA construct comprises DNA encoding RNA, and the RNA has a sequence that is substantially identical or substantially complementary to at least 21 or more consecutive nucleotides of the target gene, wherein the target gene is an insect nymph and/or adult stage regulation-related gene, selected from the group consisting of DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene, and a combination thereof.

12. A method for preparing a transgenic plant cell, comprising the steps: (i) introducing or transfecting a recombinant DNA construct into a plant cell so that the plant cell contains the construct, thereby producing the transgenic plant cell, wherein the recombinant DNA construct contains DNA encoding RNA, the RNA has a sequence that is substantially identical or substantially complementary to at least 21 or more consecutive nucleotides of the target gene, wherein the target gene is an insect nymph and/or adult stage regulation-related gene, selected from the group consisting of DS7 Gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene, and a combination thereof.

13. A method for preparing a transgenic plant, comprising the steps: regenerating a transgenic plant cell prepared by the method of claim 12 into a plant body, thereby obtaining the transgenic plant.

14. A composition comprising the dsRNA of claim 2, and an acceptable carrier for insect feeding.

15. A method of: (1) improving the control effect of aphids; (2) increasing the dropping rate of insect population; (3) decreasing the expression level of nymph and/or adult stage regulation-related gene; (4) reducing the initial number of insect population; (5) reducing plant damage rate; and/or (6) reducing crop damage degree and improving the quality of crop products, the method comprising administering the dsRNA construct of claim 2.

16. A method of: (1) improving the control effect of aphids; (2) increasing the dropping rate of insect population; (3) decreasing the expression level of nymph and/or adult stage regulation-related gene; (4) reducing the initial number of insect population; (5) reducing plant damage rate; and/or (6) reducing crop damage degree and improving the quality of crop products, the method comprising administering the host cell of claim 5.

17. A method of: (1) improving the control effect of aphids; (2) increasing the dropping rate of insect population; (3) decreasing the expression level of nymph and/or adult stage regulation-related gene; (4) reducing the initial number of insect population; (5) reducing plant damage rate; and/or (6) reducing crop damage degree and improving the quality of crop products, the method comprising administering the composition of claim 6.

18. A method of: (1) improving the control effect of aphids; (2) increasing the dropping rate of insect population; (3) decreasing the expression level of nymph and/or adult stage regulation-related gene; (4) reducing the initial number of insect population; (5) reducing plant damage rate; and/or (6) reducing crop damage degree and improving the quality of crop products, the method comprising administering the composition of claim 14.

19. A method for preparing an insect prevention and control reagent comprising the steps of: spraying the dsRNA of claim 2 on the surface of the plant, thereby producing the insect prevention and control agent.

20. A method for preparing an insect prevention and control reagent comprising the steps of: spraying the composition of claim 6 on the surface of the plant, thereby producing the insect prevention and control agent.

Description

DESCRIPTION OF DRAWINGS

[0159] FIG. 1 shows the control effect of target genes on aphids.

[0160] FIG. 2 shows the detection results of the relative expression levels of target genes.

[0161] FIG. 3 shows the control effect of the three target genes of Myzus persicae in the field.

[0162] FIG. 4 shows the results of statistical analysis of the field control effect and the dropping rate of insect of Myzus persicae.

DETAILED DESCRIPTION OF INVENTION

[0163] After extensive and intensive research, the inventors have screened the aphid nymph and/or adult stage regulation-related gene fragments, and unexpectedly found that for the DS7 gene as shown in SEQ ID NO. 1 or 24, the DS9 gene as shown in SEQ ID NO. 2 or 25, the DS15 gene as shown in SEQ ID NO. 3 or 26, DS25 gene as shown in SEQ ID NO. 4 or 27, DS27 gene as shown in SEQ ID NO. 5 or 28, DS45 gene as shown in SEQ ID NO. 6 or 29, interfering RNA (dsRNA) is synthesized, and the dsRNA is fed by phytophagous insects (such as Aphis) or directly sprayed on the surface of the phytophagous insects, thereby interfering with target genes, inhibiting the expression of target genes, and finally killing aphids. The present invention can also construct plants that can improve insect resistance, and the method of the present invention can also effectively kill aphids, the control effect of aphids is ≥80%, and the dropping rate of insect is ≥70%. On this basis, the present inventor has completed the present invention.

[0164] Terms

[0165] As used herein, the “crop” refers to various plants cultivated in agriculture, including food crops, economic crop (oil crops, vegetable crops, flowers, grasses, trees), industrial crops, feed crops, herb crops, etc., and can grow into large quantities or harvest large areas for profit or provisions (such as cereal, vegetables, cotton, flax, etc.).

[0166] Among them, food crops are mainly rice, corn, beans, potatoes, highland barley, broad beans and wheat; oil crops are mainly oilseeds, vines, big mustard, peanuts, flax, hemp, sunflower, etc.; Vegetable crops mainly include radishes, Chinese cabbage, celery, leeks, garlic, Green onions, carrots, Cucumis melo var flexuosus, lotus vegetables, Jerusalem artichokes, sword bean, coriander, asparagus lettuce, citron day-lily, peppers, cucumbers, tomatoes, coriander, etc.; fruits include pears, green plums, apples, peaches, Apricots, walnuts, plums, cherries, strawberries, crabapple, red dates and other varieties; wild fruits include Pyrus ussuriensis, Armeniaca vulgaris Lam, wild peach, Ziziphus jujuba var. spinosa, prunus maackii, sea-buckthorn, etc.; feed crops are such as corn, green manure, Astragalus sinicus, etc.; medicinal crops are ginseng, Angelica sinensis, Lonicera japonica, mint, mugwort, etc.

[0167] RNA Interference (RNAi)

[0168] As used herein, the term “RNA interfering (RNAi)” refers to: some small double-stranded RNA can efficiently and specifically block the expression of specific genes in vivo, promote mRNA degradation, and induce cells to show a phenotype with a specific gene deletion, which is also called RNA intervention or RNA interference. RNA interference is a highly specific gene silencing mechanism at the mRNA level.

[0169] As used herein, the term “small interfering RNA (siRNA)” refers to a short double-stranded RNA molecule that can target mRNA with homologous complementary sequences to degrade specific mRNA. This process is the RNA interference pathway.

[0170] In the present invention, the basic principle of RNA interference is: using plants as a medium to make insects eat small interfering RNA (siRNA) that can interfere with their gene (such as DS7 genes, DS9 genes, DS15 genes, DS25 genes, DS27 genes, DS45 genes) expression, thereby inhibiting the growth of insects.

[0171] Specifically, the principle is: through aphids' herbivorous feeding or spraying interfering substances on aphids, RNAi enters the insect body and interferes with the RNA of the target gene and inhibits the expression of the target gene, thereby interfering with the normal growth and development of the insect, causing the death of aphids.

[0172] As a preferred way, an intron sequence is used to connect complementary gene sequences at both ends. After being introduced into the cell, a “neck-loop” structure can be produced, and the “neck”-shaped part can be processed into small RNAs of about 21-25nt in the insect body, which can effectively inhibit the expression of target genes.

[0173] As another preferred way, using the T7 primers in Table 1 to amplify respectively, the double-stranded RNA is formed by complementary transcription, and this double-stranded RNA can be directly used to inhibit the expression of the target gene.

[0174] Insect Gene

[0175] As used herein, the term “insect gene” refers to a gene related to insect nymph and/or adult stage regulation. In a preferred embodiment of the present invention, the insect gene is DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, and/or DS45 gene. Low or non-expression of the gene will cause abnormalities in the growth, development, metabolism, reproduction and other processes of the insects, and even lead to the death of the insects.

[0176] As a preferred mode of the present invention, the length of the preferred insect gene fragment of the present invention is at least 21 bp, such as may be 30 bp, 50 bp, 60 bp, 80 bp, 100 bp, 200 bp, 500 bp, 1000 bp or the full length of the gene. When the gene is used in the present invention, it can be a full-length gene or a gene fragment. Preferably, the fragment for the DS7 gene is shown in SEQ ID NO: 24, the fragment for the DS9 gene is shown in SEQ ID NO: 25, the fragment for the DS15 gene is shown in SEQ ID NO. 26, the fragment for the DS25 gene is shown in SEQ ID NO. 27, the fragment for the DS27 gene is shown in SEQ ID NO. 28, the fragment for the DS45 gene is shown in SEQ ID NO. 29. The similarities between these fragments and these genes are 85%-100%, respectively, which can produce the same insecticidal effect.

[0177] The present invention also provides dsRNA for the DS50 gene, the sequence of the DS50 gene is shown in SEQ ID NO:23. Compared with DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, and/or DS45 gene, the control effect of DS50 gene is not good, the maximum is only about 23%.

[0178] The present invention provides interfering RNA targeting insect nymph and/or adult stage regulation-related genes. Insects can take up the interfering RNAi by oral administration of plants sprayed with RNAi or expressing dsRNA constructs or dsRNA, or spraying interfering RNAi directly on the surface of insects.

[0179] The dsRNA construct shown in the present invention is shown in Formula I, and the dsRNA is shown in Formula II. The length of the intervening sequence X used is not particularly limited, as long as it forms a construct with the forward sequence and the reverse sequence and when introduced into the body, it can form a dsRNA represented by Formula II. As a preferred mode of the present invention, the length of the intervening sequence of the present invention is 80-300 bp; more preferably 100-250 bp.

[0180] In a preferred embodiment of the present invention, the construct for expressing insect gene dsRNA is introduced into a host cell. The host cell can be a plant cell, tissue or organ, and the construct can express an insect gene dsRNA in a plant, and dsRNA is processed into siRNA. Generally, the length of siRNA is about 21-25 nt.

[0181] Usually, the construct is located on an expression vector. The expression vector usually also contains a promoter, an origin of replication, and/or a marker gene, etc. Methods well known to those skilled in the art can be used to construct the expression vector required by the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology, etc. The expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as kamamycin, gentamicin, hygromycin, and ampicillin resistance.

[0182] A vector containing the above-mentioned appropriate gene sequence and appropriate promoter or control sequence can be used to transform an appropriate host. In the method of the present invention, the host may be any host suitable for carrying the expression vector and capable of delivering the expression vector to plant cells. Preferably, the host is Agrobacterium.

[0183] Although the insects exemplified in the examples of the present invention are aphids. However, it should be understood that the present invention has no particular limitations on the insects applicable to the present invention. The insects may be any phytophagous insects that can feed on plants, for example, they may be Hemiptera insects.

[0184] The present invention has no particular limitations on the plants applicable to the present invention. Plants eaten by aphids are preferred, such as soybeans, radishes, peach trees, tobacco and the like.

[0185] DS7 Gene

[0186] As used herein, the terms “DS7 gene”, “tubulin alpha chain-like”, and “tubulin a chain” can be used interchangeably, and they are all widely distributed globular proteins and are the basic structural unit of microtubules in cells. It plays an important role in cell movement and division, and is expressed in the nymph stage.

[0187] In the present invention, some glutamic acid residues at the C-terminus of the protein are polyglutamylated, resulting in a polyglutamic acid chain on the γ-carboxyl group. Polyglutamylation plays a key role in spastin (SPAST) microtubule cutting. SPAST preferentially recognizes and acts on microtubules modified with short polyglutamic acid tails: the cleavage activity of SPAST increases as the number of glutamate per tubulin increasing from 1 to 8, but the decrease exceeds the glutamylation threshold.

[0188] Some glutamic acid residues at the C-terminus are monoglycosylated, but not polyglycerinated. Monoglycination is mainly limited to tubulin (cilia and flagella) incorporated into axoneme. Both polypentanoylation and monoglycination can coexist on the same protein on adjacent residues, and reducing the level of glycylation can increase polypentanoylation and interact with each other.

[0189] In one embodiment of the present invention, based on RNAi technology, the DS7 gene is used as a target to screen interfering RNA fragments against the DS7 gene. Preferably, the sequence of the DS7 gene fragment is shown in SEQ ID NO:1 or 24:

TABLE-US-00001 (SEQ ID NO.: 1) ATGCGTGAATGTATCTCTGTACACGTTGGCCAAGCTGGTGTTCAAAT CGGTAATGCCTGCTGGGAATTGTACTGTTTGGAACATGGAATTGCTCCAG ATGGTCAAATGCCATCTGACAAGACCATTGGAGGTGGAGACGACAGCTTC AACACCTTCTTCAGCGAAACTGGCTCAGGCAAACATGTGCCAAGAGCTGT GTTCGTTGATCTCGAACCAACTGTTGTTGATGAGGTAAGAACTGGAACAT ACCGCCAGTTGTTCCACCCTGAACAATTGATCACTGGTAAGGAAGATGCC GCCAACAACTACGCACGTGGACACTACACTATCGGAAAAGAGATTGTTG ATGTTGTTTTGGACCGAATCAGGAAATTGGCTGATCAGTGCACTGGTCTT CAAGGTTTCCTGATCTTCCACTCTTTCGGAGGTGGTACTGGATCTGGTTTC ACATCTTTGTTGATGGAAAGACTCAGCGTTGACTACGGAAAGAAGAGTAA ATTAGAATTCGCCATCTACCCAGCCCCTCAAGTATCCACAGCTGTAGTTG AGCCATACAACTCCATCTTGACCACACATACAACTCTTGAACACAGTGAC TGTGCATTCATGGTCGATAATGAAGCCATCTATGACATCTGCCGTCGTAA TCTCGATATTGAACGTCCAACTTACACTAACTTGAATCGTCTTATTGGCCA GATTGTTTCTTCAATCACAGCTTCTCTCCGTTTCGATGGTGCCCTCAATGT TGACTTGACTGAATTCCAGACCAATTTGGTCCCATACCCCCGTATTCATTT CCCATTGGTCACCTATGCACCAGTCATCTCCGCTGAAAAGGCTTACCATG AACAATTGTCCGTATCAGAAATCACTAACGCTTGTTTTGAACCAGCCAAC CAAATGGTGAAATGTGATCCACGTCATGGCAAATACATGGCTTGTTGCAT GTTGTACCGTGGTGATGTTGTACCCAAAGACGTCAACGCTGCCATTGCTT CCATCAAGACCAAGAGAACAATTCAGTTTGTTGACTGGTGTCCAACTGGT TTCAAAGTTGGTATCAACTACCAACCCCCAACCGTGGTACCCGGTGGTGA CTTGGCTAAGGTACAACGTGCCGTCTGCATGTTGTCCAACACTACAGCTA TTGCTGAAGCTTGGGCTAGGTTGGACCACAAGTTCGACTTGATGTACGCC AAACGTGCTTTCGTCCATTGGTATGTTGGAGAAGGTATGGAAGAAGGAGA ATTCTCTGAAGCTCGTGAGGATTTGGCTGCTCTAGAGAAAGATTACGAAG AGGTTGGCATGGACTCCGTCGAAGGCGAAGGCGAAGGTGGTGAAGAATA C (SEQ ID NO.: 24) TAATACGACTCACTATAGGGAGATCGCCATCTACCCAGCCCCTCAAG TATCCACAGCTGTAGTTGAGCCATACAACTCCATCTTGACCACACATACA ACTCTTGAACACAGTGACTGTGCATTCATGGTCGATAATGAAGCCATCTA TGACATCTGCCGTCGTAATCTCGATATTGAACGTCCAACTTACACTAACTT GAATCGTCTTATTGGCCAGATTGTTTCTTCAATCACAGCTTCTCTCCGTTT CGATGGTGCCCTCAATGTTGACTTGACTGAATTCCAGACCAATTTGGTCC CATACCCCCGTATTCATTTCCCATTGGTCACCTATGCACCAGTCATCTCCG CTGAAAAGGCTTACCATGAACAATTGTCCGTATCAGAAATCACTAACGCT TGTTTTGAACCAGCCAACCAAATGGTGAAATGTGATCCACGTCATGGCAA ATACATGGCTTGTTGCATGTTGTACCGTGGTGATGTTGTACCCAAAGACG TCAACGCTGCCATTGCTTCCATCAAGACCAAGAGAACAATTCAGTTTGTT GACTGGTGTCCAACTGGTTTCAAAGTTGGTATCAACTACCAACCCCCAAC CGTGGTACCCGAGAGGGATATCACTCAGCATAAT

[0190] DS9 gene

[0191] As used herein, the terms “DS9 gene”, “ADP/ATP translocase 3-like”, and “ADP/ATP carrier protein (AAC)” can be used interchangeably, and are responsible for transporting phosphorylated synthesis of ATP to the cytoplasm as the main ability supply of cells, providing power for thermodynamic reaction, which is expressed in the nymph stage.

[0192] In the present invention, this protein is a transport protein that allows the intracellular exchange of adenosine diphosphate (ADP) and mitochondrial adenosine triphosphate (ATP) to cross the mitochondrial inner membrane. Free ADP is transported from the cytoplasm to the mitochondrial matrix, while ATP produced by oxidative phosphorylation is transported from the mitochondrial matrix to the cytoplasm, thereby providing the cell with the main energy.

[0193] In one embodiment of the present invention, based on the RNAi technology, the DS9 gene is used as a target to screen the RNA fragments against the DS9 gene. Preferably, the sequence of the DS9 gene fragment is shown in SEQ ID NO: 2 or 25:

TABLE-US-00002 (SEQ ID NO.: 2) ATGGCCGAAACCAAAGCGCCGAAGGACCCGTATGGTTTCTTGAAGG ACTTCATGGCCGGTGGTATCTCCGCTGCCGTGTCGAAGACCGCCGTGGCT CCGATCGAGCGCGTCAAGCTTATCCTGCAAGTGCAGGCCGCTTCCACGCA GATCGCCGCCGACCAACAGTACAAAGGAATTATGGACTGTTTGGTGAGA ATCCCAAAAGAACAAGGATTTGCCAGTTTCTGGAGAGGTAACTTTGCCAA TGTCATCAGGTACTTCCCAACACAAGCATTGAACTTTGCTTTCAAGGATG TCTACAAACAGGTGTTTATGGACGGTGTGGATAAAAAGACTCAATTCTGG CGGTATTTTGCTGGTAACTTGGCATCTGGTGGTGCTGCTGGAGCAACATC TTTGTGCTTTGTATACCCCCTCGATTACGCACGTACACGATTAGGAGCTGA TGTCGGTAAAGGACCAGCTGAAAGGCAGTTCAAAGGTCTTGGTGATTGTT TAGCCAAAACCGTCAAGTCTGATGGTCCCATTGGTTTGTACCGTGGTTTC ATTGTATCAGTACAGGGTATCATCATCTACCGTGCTGCATACTTTGGATTT TTCGACACAGCTAAGGGAATGTTGCCAGACCCCAAGAATACTCCATTCTT AGTTTCATGGGGTATCGCCCAATTTGTAACAACATTCGCTGGTATTATGTC CTATCCATTTGACACAGTCAGACGTCGTATGATGATGCAATCTGGCCGTG CTGCTGACCAACGCATGTACAAGAGCACATTGGACTGCTGGGGTAAACTT TACAAGAATGAAGGTACATCTGCTTTCTTCAAGGGTGCATTCTCCAACGT ACTCAGAGGTACTGGTGGTGCCTTGGTGTTGGTCTTCTACGACGAACTCA AAAACCTCATG   (SEQ ID NO.: 25) TAATACGACTCACTATAGGGAGAGCCGGTGGTATCTCCGCTGCCGTG TCGAAGACCGCCGTGGCTCCGATCGAGCGCGTCAAGCTTATCCTGCAAGT GCAGGCCGCTTCCACGCAGATCGCCGCCGACCAACAGTACAAAGGAATT ATGGACTGTTTGGTGAGAATCCCAAAAGAACAAGGATTTGCCAGTTTCTG GAGAGGTAACTTTGCCAATGTCATCAGGTACTTCCCAACACAAGCATTGA ACTTTGCTTTCAAGGATGTCTACAAACAGGTGTTTATGGACGGTGTGGAT AAAAAGACTCAATTCTGGCGGTATTTTGCTGGTAACTTGGCATCTGGTGG TGCTGCTGGAGCAACATCTTTGTGCTTTGTATACCCCCTCGATTACGCACG TACACGATTAGGAGCTGATGTCGGTAAAGGACCAGCTGAAAGGCAGTTC AAAGGTCTTGGTGATTGTTTAGCCAAAACCGTCAAGTCTGATGGTCCCAT TGGTTTGTACCGTGGTTTCATTGTATCAGTACAGGGTATCATCATCTACCG TGCTGCATACTTTGGATTTTTCGACACAGCTAAGGGAATGTTGCCAGACC CCA AGAGGGATATCACTCAGCATAAT

[0194] DS15 Gene

[0195] As used herein, the terms “DS15 gene”, “heat shock protein 83-like”, and “heat shock protein 83” can be used interchangeably. They are intracellular molecular chaperone proteins that play an important role in protein interactions, such as assisting in folding and assisting in the establishment of a suitable protein conformation, which is expressed in the nymph stage.

[0196] In the present invention, heat shock proteins (HSP) are a family of proteins produced by cells in response to exposure to stress conditions. They are first associated with heat shock, but are now known in other stresses, including exposure to cold, and in wound healing or tissue remodeling. Many members of this group perform chaperone molecular functions by stabilizing new proteins to ensure proper folding or by helping to fold proteins damaged by cellular stress. Increasement is the regulation of transcription. The significant up-regulation of heat shock proteins is a key part of the heat shock response, which is mainly induced by heat shock factor (HSF).

[0197] In one embodiment of the present invention, based on RNAi technology, the DS15 gene is used as a target to screen RNA fragments against the DS15 gene. Preferably, the sequence of the DS15 gene fragment is shown in SEQ ID NO: 3 or 26:

TABLE-US-00003 (SEQ ID NO.: 3) ATGCCTGAAGACGTTACCATGACTGCATCTGATGATGTTGAGACCTT CGCTTTCCAAGCTGAGATCGCTCAGCTTATGTCCCTCATCATCAACACCTT CTACTCGAACAAAGAAATCTTTTTGCGAGAATTGGTATCCAATTCTTCTG ATGCATTGGACAAAATTCGTTATGAGTCATTGACTGATCCATCCAAATTG GAATCTGGCAAAGATTTACACATTAAAATCATCCCCAATGCGGAAGAAA AAACTCTGACCATTATTGACACTGGTATCGGTATGACCAAAGCTGATCTA GTCAACAACTTGGGAACCATTGCTAAATCTGGTACTAAGGCTTTCATGGA AGCTTTACAAGCTGGAGCTGATATTTCCATGATTGGTCAATTTGGTGTGG GTTTCTATTCCGCCTATCTGGTAGCTGACAAAGTCACTGTTGTTTCCAAAC ACAACGACGATGAACAATATTTGTGGGAATCTGCTGCCGGAGGTTCATTC ACCATCCGTACTGATCCTGGTGAACCATTGGGCCGTGGTACCAAAATTGT CCTTCAAATCAAAGAAGATCAAGCTGAGTTCCTCCAACAAGAAAAAATTA CCAGCATCATCAAGAAGCACTCTCAATTCATTGGCTACCCAATCAAATTA ATCGTTGAGAATGAACGTACCAAAGAAGTCAGCGATGATGAAGCTGAAG AAGAAAAGAAAGATGAAGTTGAAGGTGAAACTGAAGAAGACAAAAAAC CCAAAATTGAGGATGTTGGTGAGGATGAAGACGAAGACAAAAAAGATGA AGACAAAGACAAAAAGAAGAAGAAGACTATTAAAGAAAAGTACTTGGAT GAAGAGGTCTTGAACAAGACAAAACCAATCTGGACACGCAACCCTGATG ATATCAGCCAAGATGAATATGGTGAATTCTACAAATCCTTAACCAATGAC TGGGAAGATCATTTAGCCGTCAAACATTTCTCTGTGGAAGGACAACTTGA ATTCAGAGCATTGTTATTCATTCCCAAGCGTGCGCCTTATGACATGTTTGA GAACAAGAAGAAGAAGAACAACATTAAATTATATGTCCGTCGTGTCTTCA TCATGGACAACTGCGAAGACCTCATGCCAGAATACTTGAACTTCATCAAG GGTGTTGTTGACAGTGAGGATTTGCCGTTGAACATCTCCCGTGAAATGCT CCAACAAAACAAGATCTTGAAAGTTATCAGGAAGAATTTGGTTAAGAAA TGTTTGGAATTGTTCGAGGAATTGGCTGAAGACAAGGACAACTACAAGA AATTGTACGAACAGTTCAGCAAGAACTTGAAACTTGGAATCCACGAAGAT AGCCAAAACAGAAAGAAACTCTCAGACTTGTTGAGATTCCACTCCTCAGC CAGTGGTGACGAATCATGCTCCCTTAAGGAGTATGTTGCACGTATGAAGC CAAATCAAACCCACATTTACTACATCACAGGTGAAAGCCGTGAACAAGTA TCCAACTCTTCATTCGTTGAACGTGTCAAGAAACGTGGTTTTGAAGTTATT TACATGACTGAACCCATTGATGAATACGTTGTCCAACAAATGAAAGAATA TGACGGCAAGAACTTGGTATCTGTCACTAAAGAAGGTTTGGACTTGCCTG AAACCGATGAAGAAAAGAAGAAGCGCGAGGATGATCAATCCAGATTTGA AAAATTGTGCAAAGTTGTTAAGGACATTTTGGACAAGAAAGTTGAGAAG GTTGTCATCAGTAACAGACTTGTTGAGTCTCCCTGTTGCATTGTCACATCT CAGTATGGTTGGACTGCCAACATGGAACGTATCATGAAGGCACAAGCACT CAGAGATTCATCTACCATGGGTTATATGTCTGCCAAAAAACACTTGGAAA TCAACCCTGACCACCCGATCATTGAAACACTCAGACAAAAGGCTGAAGCT GATTGCAACGACAAGGCTGTCAGAGACTTGGTCATGCTTTTGTTCGAGAC AAGTTTGTTGTCATCTGGTTTTGGACTTGAAGACCCACAAGTTCACGCTTC TAGAATCCACAGAATGATCAAATTGGGTTTGGGCATTGATGAAGATTTGC CAGTAGTTGAAGAAAAATCTGCTGAAGTTGAAGCCTCCGAGCCTGTTGTT GAAGCTGATGCTGAAGATTCTTCTCGCATGGAAGAAGTTGAT (SEQ ID NO.: 26) TAATACGACTCACTATAGGGAGATGGTGAACCATTGGGCCGTGGTAC CAAAATTGTCCTTCAAATCAAAGAAGATCAAGCTGAGTTCCTCCAACAAG AAAAAATTACCAGCATCATCAAGAAGCACTCTCAATTCATTGGCTACCCA ATCAAATTAATCGTTGAGAATGAACGTACCAAAGAAGTCAGCGATGATG AAGCTGAAGAAGAAAAGAAAGATGAAGTTGAAGGTGAAACTGAAGAAG ACAAAAAACCCAAAATTGAGGATGTTGGTGAGGATGAAGACGAAGACAA AAAAGATGAAGACAAAGACAAAAAGAAGAAGAAGACTATTAAAGAAAA GTACTTGGATGAAGAGGTCTTGAACAAGACAAAACCAATCTGGACACGC AACCCTGATGATATCAGCCAAGATGAATATGGTGAATTCTACAAATCCTT AACCAATGACTGGGAAGATCATTTAGCCGTCAAACATTTCTCTGTGGAAG GACAACTTGAATTCAGAGCATTGTTATTCATTCCCAAGCGTGCGCCT AGAGGGATATCACTCAGCATAAT

[0198] DS25 Gene

[0199] As used herein, the terms “DS25 gene”, “eukaryotic initiation factor 4A-like”, and “eukaryotic initiation factor complex type of 4A” can be used interchangeably, it is a helicase that unwinds double-stranded RNA and is also a functional protein necessary for ribosomal subunit binding, which is expressed in the nymph stage.

[0200] In the present invention, the eukaryotic initiation factor complex forms a ternary complex with GTP and the initiator Met-tRNA. This process is regulated by guanine nucleotide exchange and phosphorylation, and is the main regulatory element of the bottleneck of gene expression. Before the translation progresses to the extension stage, many initiation factors must promote the synergy of ribosomes and mRNA, and ensure that the 5′UTR of the mRNA is sufficiently lacking in secondary structure. The fourth group of eukaryotic initiation factors promotes this combination; it is of significance in the normal regulation of translation and the transformation and progression of cancer cells.

[0201] In one embodiment of the present invention, based on RNAi technology, the DS25 gene is used as a target to screen RNA fragments against the DS25 gene. Preferably, the sequence of the DS25 gene fragment is shown in SEQ ID NO: 4 or 27:

TABLE-US-00004 (SEQ ID NO.: 4) ATGAATGCTAATGAGACGAAAAATGGACCTCCTAGTGAAACCAATG ACTACTCGGGACCACCTGGCATGGACGTCGGTGGAACTATTGAGTCTGAC TGGAAAGAAGTGGTGGATAACTTTGATGAGATGAATTTAAAAGAAGAAT TGTTGCGTGGTATTTATGGATATGGTTTTGAAAAGCCATCAGCTATTCAAC AACGTGCTATTTTGCCGTGCATCAAGGGACATGATGTCATTGCTCAGGCC CAATCTGGTACTGGCAAGACAGCTACTTTTTCCATTTCTATTCTCCAACAA ATTGATACAAGTTTGAATGAGTGCCAAGCACTTATTTTGGCACCAACACG TGAATTGGCTCAACAGATTCAAAAGGTGGTCATTGCTTTGGGTGATTTCA TGAAAGCTGATTGTCATGCTTGCATTGGCGGTACAAACGTTCGTGATGAC ATGCGTAAGCTGGATACTGGATCCCATGTAGTTGTTGGAACTCCTGGCCG TGTTTATGACATGATTGCTAGAAAATCCCTAAGAACTCAATTTATCAAGA TATTTGTGTTGGACGAAGCTGATGAAATGTTGTCTCGAGGTTTCAAAGAT CAAATTAAAGAGGTGTTCAAGTTCCTCGAAGAAGATATTCAGGTCATTCT GTTGTCTGCTACAATGCCCGAGGACGTTTTGGATGTGAGCACTCATTTCAT GCGTAATCCAGTACGCATTCTTGTTCAAAAGGAAGAACTGACATTGGAAG GTATCAAACAGTTTTACATCAATGTTACCAAAGAAGAATGGAAGTTTGAC ACTCTATGTGATTTGTACGACACTCTTAGTATCACCCAGGCTGTGATCTTC TGTAACACACGTCGTAAGGTAGAGTGGTTGACTGAAAATATGCGTTTGAA AACATTTACTGTATCAGCTATGCATGGAGAAATGGACCAACGTCAACGTG AGCTAATTATGCGTCAATTCCGTTCTGGCTCTAGTCGTGTTCTAATTACCA CTGATTTGTTGGCTCGAGGCATTGATGTACAACAAGTTTCTCTGGTCATCA ATTACGATTTGCCGTCCAATCGTGAAAACTATATTCACAGGATTGGACGT TCTGGCCGTTTCGGTCGTAAAGGAGTCGCCATTAATTTTATCACCGAAGA CGACAAAAGAGCTATGAAGGATATTGAATCATTTTACAACACTCACGTGC TCGAGATGCCACAGAATGTGGCCGATTTGCTG (SEQ ID NO.: 27) TAATACGACTCACTATAGGGAGACCACCTGGCATGGACGTCGGTGG AACTATTGAGTCTGACTGGAAAGAAGTGGTGGATAACTTTGATGAGATGA ATTTAAAAGAAGAATTGTTGCGTGGTATTTATGGATATGGTTTTGAAAAG CCATCAGCTATTCAACAACGTGCTATTTTGCCGTGCATCAAGGGACATGA TGTCATTGCTCAGGCCCAATCTGGTACTGGCAAGACAGCTACTTTTTCCAT TTCTATTCTCCAACAAATTGATACAAGTTTGAATGAGTGCCAAGCACTTA TTTTGGCACCAACACGTGAATTGGCTCAACAGATTCAAAAGGTGGTCATT GCTTTGGGTGATTTCATGAAAGCTGATTGTCATGCTTGCATTGGCGGTAC AAACGTTCGTGATGACATGCGTAAGCTGGATACTGGATCCCATGTAGTTG TTGGAACTCCTGGCCGTGTTTATGACATGATTGCTAGAAAATCCCTAAGA ACTCAATTTATCAAGATATTTGTGTTGGACGAAGCTGATGAAATGTTGTC TCGAGGTTTCAAAGATCAAATTAAAGAGGTGTTCAAGTTCCTCGAAGAAG ATATTCAGGTCATTCTGTTGTCTGCTACAATGCCCGAGGACGT AGAGGGATATCACTCAGCATAAT

[0202] DS27 Gene

[0203] As used herein, the terms “DS27”, “troponin T-like isoform 3”, and “troponin type 3” can be used interchangeably. It mediates Ca ion channels and regulates the contraction regulation function of insect striated muscle, which is expressed in the nymph and adult stages.

[0204] In the present invention, troponin is attached to the protein tropomyosin and is located in the grooves between actin filaments in muscle tissue. In a relaxed muscle, tropomyosin blocks the attachment site of the myosin cross bridge, thereby preventing contraction. When muscle cells are stimulated to contract by an action potential, calcium channels open in the sarcoplasm membrane and release calcium into the sarcoplasm. Some of this calcium attaches 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 bridges to form and start to contract muscles.

[0205] Troponin activation. Troponin C (red) binds to Ca2.sup.+ and stabilizes the activated state, wherein troponin I (yellow) no longer binds to actin. Troponin T (blue) fixes the complex to tropomyosin.

[0206] Troponin is found in skeletal muscle and heart muscle, but the specific version of troponin differs in different types of muscles. The main difference is that the TnC subunit of troponin has four calcium binding sites in skeletal muscle, but only three in cardiac muscle. Opinions on the actual content of calcium bound to troponin vary from expert to source.

[0207] In one embodiment of the present invention, based on the RNAi technology, the DS27 gene is used as a target to screen the RNA fragments against the DS27 gene. Preferably, the sequence of the DS27 gene fragment is shown in SEQ ID NO: 5 or 28:

TABLE-US-00005 (SEQ ID NO.: 5) ATGTCCGACGAAGAAGAAGTGTACACTGATTCCGAAGAAGAAACGC AACCGGAGCCTGAAAAAAGCAAAGATGGAGATGGAGATCCCGAATTCGT TAAGAGGCAAGAATTAAAATCTTCAGCCTTAGACGAACAGCTTAAAGAG TACATCCAAGAATGGCGCAAACAGCGGTCAAAGGAAGAAGACGACTTAA AGAAGTTGAAGGAAAAACAGGCCAAGCGCAAGGTTATGCGAGCGGAAG AAGAGAAGAGAATGGCCGAGAGAAAGAAGCAAGAAGAAGAACGCAGAC AGAGAGAAGTCGAGGAAAAGAAACAAAAGGACATCGAAGAAAAACGTA AACGTCTAGAAGAGGCCGAGAAAAAACGGCAAGCTATGATGGCTGCTCT TAAGGAACAAACCAATAAATCTAAAGGACCAAATTTCACCATCAGCAAA AAAGAAGGTGCGTTGAGTATGACTTCTGCCCAACTTGAACGCAATAAAAC CAGAGAACAGATCGAAGAAGAAAAGAAAATATCGTTGAGCTTCAGAATC AAACCTTTGAATATTGAAGGATTCTCTGTGCAAAAACTCCAATTCAAAGC TACCGAACTCTGGGACCAGATCATCAAGTTGGAAACAGAAAAATACGAT TTGGAGGAAAGGCAAAAGAGACAAGATTACGACTTGAAAGAGTTGAAAG AACGTCAGAAGCAACAACTCCGCCACAAGGCTCTGAAGAAAGGTCTCGA CCCCGAAGCCCTAACCGGCAAATACCCACCCAAGATCCAAGTCGCTTCCA AGTACGAGAGGCGAGTTGACACGAGGTCTTATGATGACAAAAAGAAGCT GTTCGAAGGAGGTTATATGGAAACCACTAAAGAATCAATGGAAAAACAA TGGACAGAAAAAAGTGACCAATTCGGTGGCCGCGCTAAAGGACGATTAC CGAAATGGTTCGGCGAACGTCCGGGCAAGAAGAAGGATGACCCAGACAC ACCCGAAGAGGAAGAGCTCAAGAAAAACGAGGAAGACGAAGAACCGTT TGGCCTCGACGACGAAGAAGCTGAAGAAGAAGTTGAAGAGGAAGAAGA GGAGGAAGAAGAAGAGGAAGAGGAGGAGGAAGAGGAAGAAGAGGAAG AAGAAGAAGAGGAAGAGGAAGAAGAAGAAGAA (SEQ ID NO.: 28) TAATACGACTCACTATAGGGAGAGCGCAAGGTTATGCGAGCGGAAG AAGAGAAGAGAATGGCCGAGAGAAAGAAGCAAGAAGAAGAACGCAGAC AGAGAGAAGTCGAGGAAAAGAAACAAAAGGACATCGAAGAAAAACGTA AACGTCTAGAAGAGGCCGAGAAAAAACGGCAAGCTATGATGGCTGCTCT TAAGGAACAAACCAATAAATCTAAAGGACCAAATTTCACCATCAGCAAA AAAGAAGGTGCGTTGAGTATGACTTCTGCCCAACTTGAACGCAATAAAAC CAGAGAACAGATCGAAGAAGAAAAGAAAATATCGTTGAGCTTCAGAATC AAACCTTTGAATATTGAAGGATTCTCTGTGCAAAAACTCCAATTCAAAGC TACCGAACTCTGGGACCAGATCATCAAGTTGGAAACAGAAAAATACGAT TTGGAGGAAAGGCAAAAGAGACAAGATTACGACTTGAAAGAGTTGAAAG AACGTCAGAAGCAACAACTCCGCCACAAGGCTCTGAAGAAAGGTCTCGA CCCCGAAGCCCTAACCGAGAGGGATATCACTCAGCATAAT

[0208] DS45 Gene

[0209] As used herein, the terms “DS45 gene”, “Y-box protein Ct-p40-like”, and “y box binding protein Ct-p40-like” can be used interchangeably, and affect cell differentiation and cytoskeleton formation. Deletion of it will inhibit signal transduction pathways inside and outside the cell, involved in DNA damage repair and transcription, and it is expressed in the nymph stage.

[0210] In one embodiment of the present invention, based on RNAi technology, the DS45 gene is used as a target to screen RNA fragments directed against the DS45 gene. Preferably, the sequence of the DS45 gene fragment is shown in SEQ ID NO: 6 or 29:

TABLE-US-00006 (SEQ ID NO.: 6) ATGGCGGAACAAGTCGGCGAGAGGAGGACGGAACGGCCGCCGCAG AAGCCCGTGGCCCAAAAGCCGGTCATATCTGTGAAAGTCACCGGCGTTGT TAAATGGTTCAACGTCAAAAGCGGTTATGGTTTTATTAATCGTAATGATA CAAAAGAAGATATATTTGTACATCAGTCTGCTATTATCAAGAACAACCCT AAGAAAATTGTACGCAGTGTCGGTGATGGAGAAACTGTAGAATTTGACGT TGTTGAGGGCGAAAAAGGTCACGAAGCAGCAAATGTTACTGGTCCAGAT GGAGAAGCTGTTAAAGGATCACCTTATGCAGCTGAAAGAAGAAGAAATA ACTATCGTCAGTGGTTTTATGGACGCCGTCCTAATACCCGTCCAAGAAAT GGTGGTCAACCTCCAAGAGATGGTAGTCCAAGTGGTGACAAGGAAGAAA CTGAAAATGAAGTAGGAGAACAACCAAGACGTTACCGCCAGCCACGTCA ACAGAATTGGTATAATAGCTATCGTGGAAATCGAAGAGGTCCACCACCA AATAGAGGAGAAGGTGGTGATTACAATGGTGGAGATAATTATGGATATG ATAGTTCACCTCCTGGTAGAGGCAGAGGTCGTGGGATGGGTGCGCCTAGA CGTTTCTTTAGACGTGGCAGTGGATTTAGAGGGAGCCGTGGAACAGGTGG TCCACCCAGAAGACCATATCAAGATGAAAATCAGGACAATGAATATAAT CAAAGTGATGAAAATGGAGCAAATAGACCTCGTCCTCGCTATCGCCGCCG CAATAATCGTTCTAGAGCGAGAAGTGATGGTCCTCCAAGAGCCAATAGCC AAAGTGACAATGAATCTAAACAAAAAAACTTTGGAGGAGAAGCATTGGA ACTGGATGAAAGTAGTCATGCT (SEQ ID NO.: 29) TAATACGACTCACTATAGGGAGAGCAGAAGCCCGTGGCCCAAAAGC CGGTCATATCTGTGAAAGTCACCGGCGTTGTTAAATGGTTCAACGTCAAA AGCGGTTATGGTTTTATTAATCGTAATGATACAAAAGAAGATATATTTGT ACATCAGTCTGCTATTATCAAGAACAACCCTAAGAAAATTGTACGCAGTG TCGGTGATGGAGAAACTGTAGAATTTGACGTTGTTGAGGGCGAAAAAGG TCACGAAGCAGCAAATGTTACTGGTCCAGATGGAGAAGCTGTTAAAGGA TCACCTTATGCAGCTGAAAGAAGAAGAAATAACTATCGTCAGTGGTTTTA TGGACGCCGTCCTAATACCCGTCCAAGAAATGGTGGTCAACCTCCAAGAG ATGGTAGTCCAAGTGGTGACAAGGAAGAAACTGAAAATGAAGTAGGAGA ACAACCAAGACGTTACCGCCAGCCACGTCAACAGAATTGGTATAATAGCT ATCGTGGAAATCGAAGAGGTCCACCACCAAATAGAGGAGAAGGTGGTGA TTACAATGGTGGAGATAATTATGGATATGATAGTTCACCTCCTGGTAGAG GCAGAGGTCGTGGGATGGGTGCGCCTAAGAGGGATATCACTCAGCATAA T

[0211] dsRNA Construct and its Application

[0212] The present invention provides a dsRNA construct. The dsRNA construct is double-stranded, and its positive or negative strand contains a structure as shown in Formula I:

Seq.sub.forward-X-Seq.sub.reverse  Formula I

[0213] wherein

[0214] Seq.sub.forward is a nucleotide sequence of insect nymph and/or adult stage regulation-related gene or fragment;

[0215] Seq.sub.reverse is a nucleotide sequence that is basically complementary to Seq.sub.forward;

[0216] X is an intervening sequence located between the Seq.sub.forward and the Seq.sub.reverse, and the intervening sequence is not complementary to the Seq.sub.forward and the Seq.sub.reverse,

[0217] wherein the insect nymph and/or adult stage regulation-related gene is selected from the group consisting of DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene and a combination thereof.

[0218] In a preferred embodiment of the present invention, the length of the Seq.sub.forward and Seq.sub.reverse is at least 50 bp.

[0219] In a preferred embodiment of the present invention, the dsRNA construct is ingested by insects (such as aphids) to form a dsRNA of Formula II,

##STR00004##

[0220] wherein

[0221] Seq′.sub.forward is a RNA sequence or sequence fragment corresponding to the Seq.sub.forward sequence;

[0222] Seq′.sub.reverse is a sequence that is basically complementary to the Seq′.sub.forward;

[0223] X′ is none; or is an intervening sequence located between Seq′.sub.forward and Seq′.sub.reverse, and the intervening sequence is not complementary to Seq′.sub.forward and Seq′.sub.reverse,

[0224] ∥ represents the hydrogen bond formed between Seq.sub.forward and Seq.sub.reverse.

[0225] The present invention also provides the use of the dsRNA construct, which is used to: (1) improve the control effect of aphids; and/or (2) increase the dropping rate of insect population; and/or (3) reduce the expression level of nymph and/or adult stage regulation-related gene; (4) reduce the initial number of insect population; and/or (5) reduce the damage rate of plants; and/or (6) reduce the damage degree of crops and improve the quality of crop products.

[0226] dsRNA and its Applications

[0227] The present invention also provides a dsRNA as shown in Formula II,

##STR00005##

[0228] wherein

[0229] Seq′.sub.forward is a RNA sequence or sequence fragment corresponding to Seq′.sub.forward sequence;

[0230] Seq′.sub.reverse is a sequence that is basically complementary to the Seq′.sub.forward;

[0231] X′ is none; or is an intervening sequence located between Seq′.sub.forward and Seq′.sub.reverse, and the intervening sequence is not complementary to Seq′.sub.forward and Seq′.sub.reverse;

[0232] ∥ represents the hydrogen bond formed between Seq.sub.forward and Seq.sub.reverse.

[0233] In another preferred embodiment, the length of the intervening sequence X is 0-300 bp, preferably 100 bp.

[0234] 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 DS45 gene is shown in SEQ ID NO. 6.

[0235] In another preferred embodiment, the insects are phytophagous insects, preferably from Hemiptera insects, and most preferably from Aphis.

[0236] The present invention also provides the use of the dsRNA, which is used to: (1) improve the control effect of aphids; and/or (2) increase the dropping rate of insect population; and/or (3) reduce the expression level of nymph and/or adult stage regulation-related gene; and/or (4) reduce the initial number of insect population; and/or (5) reduce the damage rate of plants.

[0237] Composition and its Application

[0238] The present invention also provides a composition. In response to the problem of efficiently killing aphids, the inventor has developed RNAi fragments for target genes based on RNAi technology, and improved the control effect of aphids and the dropping rate of insect population by feeding insects or spraying insects directly, making RNAi have the effect of inhibiting gene expression, and finally achieving the purpose of efficiently killing aphids. The method of the present invention is efficient, convenient, fast, accurate and pollution-free.

[0239] The composition includes a dsRNA construct and/or dsRNA, and an effective amount of a carrier acceptable for insect feeding. In another preferred embodiment, the composition is a composition used to induce or cause the death of aphid nymphs and/or adult stage.

[0240] In another preferred embodiment, the dsRNA has the following sequence:

[0241] dsRNA1: having a sequence corresponding to SEQ ID NO. 1 or 24;

[0242] dsRNA2: having a sequence corresponding to SEQ ID NO. 2 or 25;

[0243] dsRNA3: having a sequence corresponding to SEQ ID NO. 3 or 26;

[0244] dsRNA4: having a sequence corresponding to SEQ ID NO. 4 or 27;

[0245] dsRNA5: having a sequence corresponding to SEQ ID NO. 5 or 28;

[0246] dsRNA6: having a sequence corresponding to SEQ ID NO. 6 or 29.

[0247] The present invention also provides a use of the composition, which is selected from the following group:

[0248] (1) improving the control effect of aphids; and/or

[0249] (2) increasing the dropping rate of insect population; and/or

[0250] (3) decreasing the expression level of nymph and/or adult stage regulation-related gene; and/or

[0251] (4) reducing the initial number of insect population; and/or

[0252] (5) reducing plant damage rate; and/or

[0253] (6) reducing crop damage degree and improving the quality of crop products.

[0254] In a preferred embodiment of the present invention, the composition is an aqueous solution, and the pH is usually about 5-8, preferably, the pH is about 6-8.

[0255] As used herein, the term “effective amount” or “effective dose” refers to an amount that can produce function or activity for feeding the insect and can be accepted by the insect. Preferably, the content of dsRNA1 is about 1-500 ng/μl, preferably, 5-300 ng/μl, more preferably, 50-150 ng/μl; the content of dsRNA2 is about 1-500 ng/μl, preferably, 5-300 ng/μl, more preferably, 50-150 ng/μl; the content of dsRNA3 is about 1-500 ng/μl, preferably, 5-300 ng/μl, more preferably, 50-150 ng/μl; the content of dsRNA4 is about 1-500 ng/μl, preferably, 5-300 ng/μl, more preferably, 50-150 ng/μl; the content of dsRNA5 is about 1-500 ng/μl, preferably, 5-300 ng/μl, more preferably, 50-150 ng/μl; the content of dsRNA6 is about 1-500 ng/μl, preferably, 5-300 ng/μl, more preferably, 50-150 ng/μl. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through a feeding experiment or a spray experiment).

[0256] As used herein, “insect feeding acceptable” ingredients are suitable for the insects without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit/risk ratio.

[0257] As used herein, the term “carrier” includes various excipients and diluents. Such carriers include (but are not limited to): water, saline, buffer, glucose, glycerol, ethanol, and combinations thereof.

[0258] The composition of the present invention can be directly sprayed, fed, or made into an injection form, for example, prepared by conventional methods with water, physiological saline or an aqueous solution containing glucose and other adjuvants. The composition is preferably manufactured under sterile or RNase-free conditions.

[0259] The main advantages of the present invention include:

[0260] 1) The dsRNA designed for specific target genes of the present invention can effectively kill aphids, improve the control effect of aphids (≥80%) and the dropping rate of insect population (≥70%);

[0261] 2) The obtained dsRNA can be directly used to kill aphids and is convenient to use;

[0262] 3) Low production cost, good stability, suitable for mass production;

[0263] 4) Good environmental compatibility, green and pollution-free, and safe for humans and animals.

[0264] The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental methods in the following examples without particular conditions, they are performed under routine conditions (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or as instructed by the manufacturer. Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.

[0265] General Methods and Materials

[0266] 1. Aphid Breeding and Biological Testing

[0267] The green peach aphid (Myzus persicae) was cultivated and tested on radish seedlings cultivated in indoor greenhouses or plastic greenhouses, and the soybean aphid (Aphid glycine) was cultivated and tested on soybean seedlings cultivated in indoor greenhouses or plastic greenhouses. The temperature of the incubation room was 25±1° C., the relative humidity was 40-60%, and the photoperiod was 12 h: 12 h.

[0268] Before the test, a certain number of aphids were inoculated on the target plant and counted. After spraying a certain concentration of dsRNA, the counting was performed on day 1, 3, and 5 respectively. The test for each gene was repeated 10 times. According to the counting results, the control effect of the target gene was determined.

[0269] 2. Statistical Methods of Control Effect

[0270] In this study, two statistical methods were used to evaluate the effect of target dsRNA on aphids.

[0271] The first method, the control effect, is calculated as follows:

Control effect (%)=(1−CK0×PT1/CK1×PT0)×100

[0272] wherein PT0: the number of insects before drug administration in the treatment area; PT1: the number of insects after drug administration in the treatment area; [0273] CK0: the number of insects before drug administration in the control area; CK1: the number of insects after drug administration in the control area.

[0274] The second method, the dropping rate of insect population, is calculated as follows:

the dropping rate of insect population (%)=[(number of insects before drug administration−number of insects after drug administration)/number of insects before drug administration]×100

[0275] 3. RNA Extraction and Quality Test

[0276] The total RNA was extracted using TRIzol® Reagent (Invitrogen), and the operation was performed according to the instructions: 1) adding 50-100 mg of Ostrinia nubilalis sample that was well ground into 1 mL of TRIzol, mixed well, and placed at room temperature for 5 minutes. 2) adding 200 μL of chloroform, shaked and mixed, and placed at room temperature for 3 minutes. 3) centrifuged at 12,000 rpm (4° C.) for 15 minutes, transferring the upper aqueous phase to another new centrifuge tube, adding 500 μL of pre-cooled isopropanol, shaked and mixed, and placed at room temperature for 10 minutes. 4) centrifuged at 12 000 rpm (4° C.) for 15 minutes, and carefully aspirating the supernatant. 5) washed with 500 μL of pre-cooled 75% ethanol and mixed gently with a vortex for 10 sec. 6) centrifuged at 12 000 rpm (4° C.) for 2 minutes, carefully aspirating the supernatant and drying it at room temperature for 5 minutes, adding an appropriate amount of DEPC sterilized water to dissolve it, and obtaining a total RNA sample. Detecting the absorbance under a spectrophotometer, detecting the total RNA quality by 1% agar gel electrophoresis, and storing it at −80° C., ready for use.

[0277] 4. dsRNA Synthesis

[0278] Using the kit MEGAscript® RNAi Kit (Ambion) to synthesize dsRNA, and performing experimental operations according to the instructions. A T7 promoter sequence was added to the 5′end of the primer of the amplification template to facilitate subsequent dsRNA synthesis. Using pPigbac A3 EGFP as a template for the synthesis of control group dsEGFP, dsEGFP was used as a negative control to participate in the treatment of the experimental group in subsequent experiments. See Appendix S3 for the primers used to synthesize dsRNAs. During the synthesis process, template DNA and single-stranded RNA were removed with DNase and RNase, respectively.

[0279] 5. Detection of Gene Expression (q-RT-PCR)

[0280] Using TRIzol® reagent (Invitrogen) for total RNA extraction, and the steps were strictly in accordance with the operation manual. Taking 1 μg of total RNA and using the kit ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO) to synthesize the first strand of cDNA. The kit used for the RT-qPCR reaction was SYBR® Premix Ex Taq™ II (Takara), and the primers were detailed in Appendix S3. For each gene sample, the detection was repeated 3 times, the expression level analysis selected the expression level of 18S rRNA for normalization. Data analysis was referred to 2.sup.−ΔΔCT Method (Livak & Schmittgen, 2001). The corresponding value was obtained by calculating the mean value and standard error. In order to eliminate individual differences, the samples of each experimental group were a sample pool formed by 2 surviving larvae after treatment, and each experimental group was subjected to three biological replicates.

Example 1 Target Gene Sequence and dsRNA Synthesis

[0281] In order to screen effective target genes of aphids based on RNA interference technology, transcriptome sequencing was performed on the green peach aphid (Myzus persicae) and the soybean aphid (Aphid glycine) (the sampling and sequencing analysis methods of the two aphids were same). After extracting total RNA from aphids at different developmental stages, the same amount of RNA was taken and mixed to form the total RNA for the entire developmental stage of aphids and sent to Shenzhen BGI Technology Services Co. LTD for transcriptome sequencing using the Illumina Hiseq2000 platform. After removing the adapters from the sequencing results, using the denove program to assemble, and then performing functional annotations on Unigene. In this study, target gene fragments were selected from these functionally annotated Unigenes for amplification and dsRNA was synthesized. Through a large number of screenings, the primers of the present invention for the amplification and synthesis of 6 target genes, the exogenous control gene GFP and the endogenous control gene DS50 from soybean aphid were shown in Table 1. The DNA sequences of the 6 gene fragments were shown in Table 2. Wherein ds7, ds9, and ds15 were against aphids, especially the green peach aphid, and ds25, ds27, and ds45 were against aphids, especially the soybean aphid.

TABLE-US-00007 TABLE 1 Amplification and synthesis of the primer sequence of the target gene dsRNA. SEQ SEQ ID ID Name Primer F NO.: Primer R NO.: dsGFP TAATAC GACTCA CTATAG GGAGA 7 TAATAC GACTCA CTATAG GGAGA 8 GACGAC GGCAAC TACA ACTCCA GCAGGA CCAT ds7 TAATAC GACTCA CTATAG GGAGA 9 TAATAC GACTCA CTATAG GGAGA 10 TCGCCA TCTACC CAGCCC CT CGGGTA CCACGG TTGGGG GT ds9 TAATAC GACTCA CTATAG GGAGA 11 TAATAC GACTCA CTATAG GGAGA 12 GCCGGT GGTATC TCCGCT GC TGGGGT CTGGCA ACATTC CCT ds15 TAATAC GACTCA CTATAG GGAGA 13 TAATAC GACTCA CTATAG GGAGA 14 TGGTGA ACCATT GGGCCG TGG AGGCGC ACGCTT GGGAAT GA ds25 TAATAC GACTCA CTATAG GGAGA 15 TAATAC GACTCA CTATAG GGAGA 16 CCACCT GGCATG GACGTC GG ACGTCC TCGGGC ATTGTA GCA ds27 TAATAC GACTCA CTATAG GGAGA 17 TAATAC GACTCA CTATAG GGAGA 18 GCGCAA GGTTAT GCGAGC GG CGGTTA GGGCTT CGGGGT CG ds45 TAATAC GACTCA CTATAG GGAGA 19 TAATAC GACTCA CTATAG GGAGA 20 GCAGAA GCCCGT GGCCCA AA TAGGCG CACCCA TCCCAC GA ds50 TAATAC GACTCA CTATAG GGAGA 21 TAATAC GACTCA CTATAG GGAGA 22 CGTGTC TGAGGC GGTTGC CA TGATCT TGGCCC GGAGAG CCGG

TABLE-US-00008 TABLE 2 Sequence fragments of 6 target genes. SEQ ID Name Sequence NO.: ds7 >CL1054.Contig1_TY tubulin alpha 1 chain-like ATGCGTGAATGTATCTCTGTACACGTTGGCCAA GCTGGTGTTCAAATCGGTAATGCCTGCTGGGAA TTGTACTGTTTGGAACATGGAATTGCTCCAGAT GGTCAAATGCCATCTGACAAGACCATTGGAGG TGGAGACGACAGCTTCAACACCTTCTTCAGCGA AACTGGCTCAGGCAAACATGTGCCAAGAGCTG TGTTCGTTGATCTCGAACCAACTGTTGTTGATG AGGTAAGAACTGGAACATACCGCCAGTTGTTCC ACCCTGAACAATTGATCACTGGTAAGGAAGAT GCCGCCAACAACTACGCACGTGGACACTACAC TATCGGAAAAGAGATTGTTGATGTTGTTTTGGA CCGAATCAGGAAATTGGCTGATCAGTGCACTG GTCTTCAAGGTTTCCTGATCTTCCACTCTTTCGG AGGTGGTACTGGATCTGGTTTCACATCTTTGTT GATGGAAAGACTCAGCGTTGACTACGGAAAGA AGAGTAAATTAGAATTCGCCATCTACCCAGCCC CTCAAGTATCCACAGCTGTAGTTGAGCCATACA ACTCCATCTTGACCACACATACAACTCTTGAAC ACAGTGACTGTGCATTCATGGTCGATAATGAAG CCATCTATGACATCTGCCGTCGTAATCTCGATA TTGAACGTCCAACTTACACTAACTTGAATCGTC TTATTGGCCAGATTGTTTCTTCAATCACAGCTTC TCTCCGTTTCGATGGTGCCCTCAATGTTGACTTG ACTGAATTCCAGACCAATTTGGTCCCATACCCC CGTATTCATTTCCCATTGGTCACCTATGCACCA GTCATCTCCGCTGAAAAGGCTTACCATGAACAA TTGTCCGTATCAGAAATCACTAACGCTTGTTTT GAACCAGCCAACCAAATGGTGAAATGTGATCC ACGTCATGGCAAATACATGGCTTGTTGCATGTT GTACCGTGGTGATGTTGTACCCAAAGACGTCAA CGCTGCCATTGCTTCCATCAAGACCAAGAGAAC AATTCAGTTTGTTGACTGGTGTCCAACTGGTTT CAAAGTTGGTATCAACTACCAACCCCCAACCGT GGTACCCGGTGGTGACTTGGCTAAGGTACAAC GTGCCGTCTGCATGTTGTCCAACACTACAGCTA TTGCTGAAGCTTGGGCTAGGTTGGACCACAAGT TCGACTTGATGTACGCCAAACGTGCTTTCGTCC ATTGGTATGTTGGAGAAGGTATGGAAGAAGGA GAATTCTCTGAAGCTCGTGAGGATTTGGCTGCT CTAGAGAAAGATTACGAAGAGGTTGGCATGGA CTCCGTCGAAGGCGAAGGCGAAGGTGGTGAAG AATAC ds9 >CL3025.Contig1_TY ADP/ATP 2 translocase 3-like ATGGCCGAAACCAAAGCGCCGAAGGACCCGTA TGGTTTCTTGAAGGACTTCATGGCCGGTGGTAT CTCCGCTGCCGTGTCGAAGACCGCCGTGGCTCC GATCGAGCGCGTCAAGCTTATCCTGCAAGTGCA GGCCGCTTCCACGCAGATCGCCGCCGACCAAC AGTACAAAGGAATTATGGACTGTTTGGTGAGA ATCCCAAAAGAACAAGGATTTGCCAGTTTCTGG AGAGGTAACTTTGCCAATGTCATCAGGTACTTC CCAACACAAGCATTGAACTTTGCTTTCAAGGAT GTCTACAAACAGGTGTTTATGGACGGTGTGGAT AAAAAGACTCAATTCTGGCGGTATTTTGCTGGT AACTTGGCATCTGGTGGTGCTGCTGGAGCAACA TCTTTGTGCTTTGTATACCCCCTCGATTACGCAC GTACACGATTAGGAGCTGATGTCGGTAAAGGA CCAGCTGAAAGGCAGTTCAAAGGTCTTGGTGAT TGTTTAGCCAAAACCGTCAAGTCTGATGGTCCC ATTGGTTTGTACCGTGGTTTCATTGTATCAGTAC AGGGTATCATCATCTACCGTGCTGCATACTTTG GATTTTTCGACACAGCTAAGGGAATGTTGCCAG ACCCCAAGAATACTCCATTCTTAGTTTCATGGG GTATCGCCCAATTTGTAACAACATTCGCTGGTA TTATGTCCTATCCATTTGACACAGTCAGACGTC GTATGATGATGCAATCTGGCCGTGCTGCTGACC AACGCATGTACAAGAGCACATTGGACTGCTGG GGTAAACTTTACAAGAATGAAGGTACATCTGCT TTCTTCAAGGGTGCATTCTCCAACGTACTCAGA GGTACTGGTGGTGCCTTGGTGTTGGTCTTCTAC GACGAACTCAAAAACCTCATG ds15 >CL597.Contig1_TY heat shock 3 protein 83-like ATGCCTGAAGACGTTACCATGACTGCATCTGAT GATGTTGAGACCTTCGCTTTCCAAGCTGAGATC GCTCAGCTTATGTCCCTCATCATCAACACCTTCT ACTCGAACAAAGAAATCTTTTTGCGAGAATTGG TATCCAATTCTTCTGATGCATTGGACAAAATTC GTTATGAGTCATTGACTGATCCATCCAAATTGG AATCTGGCAAAGATTTACACATTAAAATCATCC CCAATGCGGAAGAAAAAACTCTGACCATTATT GACACTGGTATCGGTATGACCAAAGCTGATCTA GTCAACAACTTGGGAACCATTGCTAAATCTGGT ACTAAGGCTTTCATGGAAGCTTTACAAGCTGGA GCTGATATTTCCATGATTGGTCAATTTGGTGTG GGTTTCTATTCCGCCTATCTGGTAGCTGACAAA GTCACTGTTGTTTCCAAACACAACGACGATGAA CAATATTTGTGGGAATCTGCTGCCGGAGGTTCA TTCACCATCCGTACTGATCCTGGTGAACCATTG GGCCGTGGTACCAAAATTGTCCTTCAAATCAAA GAAGATCAAGCTGAGTTCCTCCAACAAGAAAA AATTACCAGCATCATCAAGAAGCACTCTCAATT CATTGGCTACCCAATCAAATTAATCGTTGAGAA TGAACGTACCAAAGAAGTCAGCGATGATGAAG CTGAAGAAGAAAAGAAAGATGAAGTTGAAGGT GAAACTGAAGAAGACAAAAAACCCAAAATTGA GGATGTTGGTGAGGATGAAGACGAAGACAAAA AAGATGAAGACAAAGACAAAAAGAAGAAGAA GACTATTAAAGAAAAGTACTTGGATGAAGAGG TCTTGAACAAGACAAAACCAATCTGGACACGC AACCCTGATGATATCAGCCAAGATGAATATGGT GAATTCTACAAATCCTTAACCAATGACTGGGAA GATCATTTAGCCGTCAAACATTTCTCTGTGGAA GGACAACTTGAATTCAGAGCATTGTTATTCATT CCCAAGCGTGCGCCTTATGACATGTTTGAGAAC AAGAAGAAGAAGAACAACATTAAATTATATGT CCGTCGTGTCTTCATCATGGACAACTGCGAAGA CCTCATGCCAGAATACTTGAACTTCATCAAGGG TGTTGTTGACAGTGAGGATTTGCCGTTGAACAT CTCCCGTGAAATGCTCCAACAAAACAAGATCTT GAAAGTTATCAGGAAGAATTTGGTTAAGAAAT GTTTGGAATTGTTCGAGGAATTGGCTGAAGACA AGGACAACTACAAGAAATTGTACGAACAGTTC AGCAAGAACTTGAAACTTGGAATCCACGAAGA TAGCCAAAACAGAAAGAAACTCTCAGACTTGT TGAGATTCCACTCCTCAGCCAGTGGTGACGAAT CATGCTCCCTTAAGGAGTATGTTGCACGTATGA AGCCAAATCAAACCCACATTTACTACATCACAG GTGAAAGCCGTGAACAAGTATCCAACTCTTCAT TCGTTGAACGTGTCAAGAAACGTGGTTTTGAAG TTATTTACATGACTGAACCCATTGATGAATACG TTGTCCAACAAATGAAAGAATATGACGGCAAG AACTTGGTATCTGTCACTAAAGAAGGTTTGGAC TTGCCTGAAACCGATGAAGAAAAGAAGAAGCG CGAGGATGATCAATCCAGATTTGAAAAATTGTG CAAAGTTGTTAAGGACATTTTGGACAAGAAAG TTGAGAAGGTTGTCATCAGTAACAGACTTGTTG AGTCTCCCTGTTGCATTGTCACATCTCAGTATG GTTGGACTGCCAACATGGAACGTATCATGAAG GCACAAGCACTCAGAGATTCATCTACCATGGGT TATATGTCTGCCAAAAAACACTTGGAAATCAAC CCTGACCACCCGATCATTGAAACACTCAGACAA AAGGCTGAAGCTGATTGCAACGACAAGGCTGT CAGAGACTTGGTCATGCTTTTGTTCGAGACAAG TTTGTTGTCATCTGGTTTTGGACTTGAAGACCC ACAAGTTCACGCTTCTAGAATCCACAGAATGAT CAAATTGGGTTTGGGCATTGATGAAGATTTGCC AGTAGTTGAAGAAAAATCTGCTGAAGTTGAAG CCTCCGAGCCTGTTGTTGAAGCTGATGCTGAAG ATTCTTCTCGCATGGAAGAAGTTGAT ds25 >CL5923.Contig1_Ag_all eukaryotic 4 initiation factor 4A-like ATGAATGCTAATGAGACGAAAAATGGACCTCC TAGTGAAACCAATGACTACTCGGGACCACCTG GCATGGACGTCGGTGGAACTATTGAGTCTGACT GGAAAGAAGTGGTGGATAACTTTGATGAGATG AATTTAAAAGAAGAATTGTTGCGTGGTATTTAT GGATATGGTTTTGAAAAGCCATCAGCTATTCAA CAACGTGCTATTTTGCCGTGCATCAAGGGACAT GATGTCATTGCTCAGGCCCAATCTGGTACTGGC AAGACAGCTACTTTTTCCATTTCTATTCTCCAAC AAATTGATACAAGTTTGAATGAGTGCCAAGCA CTTATTTTGGCACCAACACGTGAATTGGCTCAA CAGATTCAAAAGGTGGTCATTGCTTTGGGTGAT TTCATGAAAGCTGATTGTCATGCTTGCATTGGC GGTACAAACGTTCGTGATGACATGCGTAAGCTG GATACTGGATCCCATGTAGTTGTTGGAACTCCT GGCCGTGTTTATGACATGATTGCTAGAAAATCC CTAAGAACTCAATTTATCAAGATATTTGTGTTG GACGAAGCTGATGAAATGTTGTCTCGAGGTTTC AAAGATCAAATTAAAGAGGTGTTCAAGTTCCTC GAAGAAGATATTCAGGTCATTCTGTTGTCTGCT ACAATGCCCGAGGACGTTTTGGATGTGAGCACT CATTTCATGCGTAATCCAGTACGCATTCTTGTTC AAAAGGAAGAACTGACATTGGAAGGTATCAAA CAGTTTTACATCAATGTTACCAAAGAAGAATGG AAGTTTGACACTCTATGTGATTTGTACGACACT CTTAGTATCACCCAGGCTGTGATCTTCTGTAAC ACACGTCGTAAGGTAGAGTGGTTGACTGAAAA TATGCGTTTGAAAACATTTACTGTATCAGCTAT GCATGGAGAAATGGACCAACGTCAACGTGAGC TAATTATGCGTCAATTCCGTTCTGGCTCTAGTC GTGTTCTAATTACCACTGATTTGTTGGCTCGAG GCATTGATGTACAACAAGTTTCTCTGGTCATCA ATTACGATTTGCCGTCCAATCGTGAAAACTATA TTCACAGGATTGGACGTTCTGGCCGTTTCGGTC GTAAAGGAGTCGCCATTAATTTTATCACCGAAG ACGACAAAAGAGCTATGAAGGATATTGAATCA TTTTACAACACTCACGTGCTCGAGATGCCACAG AATGTGGCCGATTTGCTG ds27 >CL6080.Contig1_Ag_all troponin 5 T-like isoform 3 ATGTCCGACGAAGAAGAAGTGTACACTGATTC CGAAGAAGAAACGCAACCGGAGCCTGAAAAAA GCAAAGATGGAGATGGAGATCCCGAATTCGTT AAGAGGCAAGAATTAAAATCTTCAGCCTTAGA CGAACAGCTTAAAGAGTACATCCAAGAATGGC GCAAACAGCGGTCAAAGGAAGAAGACGACTTA AAGAAGTTGAAGGAAAAACAGGCCAAGCGCAA GGTTATGCGAGCGGAAGAAGAGAAGAGAATGG CCGAGAGAAAGAAGCAAGAAGAAGAACGCAG ACAGAGAGAAGTCGAGGAAAAGAAACAAAAG GACATCGAAGAAAAACGTAAACGTCTAGAAGA GGCCGAGAAAAAACGGCAAGCTATGATGGCTG CTCTTAAGGAACAAACCAATAAATCTAAAGGA CCAAATTTCACCATCAGCAAAAAAGAAGGTGC GTTGAGTATGACTTCTGCCCAACTTGAACGCAA TAAAACCAGAGAACAGATCGAAGAAGAAAAGA AAATATCGTTGAGCTTCAGAATCAAACCTTTGA ATATTGAAGGATTCTCTGTGCAAAAACTCCAAT TCAAAGCTACCGAACTCTGGGACCAGATCATCA AGTTGGAAACAGAAAAATACGATTTGGAGGAA AGGCAAAAGAGACAAGATTACGACTTGAAAGA GTTGAAAGAACGTCAGAAGCAACAACTCCGCC ACAAGGCTCTGAAGAAAGGTCTCGACCCCGAA GCCCTAACCGGCAAATACCCACCCAAGATCCA AGTCGCTTCCAAGTACGAGAGGCGAGTTGACA CGAGGTCTTATGATGACAAAAAGAAGCTGTTC GAAGGAGGTTATATGGAAACCACTAAAGAATC AATGGAAAAACAATGGACAGAAAAAAGTGACC AATTCGGTGGCCGCGCTAAAGGACGATTACCG AAATGGTTCGGCGAACGTCCGGGCAAGAAGAA GGATGACCCAGACACACCCGAAGAGGAAGAGC TCAAGAAAAACGAGGAAGACGAAGAACCGTTT GGCCTCGACGACGAAGAAGCTGAAGAAGAAGT TGAAGAGGAAGAAGAGGAGGAAGAAGAAGAG GAAGAGGAGGAGGAAGAGGAAGAAGAGGAAG AAGAAGAAGAGGAAGAGGAAGAAGAAGAAGA A ds45 >CL2125.Contig1_Ag_all Y-box 6 protein Ct-p40-like ATGGCGGAACAAGTCGGCGAGAGGAGGACGGA ACGGCCGCCGCAGAAGCCCGTGGCCCAAAAGC CGGTCATATCTGTGAAAGTCACCGGCGTTGTTA AATGGTTCAACGTCAAAAGCGGTTATGGTTTTA TTAATCGTAATGATACAAAAGAAGATATATTTG TACATCAGTCTGCTATTATCAAGAACAACCCTA AGAAAATTGTACGCAGTGTCGGTGATGGAGAA ACTGTAGAATTTGACGTTGTTGAGGGCGAAAA AGGTCACGAAGCAGCAAATGTTACTGGTCCAG ATGGAGAAGCTGTTAAAGGATCACCTTATGCA GCTGAAAGAAGAAGAAATAACTATCGTCAGTG GTTTTATGGACGCCGTCCTAATACCCGTCCAAG AAATGGTGGTCAACCTCCAAGAGATGGTAGTC CAAGTGGTGACAAGGAAGAAACTGAAAATGAA GTAGGAGAACAACCAAGACGTTACCGCCAGCC ACGTCAACAGAATTGGTATAATAGCTATCGTGG AAATCGAAGAGGTCCACCACCAAATAGAGGAG AAGGTGGTGATTACAATGGTGGAGATAATTAT GGATATGATAGTTCACCTCCTGGTAGAGGCAGA GGTCGTGGGATGGGTGCGCCTAGACGTTTCTTT AGACGTGGCAGTGGATTTAGAGGGAGCCGTGG AACAGGTGGTCCACCCAGAAGACCATATCAAG ATGAAAATCAGGACAATGAATATAATCAAAGT GATGAAAATGGAGCAAATAGACCTCGTCCTCG CTATCGCCGCCGCAATAATCGTTCTAGAGCGAG AAGTGATGGTCCTCCAAGAGCCAATAGCCAAA GTGACAATGAATCTAAACAAAAAAACTTTGGA GGAGAAGCATTGGAACTGGATGAAAGTAGTCA TGCT

Example 2 the Control Effect of Target Gene dsRNA on Aphids

[0282] Inoculating a certain number of green peach aphid or soybean aphid on radish seedlings or soybean seedlings, first, recording the number of aphids inoculated on each plant respectively, and dissolving the synthesized dsRNA into 2% Tween-80, the dsRNA concentrations of the 6 target genes were shown in Table 3. Then spraying 1 ml of dsRNA on the plants inoculated with aphids, and counting on the next day as the statistical results of the first day after dsRNA treatment. Then counting every other day for a total of 3 times and recording as the results of the first day, the third day and the fifth day after treatment, using 2% Tween-80 and dsGFP as a control. The statistical results show that, compared with the control spraying only 2% Tween-80, the control effects of the 3 target genes of the green peach aphid and the 3 target genes of the soybean aphid on the two kinds of aphids all have exceeded 80% (FIG. 1, A, B).

TABLE-US-00009 TABLE 3 Spraying concentration of target gene dsRNA Gene name of green peach Concentration Gene name of Concentration aphid (ng/μl) soybean aphid (ng/μl) dsGFP 295 dsGFP 265 ds7 233 ds25 282 ds9 241 ds27 257 ds15 279 ds45 242

Example 3 Statistics of the Dropping Rate of Insect Population of Aphids by Target Genes

[0283] Aphids are virginopara insects, born as first-instar newborn aphids. The period from the first instar aphid to the time it can give birth is about 5-7 days (affected by environmental temperature). There are obvious alternation of generations in aphids on a plant, that is, insects of different generations and sizes (different instars) exist at the same time. Therefore, when the test plants are inoculated, there will be aphids of various instars (such as 2th-4th, and there may be adults). In this way, after various test treatments, the aphids quickly begin to reproduce and produce the next generation, resulting in the number of aphids on the tested plant being increased after counting before drug spraying. This is a great interference to the judgment of the control effect of aphids insecticides. Therefore, there is a more rigorous or relatively accurate calculation method for the control effect of aphids, that is, the dropping rate of insect population (see the general methods and materials section for the calculation formula).

[0284] The statistical results of the present invention show that after spraying the dsRNA of the 3 green peach aphid target genes, the dropping rates of insect population of the green peach aphid population at 1 day, 3 days and 5 days after treatment are shown in Table 4. The dropping rates of insect population have all reached more than 70% on the 5th day after spraying.

[0285] After spraying the dsRNA of the 3 soybean aphid target genes, the dropping rates of insect population of soybean aphid population at 1 day, 3 days and 5 days after treatment are shown in Table 5. The dropping rate of insect population on the 5th day after spraying, except for ds45, the dropping rate of insect population of which is 67.61%, the dropping rates of insect population of the other two target genes are all above 70%.

TABLE-US-00010 TABLE 4 The dropping rate of insect population after spraying with dsRNA of target gene of green peach aphid Treatment 1 d 3 d 5 d 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

TABLE-US-00011 TABLE 5 the dropping rate of insect population after spraying with dsRNA of target gene of soybean aphid Treatment 1 d 3 d 5 d 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

Example 4 Comparison of the Control Effect of Green Peach Aphid Target and Imidacloprid

[0286] Experimental Method:

[0287] 1. Radish seedlings of 12-15 days, inoculated with 100 insects, stabilized for 1 day, sprayed with dsRNA the next day.

[0288] 2. The concentration of dsRNA used for spraying was 300 ng/μl. The synthesized dsRNA was dissolved in water and sprayed 300 μl per plant.

[0289] 3. The concentration of imidacloprid was 10,000 times solution (Germany Bayer Emerald 70% imidacloprid 3 g, water dispersible granules), sprayed 300 μl per plant.

[0290] 4. The experiment method: spray treatment, 4 replicates for each treatment.

[0291] The results are shown in Table 6, Table 7, and FIG. 3 and FIG. 4.

TABLE-US-00012 TABLE 6 Field test of green peach aphid: the dropping rate of insect population 1 d 3 d 5 d Control −116.00 ± 15.06 Aa  −297.87 ± 132.86 Aa  −518.78 ± 161.36 Aa Imidacloprid 37.33 ± 9.65 Bb  75.83 ± 15.25 Bb 84.35 ± 9.60 Bb dsGFP −67.29 ± 40.98 Aa  −241.98 ± 106.06 Aa  −369.79 ± 131.65 Aa ds7 15.75 ± 27.55 Cb 51.54 ± 10.55 Cb 81.81 ± 9.47 Bb ds9 15.85 ± 16.94 Cb 66.41 ± 11.20 Bb 72.64 ± 4.51 Cb ds15 21.15 ± 15.10 Bb 57.65 ± 18.66 Bb 79.32 ± 4.92 Bb

TABLE-US-00013 TABLE 7 Field test of green peach aphid: Control effect 1 d 3 d 5 d dsGFP 22.85 ± 15.71 Aa  12.58 ± 12.90 Aa  22.88 ± 18.49 Aa ds7 61.30 ± 11.62 Bb 86.26 ± 6.91 Bb 96.91 ± 1.49 Bb ds9 60.56 ± 10.51 Bb 91.25 ± 2.22 Cb 95.18 ± 2.07 Bb ds15 63.67 ± 4.78 Bb  89.34 ± 3.21 Cb 96.51 ± 1.12 Bb Imidacloprid 70.96 ± 4.25 Cc  93.78 ± 3.14 Cb 97.37 ± 1.88 Bb

[0292] The results show that the three genes for green peach aphid have shown obvious lethal effects on the third day, and the dropping rate of insect population is over 70% on the fifth day. Compared with imidacloprid, there is no obvious difference in the dropping rate of insect population. However, compared with the control dsGFP, the dropping rate of insect population shows a significant difference (Table 6); at the same time, the statistical analysis of the control effect shows that the control effect of these three target genes has reached more than 90%, and it can show better control effect on the third day (Table 7). Therefore, this result shows that these three target genes have a strong lethal effect on green peach aphid and can be used as target genes to control green peach aphid for pest control.

Example 5 Detection of Target Gene Expression

[0293] In order to prove that the control effect of spraying dsRNA of these target genes on aphids population is due to the inhibition of the expression of target genes, aphids on day 1, 3, and 5 after treatment with 6 target genes dsRNA were collected, and quantitative PCR (q-RT-PCR) was used to detect whether the target gene was suppressed. The test results of the three target genes of green peach aphid are shown in FIG. 2A. Except for the ds9 gene, the target gene is induced to be up-regulated after 1 day of treatment, all genes are significantly down-regulated after 3 and 5 days of treatment, indicating that the death of aphids is closely related to the level of gene expression.

[0294] The detection results of the 3 soybean aphid target genes are shown in FIG. 2B. Except for the expression of the target gene after 1 day of ds25 gene treatment is not significantly different from the control, all genes are significantly down-regulated after 3 and 5 days of treatment, indicating that the death of aphids is closely related to the level of gene expression.

Example 6

[0295] Preparation of the Composition

[0296] This example provides a composition for efficiently killing aphids. The composition is an aqueous solution and includes components: [0297] 1. The dsRNA for the DS7 gene fragment as shown in SEQ ID NO. 1 or 24 the concentration is 100 ng/μl; [0298] 2. The dsRNA for the DS9 gene fragment as shown in SEQ ID NO. 2 or 25 the concentration is 100 ng/μl. [0299] 3. The dsRNA for the DS15 gene fragment as shown in SEQ ID NO. 3 or 26 the concentration is 100 ng/μl. [0300] 4. The the dsRNA for the DS25 gene fragment as shown in SEQ ID NO. 4 or 27 the concentration is 100 ng/μl. [0301] 5. The dsRNA for the DS27 gene fragment as shown in SEQ ID NO. 5 or 28 the concentration is 100 ng/μl. [0302] 6. The dsRNA for the DS45 gene fragment as shown in SEQ ID NO. 6 or 29 the concentration is 100 ng/μl.

Comparative Example 1

[0303] The method is the same as that of Examples 1 and 2, the difference is that the target gene is DS50 and the primers used are:

TABLE-US-00014 primer F: (SEQ ID NO.: 30) TAATACGACTCACTATAGGGAGACGTGTCTGAGGCGGTTGCCA primer R: (SEQ ID NO.: 31) TAATACGACTCACTATAGGGAGATGATCTTGGCCCGGAGAGCCGG

[0304] The length of the amplified product is 578 bp.

[0305] The DS50 gene is a fatty acid synthase-like gene. The sequence is shown in SEQ ID NO. 23, which encodes the FASN gene. Fatty acid synthase is a multi-enzyme protein that catalyzes fatty acid synthesis. It is not a single enzyme, but an entire enzyme system composed of two identical 272 kDa multifunctional polypeptides, in which the substrate is submitted from one functional domain to the next, and its main function is to catalyze the synthesis of palmitate from acetyl-CoA and malonyl-CoA in the presence of NADPH.

[0306] The results show that the dsRNA designed for the DS50 gene by the method of the present invention has a very poor control effect on aphids, with a maximum of only about 23%.

[0307] The sequence of the DS50 gene fragment is shown in SEQ ID NO: 23:

TABLE-US-00015 (SEQ ID NO.: 23) TTGGAATTGATTCAACATCTAGCTCAAAGAGGAGCCCGCAAATTTGTTTTA GTGTCGAAATTGAACAACAAACCTCAGTCAGGTTACAAGACGTTGACCTTA AGACGGTTGAAGAACAAGAACGTTACCGTAGTCCTATCGTTTGCTGACCCA TCAACAGTGAGAGGCGCTGAAGACGTACTGAGAGAAGCTGTAGCCCTCGGA ACAGTCTGTGGTATTTACCACATAACCACCGCTCCGGAAACCAAACACTTG CAATCCCTGAGCGAAAAGGATTTCGCAGAGACGAAAAAAGTCGTGTCTGAG GCGGTTGCCAATTTGGACACACTGAGCAGGAGATTGATTCCTCAACTTGAA TCGTTTGTTGTCCTTGCTCCGGCCGTCGCATCAAGAGGAGCTAAAGCCAAG TCCAACTACGTTTTCGCAAACGCAGATGTTATCAGAGTCGCTGAAGTCCGT AAAGTTTCGGGCTATCCAACAGTAGTCATAGAATACGGCGCAATCGAAGGT ATTTCGAATGCGTTCAACAGTCCAAACTTCAAACCAGCGTCGATCGTTTCA GCGTTGAATGTTCTGGATGAAATTACCAAACAACCACAAAACCCAACAGTC GTGTCCTTCTCAAAATTCAACGGTCCAATTTATGAAGAAACGGATGCCGCC ACTCCATTGTTGAAGACAATTGCCAAGATTTTCGGTTACAAGACACTGTCC CAAATTGAACAGACCTTTAATCTCGCTCAACTCGGCCTGGACACGTTCCTC GCACCACGCGTTCAAGAAGCCATCAGACAACAAGCCAACGCAGTCATCGAG GTAGAAGAACTAAGAACACTGACGTTCCCGGCTCTCCGGGCCAAGATCATC GAATTACTCGCC

[0308] All literatures mentioned in the present application are incorporated by reference herein, as though individually incorporated by reference. Additionally, it should be understood that after reading the above teaching, many variations and modifications may be made by the skilled in the art, and these equivalents also fall within the scope as defined by the appended claims.