Application of inhibiting farnesoid dehydrogenase in enhancing toxicity of trifluoro-sassen ester

Abstract

The invention provides an application of inhibiting farnesol dehydrogenase in enhancing the toxicity of trifloxacin, and belongs to the technical field of pest control. The invention provides an application of inhibiting farnesol dehydrogenase in enhancing the toxicity of trifluoro-sassen ester. By inhibiting the expression of the farnesol dehydrogenase, the toxicity of the trifloxystrobin can be improved, and the pest control effect of the trifloxystrobin is improved. The result of the embodiment shows that the expression of the aedes aegypti farnesol dehydrogenase FDL22 and the expression of the green peach aphid farnesol dehydrogenase FDL242 are silenced by dsRNA, and the toxicity of the trifluorocide to the aedes aegypti and the green peach aphid can be remarkably improved. The juvenile hormone analogue S-(+)-methoprene is used in combination with the trifluoro-nematicidal ester, so that the toxicity of the trifluoro-nematicidal ester to aedes aegypti larvae can be remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of pest control, and in particular to application of inhibiting farnesol dehydrogenase in enhancing the toxicity of trifluoxetine. Background Art

[0002] Insecticides play an irreplaceable and core role in the prevention and control of field pests. The current research and application of nematicides, insecticides, and acaricides must not only accurately control harmful nematodes, insects, and mites, and address the existing problem of pesticide resistance among field pests, but also take into account environmental safety to promote sustainable agricultural ecological development. Therefore, the creation and development of insecticides with new targets and new modes of action are crucial. Trifluoxetine is a green nematicide and acaricide independently developed in my country that has a new structure and can kill both nematodes and mites. It has no cross-resistance with common nematicides, insecticides, and acaricides on the market, has long-lasting and stable efficacy, is green, low-toxic, and has no residue, and is highly ecologically safe, with broad prospects for promotion and application.

[0003] Research on enhancing the toxicity of insecticides is a key area of ​​innovation in agricultural science and technology. Exploring effective pest control strategies while reducing pesticide usage, thereby conserving resources and reducing pollution, can also delay the development of pest resistance and extend the lifecycle of existing insecticides. Currently, several patents have been published related to the toxicity enhancement of trifluoxetine (CN113875758B, CN110946146B, CN119791116A, CN109006821A, etc.). Most of these studies focus on the combination of trifluoxetine with other commercially available insecticides, limiting their scope of application. Therefore, in-depth research on the pathways and mechanisms of trifluoxetine's toxicity enhancement will facilitate its widespread promotion and application, and also provide fundamental support for research on compounding and enhancing the synergy of insecticides. Summary of the Invention

[0004] The present invention provides an application of inhibiting farnesol dehydrogenase in enhancing the toxicity of trifluoxetine. The toxicity of trifluoxetine to the pests can be significantly improved by inhibiting the expression of farnesol dehydrogenase in the pests.

[0005] The present invention provides the use of inhibiting farnesol dehydrogenase in enhancing the toxicity of trifluoxetine.

[0006] Preferably, the farnesol dehydrogenase includes any one of the following (1) to (3): (1) The amino acid sequence of Aedes aegypti farnesol dehydrogenase FDL22 is shown in SEQ ID NO. 1; the nucleotide sequence encoding the Aedes aegypti farnesol dehydrogenase FDL22 protein is shown in SEQ ID NO. 2; (2) The amino acid sequence of Myzus persicae farnesol dehydrogenase FDL242 is shown in SEQ ID NO. 13; the nucleotide sequence encoding the Myzus persicae farnesol dehydrogenase FDL242 protein is shown in SEQ ID NO. 14; (3) Homologous genes of (1) or (2) in insects other than Aedes aegypti and Myzus persicae.

[0007] Preferably, the method for inhibiting the expression of farnesol dehydrogenase comprises knocking down farnesol dehydrogenase by dsRNA and / or exogenously administering a juvenile hormone analog to inhibit the expression of farnesol dehydrogenase.

[0008] Preferably, the juvenile hormone analogue comprises S-(+)-methoprene.

[0009] The present invention provides a farnesol dehydrogenase of Aedes aegypti FDL22 The dsRNA has a nucleotide sequence as shown in SEQ ID NO.3.

[0010] The present invention provides a kind of green peach aphid farnesol dehydrogenase FDL242 The dsRNA has a nucleotide sequence as shown in SEQ ID NO.15.

[0011] The invention provides a synergistic insecticide comprising dsRNA for inhibiting the expression of farnesol dehydrogenase and trifluoxetine.

[0012] Preferably, the dsRNA that inhibits the expression of farnesol dehydrogenase includes Aedes aegypti farnesol dehydrogenase FDL22 Myzus persicae farnesol dehydrogenase FDL242 of dsRNA.

[0013] The present invention provides the use of the dsRNA described in the above technical solution or the synergistic insecticide described in the above technical solution in controlling pests.

[0014] The present invention provides a method for controlling pests using the synergistic insecticide described in the above technical solution, comprising: When the synergistic insecticide comprises a dsRNA that inhibits the expression of farnesol dehydrogenase, the dsRNA is delivered to the pest, which is then exposed to trifluoxetine.

[0015] The present invention provides the use of inhibiting farnesol dehydrogenase in enhancing the toxicity of trifluoxetine. The present invention can significantly enhance the toxicity of trifluoxetine by inhibiting the expression of farnesol dehydrogenase in insects, thereby improving the control effect of trifluoxetine on pests. The present invention shows through the results of the examples that the toxicity of trifluoxetine to Aedes aegypti larvae can be significantly enhanced by knocking down the expression of farnesol dehydrogenase FDL22 of Aedes aegypti by dsRNA; by applying the juvenile hormone analog S-(+)-methoprene in combination with trifluoxetine, the expression of farnesol dehydrogenase FDL22 of Aedes aegypti can be inhibited, and the toxicity of the mixed solution to Aedes aegypti larvae is significantly enhanced compared with the use of trifluoxetine alone; by knocking down the homologous gene of Aedes aegypti farnesol dehydrogenase FDL22 in green peach aphid by dsRNA, the green peach aphid farnesol dehydrogenase FDL242 , which can significantly increase the toxicity of triflumuride to peach aphid nymphs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For feeding and soaking ds for Aedes aegypti larvae FDL22 Significantly reduce farnesol dehydrogenase in Aedes aegypti larvae FDL22 mRNA expression level results diagram; Figure 2 Farnesol dehydrogenase in Aedes aegypti larvae FDL22 The results of the inhibition of mRNA expression level on the toxicity enhancement of 133.30 mg / L trifluoxetine to Aedes aegypti larvae; Figure 3 This is the toxicity enhancement result of a mixture of 3 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine against Aedes aegypti larvae treated for 48 hours; Figure 4 This is the toxicity enhancement result of a mixture of 6 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine against Aedes aegypti larvae treated for 24 hours; Figure 5 This is the toxicity enhancement result of a mixture of 10 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine against Aedes aegypti larvae treated for 24 hours; Figure 6 The combination of S-(+)-methoprene and trifluoxetine inhibits farnesol dehydrogenase FDL22 mRNA expression level results diagram; Figure 7 This is a schematic diagram of the alignment of the amino acid sequence of farnesol dehydrogenase FDL22 from Aedes aegypti and the amino acid sequence of farnesol dehydrogenase FDL242 from Myzus persicae; Figure 8 To inject ds FDL242 Significantly reduced the number of green peach aphid nymphs FDL242 mRNA expression level results diagram; Figure 9 Farnesol dehydrogenase in green peach aphid nymphs FDL242 The results of the inhibition of mRNA expression level on the toxicity enhancement of 128.64 mg / L trifluoxetine solution to green peach aphid nymphs; Figure 10 Farnesol dehydrogenase in green peach aphid nymphs FDL242 The results of the reduction of mRNA expression level on the toxicity enhancement of 230.16 mg / L trifluoxetine solution to green peach aphid nymphs; Figure 11 Schematic diagram of enhancing the toxicity of trifluoxetine to pests by inhibiting the expression of farnesol dehydrogenase. DETAILED DESCRIPTION

[0017] The present invention provides a method for inhibiting farnesol dehydrogenase in enhancing the toxicity of trifluoxetine. As an optional embodiment of the present invention, the farnesol dehydrogenase comprises any one of the following (1) to (3): (1) the amino acid sequence of Aedes aegypti farnesol dehydrogenase FDL22 as shown in SEQ ID NO.1; the nucleotide sequence encoding the Aedes aegypti farnesol dehydrogenase FDL22 protein as shown in SEQ ID NO.2; (2) the amino acid sequence of Peach Aphid farnesol dehydrogenase FDL242 as shown in SEQ ID NO.13; the nucleotide sequence encoding the Peach Aphid farnesol dehydrogenase FDL242 protein as shown in SEQ ID NO.14; (3) homologous genes of (1) or (2) in other pests other than Aedes aegypti and Myzus persicae.

[0018] In the present invention, the farnesol dehydrogenase comprises the Aedes aegypti farnesol dehydrogenase FDL22 having an amino acid sequence as shown in SEQ ID NO. 1. As an optional embodiment of the present invention, the Aedes aegypti farnesol dehydrogenase FDL22 The nucleotide sequence of the gene is shown in SEQ ID NO. 2. In the present invention, the Aedes aegypti farnesol dehydrogenase FDL22 The invention is a gene encoding the farnesol dehydrogenase FDL22 protein of Aedes aegypti. FDL22 The expression of trifluoxetine can significantly increase the toxicity of trifluoxetine to Aedes aegypti.

[0019] In the present invention, the farnesol dehydrogenase comprises the green peach aphid farnesol dehydrogenase FDL242 whose amino acid sequence is shown in SEQ ID NO. 13. As an optional embodiment of the present invention, the green peach aphid farnesol dehydrogenase FDL242 The nucleotide sequence of the gene is shown in SEQ ID NO. 14. The gene encoding the green peach aphid farnesol dehydrogenase FDL242 protein may also be referred to as green peach aphid farnesol dehydrogenase FDL242The present invention inhibits the farnesol dehydrogenase of green peach aphid. FDL242 The expression of trifluoxetine can significantly enhance the toxicity of trifluoxetine to peach aphid.

[0020] In the present invention, the farnesol dehydrogenase includes homologous genes of (1) or (2) in pests other than Aedes aegypti and Myzus persicae. As an optional embodiment of the present invention, the homologous gene preferably has an amino acid sequence similarity of ≥40% with the farnesol dehydrogenase FDL22 of Aedes aegypti or the farnesol dehydrogenase FDL242 of Myzus persicae. The present invention can enhance the toxicity of trifluoxetine to the corresponding insects by inhibiting the expression of the homologous genes in the corresponding insects.

[0021] As an optional embodiment of the present invention, the method for inhibiting the expression of farnesol dehydrogenase includes knocking down farnesol dehydrogenase with dsRNA and / or exogenously applying juvenile hormone analogs to inhibit the expression of farnesol dehydrogenase. In the present invention, the dsRNA includes Aedes aegypti farnesol dehydrogenase FDL22 Myzus persicae farnesol dehydrogenase FDL242 As an optional embodiment of the present invention, the Aedes aegypti farnesol dehydrogenase FDL22 The nucleotide sequence of the dsRNA is shown in SEQ ID NO.3; the peach aphid farnesol dehydrogenase FDL242 The nucleotide sequence of the dsRNA is shown in SEQ ID NO. 15. In the present invention, the juvenile hormone analogue includes S-(+)-methoprene.

[0022] In the present invention, when the juvenile hormone analogue S-(+)-methoprene enhances the toxicity of trifluoxetine, the S-(+)-methoprene and trifluoxetine can be used together as a synergistic insecticide. Specifically, the synergistic insecticide includes S-(+)-methoprene and trifluoxetine. As an optional embodiment of the present invention, the weight ratio of the trifluanid to the S-(+)-methoprene may be ≥13:1, or (13-65):1, or (13-45):1, or 13:1, 15:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, 30:1, 32:1, 34:1, 36:1, 38:1, 40:1, 41:1, 42:1, 43:1, 44:1, 44.4:1, 45:1, 46:1, 47:1, 48:1, 49:1. :1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 40:2, 45:2, 50:2, 55:2, 60:2, 65:2, 40:3, 45:3, 50:3, 55:3, 60:3, 65:3, 40:4, 45:4, 50:4, 55:4, 60:4, 65:4, 133.30:3, 133.30:6 or 133.30:10.

[0023] The present invention provides a method for controlling pests using a synergistic insecticide composed of S-(+)-methoprene and trifluoxetine, comprising: exposing the pests to a solution of a juvenile hormone analog and trifluoxetine. As an optional embodiment of the present invention, when trifluoxetine and S-(+)-methoprene are compounded, the weight ratio of the two can be ≥13:1, (13-65):1, or (13-45):1. When the synergistic insecticide obtained by compounding trifluoxetine and S-(+)-methoprene is used for controlling pests, the exposure method can be to immerse the pests in a solution containing the synergistic insecticide; the mass concentration of trifluoxetine in the solution containing the synergistic insecticide can be 133.30 mg / L, and the mass concentration of S-(+)-methoprene can be 3, 6, or 10 mg / L.

[0024] Aedes aegypti Aedes aegypti The larvae live in small water bodies, their activities are limited, and they are easy to treat with drugs, making them an excellent model for toxicology research. In this study, the larvae of Aedes aegypti were used as a research model. The method and target of the juvenile hormone, a hormone conserved in insects, and its synthesis pathway were used to find ways and targets that can significantly enhance the efficacy of trifluoxetine. The agricultural pest Myzus persicae was used as the research model. Myzus pericae It is an application case, providing support for the widespread application of trifluoxetine.

[0025] The present invention provides a farnesol dehydrogenase of Aedes aegypti FDL22 The dsRNA provided by the present invention can effectively knock down the farnesol dehydrogenase of Aedes aegypti FDL22 , silencing the expression of Aedes aegypti farnesol dehydrogenase FDL22. In the present invention, the Aedes aegypti farnesol dehydrogenase FDL22 dsRNA can also be called ds FDL22 .

[0026] The present invention provides a kind of green peach aphid farnesol dehydrogenase FDL242 The dsRNA provided by the present invention can effectively knock down the farnesol dehydrogenase of green peach aphid. FDL242 , silencing the expression of peach aphid farnesol dehydrogenase FDL242. In the present invention, the peach aphid farnesol dehydrogenase FDL242 dsRNA can also be called ds FDL242 .

[0027] The invention provides a synergistic insecticide comprising dsRNA for inhibiting the expression of farnesol dehydrogenase and trifluoxetine.

[0028] As an optional embodiment of the present invention, the dsRNA includes Aedes aegypti farnesol dehydrogenase FDL22 Myzus persicae farnesol dehydrogenase FDL242 As an optional embodiment of the present invention, the Aedes aegypti farnesol dehydrogenase FDL22 The nucleotide sequence of the dsRNA is shown in SEQ ID NO.3; the peach aphid farnesol dehydrogenase FDL242 The nucleotide sequence of the dsRNA is shown in SEQ ID NO.15.

[0029] The present invention provides the use of the dsRNA described in the above technical solution or the synergistic insecticide described in the above technical solution in pest control. The present invention can significantly increase the toxicity of trifluoxetine through the dsRNA, thereby improving the pest control effect. In the present invention, the dsRNA or juvenile hormone analog in the synergistic insecticide that inhibits the expression of farnesol dehydrogenase can inhibit farnesol dehydrogenase, thereby synergistically exerting pest control capabilities with trifluoxetine, thereby improving the pest control effect. As an optional embodiment of the present invention, the pests include mosquitoes and / or aphids; the mosquitoes include Aedes aegypti; the Aedes aegypti include Aedes aegypti larvae; the Aedes aegypti larvae include first-instar larvae and / or second-instar larvae; the aphids include green peach aphids; and the green peach aphids include adults and nymphs.

[0030] The present invention provides a method for controlling pests using the synergistic insecticide described in the above technical solution, comprising: When the synergistic insecticide comprises a dsRNA that knocks down the expression of farnesol dehydrogenase, the dsRNA is delivered to the pest, and the pest is then exposed to a solution containing trifluoxetine.

[0031] As an optional embodiment of the present invention, when the synergistic insecticide includes dsRNA that knocks down the expression of farnesol dehydrogenase; the dsRNA includes Aedes aegypti farnesol dehydrogenase FDL22 Myzus persicae farnesol dehydrogenase FDL242 As an optional embodiment of the present invention, the Aedes aegypti farnesol dehydrogenase FDL22 The nucleotide sequence of the dsRNA is shown in SEQ ID NO.3; the peach aphid farnesol dehydrogenase FDL242 The nucleotide sequence of the dsRNA is shown in SEQ ID NO.15.

[0032] In the present invention, when the dsRNA for knocking down farnesol dehydrogenase expression is Aedes aegypti farnesol dehydrogenase FDL22 Aedes aegypti farnesol dehydrogenase FDL22 The dsRNA is delivered to the body of Aedes aegypti, and then the Aedes aegypti is exposed to the solution containing trifluanid. As an optional embodiment of the present invention, the Aedes aegypti includes first-instar and / or second-instar larvae of Aedes aegypti. As an optional embodiment of the present invention, the delivery method includes feeding and soaking method. As an optional embodiment of the present invention, the feeding and soaking method includes feeding the Aedes aegypti with food containing the dsRNA, and after 24 hours, soaking the Aedes aegypti with 0.1% DEPC-treated water containing the dsRNA. As an optional embodiment of the present invention, the mass concentration of the dsRNA in the food containing the dsRNA can be 10 µg / mL; the mass concentration of the dsRNA in the 0.1% DEPC water containing the dsRNA can be 50 µg / mL. After the present invention delivers the dsRNA to the body of Aedes aegypti through the above-mentioned feeding and soaking method, it is detected. FDL22 The expression of the gene was significantly reduced, proving that the dsRNA provided by the present invention can knock down the farnesol dehydrogenase of Aedes aegypti FDL22 , silencing farnesol dehydrogenase in Aedes aegypti FDL22 Gene expression.

[0033] Knockdown of farnesol dehydrogenase in Aedes aegypti FDL22After the gene is generated, the present invention exposes the Aedes aegypti to a solution containing trifluanid. As an optional embodiment of the present invention, the exposure includes soaking the Aedes aegypti in a trifluanid solution; the soaking time can be 48 hours. As an optional embodiment of the present invention, the mass concentration of the trifluanid solution is ≥133.30 mg / L. The present invention has been verified by the effect that the knockdown of the farnesol dehydrogenase in Aedes aegypti FDL22 After the gene is expressed, the toxicity of trifluoxetine to Aedes aegypti larvae is significantly increased.

[0034] In the present invention, when the dsRNA that inhibits the expression of farnesol dehydrogenase is Myzus persicae farnesol dehydrogenase FDL242 Myzus persicae farnesol dehydrogenase FDL242 The dsRNA is delivered into the body of peach aphid, and then the peach aphid is exposed to the trifluanid. As an optional embodiment of the present invention, the peach aphid includes adults and / or nymphs of peach aphid. As an optional embodiment of the present invention, the delivery method includes injection. The present invention has no special limitation on the injection method, and any conventional injection method in the art can be used. As an optional embodiment of the present invention, the injection method can be microinjection; the injection site can be the coxa fossa of the fourth-instar nymph of peach aphid; when injecting, the concentration of the dsRNA is 10,000 ng / μL; the injection volume of the dsRNA can be 30 nL / aphid; and the injection speed can be 30 nL / s. After the dsRNA is delivered into the body of peach aphid by injection, the peach aphid farnesol dehydrogenase is detected. FDL242 The expression of α-myzus persicae was significantly reduced, proving that the dsRNA provided by the present invention can knock down the myzus persicae farnesol dehydrogenase. FDL242 , silencing Myzus persicae farnesol dehydrogenase FDL242 expression.

[0035] The present invention preferably uses green peach aphid farnesol dehydrogenase FDL242 Two days after the dsRNA is delivered to peach aphids, the aphids are exposed to the solution containing trifluoxetine. As an optional embodiment of the present invention, the mass concentration of trifluoxetine in the solution containing trifluoxetine is ≥128.64 mg / L, and can be between 128.64 and 230.16 mg / L. As an optional embodiment of the present invention, the exposure time can be 10 seconds.

[0036] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 This example uses Aedes aegypti larvae as a model and uses dsRNA interference to knock down Aedes aegypti farnesol dehydrogenase. FDL22 expression, thereby enhancing the toxicity of trifluoxetine to Aedes aegypti larvae.

[0038] The amino acid sequence of Aedes aegypti farnesol dehydrogenase FDL22 is shown in SEQ ID NO. 1, specifically: MDRWAGKVAVVTGASSGIGAAITTDLAKAGMVVVGLARRVERVEALKANLPESAKPRLHAVKCDVSKEEDITQVFKWVEEKFGGVDVLVNNAGILRQTDLLGTDNGQMLREVLDTNVMGLVLCSQKAYQSMKKRSVDGHIVHINSVVGHKVFDFPQSNIYPASKHAVTAITETMRNELRNAGSRIKVTSISPGVVRTEILPESIIEGGHSLLESEDISEAVLYVLGTPPRVQVHELTIKPVGEKF.

[0039] The gene encoding the farnesol dehydrogenase FDL22 protein of Aedes aegypti, also known as farnesol dehydrogenase FDL22The nucleotide sequence is as shown in SEQ ID NO.2, specifically: 5’-atggaccgttgggcaggaaaggtggccgtagtgaccggggcaagttcgggaattggagccgccatcactacggacctggccaaagctggaatggtggtcgttggattggcacgtcgagtggaacgggttgaagctttgaaagcaaacttgccggagtctgctaagccgcgtttgcatgcggttaagtgtgacgtatccaaggaggaagacattacccaggtgttcaaatgggttgaggagaaatttgggggagttgacgtgttggtcaacaatgcgggaatccttcggcagacggatttgctgggtaccgataatggtcaaatgctgcgagaagtcttggatacgaatgtgatgggactggtgctctgcagccagaaggcctatcaatcgatgaagaagcgctcggtcgatgggcacattgtccatataaatagcgtcgttggccataaggtttttgatttcccgcaatcgaatatatatccggcgtcgaagcacgcagtgacggccattacggaaactatgagaaacgaattgcggaatgccgggtccagaataaaggttacgagtattagccccggtgtggttcgcactgaaatccttccggagtccattattgaaggaggtcattcgctgttggaatcggaagatatatccgaagctgtattgtacgtgcttggaacacctcctcgagtgcaagtccatgaattgactataaaaccagtaggtgagaagttttga-3’。

[0040] 1. FDL22 Synthesis of dsRNA of FDL22siRNA prediction was performed using siDirect (https: / / sidirect2.rnai.jp / ). The region with the most siRNAs and low off-target efficiency was selected as the dsRNA template. The template sequence SEQ ID NO.3 is: 5'-tgggttgaggagaaatttgggggagttgacgtgttggtcaacaatgcgggaatccttcggcagacggatttgctgggtaccgataatggtcaaatgctgcgagaagtcttggatacgaatgtgatgggactggtgctctgcagccagaaggcctatcaatcgatgaagaagcgctcggtcgatgggcacattgtccatataaatagcgtcgttggccataaggtttttgatttcccgcaatcgaatatata tccggcgtcgaagcacgcagtgacggccattacggaaactatgagaaacgaattgcggaatgccgggtccagaataaaggttacgagtattagccccggtgtggttcgcactgaaatccttccggagtcca ttattgaaggaggtcattcgctgttggaatcggaagatatatccgaagctgtattgtacgtgcttggaacacctcctcgagtgcaagtccatgaattgactataaaaccagtaggtgagaagttttga-3'.

[0041] Based on the dsRNA template sequence, primers with T7 promoter were designed using the primer design online website (https: / / crm.vazyme.com / cetool / simple.html?enzymeLociFlag=Y). The primer sequences are as follows: ds FDL22 F: 5'-taatacgactcactatagggTGGGTTGAGGAGAAATTTGGG-3' (SEQ ID NO.4), ds FDL 22 R: 5'-taatacgactcactatagggTCAAAACTTCTCACCTACTGGTTTTATA-3' (SEQ ID NO. 5).

[0042] RNA was extracted from Aedes aegypti larvae using the TransZol Up Kit (Full Gold). The extracted RNA was subjected to 1% agarose gel electrophoresis, concentration determination, and quality assessment, and the quality was qualified. The HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novozymes) was used to reverse transcribe and synthesize cDNA using 1 µg of RNA as a template. The cDNA was used as a template to amplify the ds FDL22 The PCR product was verified by 1% agarose gel electrophoresis in the template region, revealing a single band. The DNA template was then extracted using the phenol / chloroform method and purified using ethanol / sodium acetate precipitation. dsRNA was synthesized using the TranscriptAid T7 High-Yield Transcription Kit (Thermo Fisher Scientific) using 1 µg of DNA as a template. Finally, the dsRNA was extracted using phenol / chloroform and purified using ethanol / sodium acetate precipitation. After electrophoresis on a 1% agarose gel and concentration determined, the dsRNA was aliquoted and stored at -80°C.

[0043] 2. GFP dsRNA (named ds GFP ) synthesis: using primer ds GFP -F: 5′-taatacgactcactatagggAAGGGCGAGGAGCTGTTCACCG-3′ (SEQ ID NO. 6) and primer ds GFP -R: 5'-taatacgactcactatagggCAGCAGGACCATGTGATCGCGC-3' (SEQ ID NO. 7) was amplified from a plasmid containing a GFP fragment. GFP The template region (SEQ ID NO. 8) was verified by 1% agarose gel electrophoresis, revealing a single band. The DNA template was then extracted using the phenol / chloroform method and purified using ethanol / sodium acetate precipitation. dsRNA was synthesized using the TranscriptAid T7 High-Yield Transcription Kit (Thermo Fisher Scientific) using 1 µg of DNA as a template. Finally, the dsRNA was extracted using phenol / chloroform and purified using ethanol / sodium acetate precipitation. After electrophoresis on a 1% agarose gel and concentration determined, the dsRNA was aliquoted and stored at -80°C.

[0044] The dsRNA of the GFP gene is called ds GFP As a control; FDL22 The dsRNA of a gene is called ds FDL22 .

[0045] The nucleotide sequence of SEQ ID NO.8 is: 5’-aagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcagctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggccaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagccaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctg-3’。

[0046] 3. Delivery of dsRNA ds GFP and ds FDL22 were introduced into Aedes aegypti larvae by the following methods respectively.

[0047] dsRNA was delivered via feeding and immersion. Equal volumes of 1% agarose were mixed with overnight fermented larval food (fish food: yeast = 6.4 mg / mL: 3.2 mg / mL) and placed in a 55°C waterbath. 20 µg of dsRNA was added to 2 mL of the mixture and then filled into one well of a six-well cell culture plate. The mixture was allowed to solidify, after which 40 newly hatched Aedes aegypti larvae were counted and introduced into one well. The next day, 20 µg of dsRNA was dissolved in 400 µL of 0.1% DEPC water and added to a single well. On the third day, half of the larvae in each well were sampled for RNA extraction to examine RNAi efficiency. The other half of the larvae in each well were used for virulence experiments.

[0048] 4. Interference efficiency test: TransZol Up kit (full gold) was used to detect the interference efficiency of ds GFP and ds FDL22 RNA was extracted from the treated larvae. The extracted RNA was subjected to 1% agarose gel electrophoresis, concentration determination, and quality assessment. After the RNA was confirmed to be of acceptable quality, 1 µg of RNA was used as a template for reverse transcription and cDNA synthesis using the HiScript III 1st Strand cDNA Synthesis Kit (with gDNA wiper) (Novozymes). The synthesized cDNA was diluted 10-fold and used for fluorescence quantitative PCR to detect the presence of cDNA. FDL22 The primers used in fluorescent quantitative PCR were designed through an online website (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome) and the sequence information is as follows: q-FDL22 F: 5'-TCGAGTGGAACGGGTTGAAG-3' (SEQ ID NO.9), q-FDL22 R: 5'-ACCTGGGTAATGTCTTCCTCCT-3' (SEQ ID NO.10); quantitative internal reference primers q-RSP17 F: 5'-AAGAAGTGGCCATCATTCCA-3' (SEQ ID NO.11), q-RSP17 R: 5'-GGTCTCCGGGTCGACTTC-3' (SEQID NO.12). qPCR reactions were performed using the ChamQ Universal SYBR qPCR MasterMix kit on a Bio-Rad Real-Time PCR system, using 2 -ΔΔCt The results are shown in Table 1 and Figure 1 shown. Figure 1 For feeding and soaking ds for Aedes aegypti larvae FDL22Significantly reduce farnesol dehydrogenase in Aedes aegypti larvae FDL22 Graph showing the results of mRNA expression levels.

[0049] Table 1 ds GFP and ds FDL22 48 hours after treatment with Aedes aegypti larvae FDL22 The relative expression level

[0050] Note: n=4-6, 4-6 biological replicates were performed; # indicates t The test analysis showed that the mean values ​​of the trifluoxetine + S-(+)-methoprene group and the trifluoxetine group were significantly different ( p <0.05), the same below.

[0051] From Table 1 and Figure 1 Available, delivered by feeding and immersion FDL22 Aedes aegypti larvae FDL22 The expression level of dsFDL22 in Aedes aegypti larvae was reduced by about 40% compared with the control group of Aedes aegypti larvae fed and immersed in dsGFP. FDL22 The expression of FDL22 in Aedes aegypti larvae was achieved, which met the requirements for functional analysis and subsequent dsRNA interference in Aedes aegypti larvae. FDL22 Requirements for the experimental enhancement of the toxicity of trifluoxetine to Aedes aegypti larvae after expression.

[0052] 5. Toxicity test for Aedes aegypti larvae Toxicity test was conducted on the second instar larvae of Aedes aegypti that had not been exposed to trifluanid. GFP Treated larvae and 25 ds FDL22 The treated larvae were immersed in 50 mL of 133.30 mg / L trifluoxetine for 48 h, and the change in mortality was used as the analysis indicator.

[0053] The results are shown in Table 2 and Figure 2 shown. Figure 2 Farnesol dehydrogenase in Aedes aegypti larvae FDL22 The results of the inhibition of mRNA expression level on the toxicity enhancement of 133.30 mg / L trifluoxetine against Aedes aegypti larvae.

[0054] Table 2 ds GFP and ds FDL22 Mortality of Aedes aegypti larvae after immersion in a solution containing 133.30 mg / L trifluoxetine for 48 hours

[0055] From Table 2 and Figure 2 Available, dsFDL22 The mortality rate of Aedes aegypti larvae pretreated for 48 hours after being immersed in 133.30 mg / L trifluoxetine for 48 hours was increased by about 20% compared with that of Aedes aegypti larvae pretreated for 48 hours with dsGFP. FDL22 Under the premise of stable expression, the toxicity of trifluoxetine to Aedes aegypti larvae can be significantly improved. The expression level of FDL22 in Aedes aegypti is negatively correlated with the toxicity of trifluoxetine to larvae.

[0056] Example 2 This example uses Aedes aegypti larvae as a model. By mixing S-(+)-methoprene and trifluoxetine and applying them together, the toxicity of trifluoxetine to Aedes aegypti larvae is significantly enhanced.

[0057] A 3000 mg / L stock solution of S-(+)-methoprene was prepared using acetone as the solvent. This solution was then added to 50 mL of a 133.30 mg / L trifluoxetine solution to achieve final concentrations of 3 mg / L, 6 mg / L, and 10 mg / L S-(+)-methoprene in the mixed solution. Toxicity was assessed in second-instar larvae of Aedes aegypti mosquitoes that had not been exposed to trifluoxetine or S-(+)-methoprene. Twenty-five second-instar larvae from each experimental group were immersed in 50 mL of a mixture of 3 mg / L, 6 mg / L, or 10 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine. Larval mortality was measured after 24 or 48 hours. At the same time, 3 mg / L, 6 mg / L and 10 mg / L S-(+)-methoprene were added as control group 1, and 133.30 mg / L trifluoxetine was added as control group 2. The mortality rate of the larvae in the experimental and control groups was used as the analysis index. The results are shown in Tables 3 to 5 and Figures 3-5 As shown, Figure 3 This is the toxicity enhancement result of a mixture of 3 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine against Aedes aegypti larvae treated for 48 hours; Figure 4 This is the toxicity enhancement result of a mixture of 6 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine against Aedes aegypti larvae treated for 24 hours; Figure 5 This figure shows the toxicity enhancement results of a mixture of 10 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine against Aedes aegypti larvae after treatment for 24 hours.

[0058] Table 3 Mortality of larvae treated with 3 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine for 48 h (%)

[0059] Note: n = 5, 5 biological replicates were performed; different letters (a, b, c) indicate significant differences between groups determined by one-way ANOVA and Tukey's test ( p <0.05), the same below.

[0060] Table 4 Mortality of larvae after 24 h treatment with 6 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine (%)

[0061] Table 5 Mortality of larvae treated with 10 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine for 24 h (%)

[0062] From Tables 3~5 and Figures 3-5 The results showed that the toxicity of a mixture of 3 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine to Aedes aegypti larvae treated for 48 hours was more than three times higher than that of trifluoxetine alone, while 3 mg / L S-(+)-methoprene had no toxicity to Aedes aegypti larvae. The toxicity of 6 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine to Aedes aegypti larvae treated for 24 hours was significantly higher than that of trifluoxetine alone. The toxicity of 10 mg / L S-(+)-methoprene and 133.30 mg / L trifluoxetine to Aedes aegypti larvae treated for 24 hours was significantly higher than that of trifluoxetine alone. In summary, S-(+)-methoprene can significantly enhance the toxicity of trifluoxetine, and the two act on Aedes aegypti larvae, producing a synergistic technical effect in the poisoning and prevention of Aedes aegypti larvae.

[0063] Example 3 This example uses Aedes aegypti larvae as a model to test the effects of S-(+)-methoprene and trifluoxetine on the larvae of Aedes aegypti. FDL22 There is down-regulated expression.

[0064] A mixture of S-(+)-methoprene and 133.30 mg / L trifluanid was applied to 50 second-instar Aedes aegypti larvae for 2 days at a final concentration of 6 mg / L. Surviving larvae were harvested and RNA was extracted from their tissue using the TransZol Up Kit (Full Gold). The extracted RNA was subjected to 1% agarose gel electrophoresis, concentration determination, and quality assessment. After verification, the RNA was reverse transcribed and synthesized into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (with gDNA wiper) (Novozymes) using 300 ng of RNA as a template. The synthesized cDNA was diluted 10-fold and used for fluorescence quantitative PCR. FDL22 The primers and method used in the fluorescence quantitative PCR were the same as those used in the 4 interference efficiency determination in Example 1. The results of fluorescence quantitative PCR are shown in Figure 6 and Table 6. Figure 6 The combination of S-(+)-methoprene and trifluoxetine inhibits farnesol dehydrogenase FDL22 Graph showing the results of mRNA expression levels.

[0065] Table 6 S-(+)-methoprene and trifluoxetine mixed application inhibited FDL22 mRNA expression levels

[0066] From Table 6 and Figure 6 It was found that when 6 mg / L S-(+)-methoprene was mixed with 133.30 mg / L trifluanid, the FDL22 mRNA expression was downregulated, based on implementation case 2, using dsRNA to interfere with the larvae of Aedes aegypti FDL22 , increasing the toxicity of trifluoxetine to Aedes aegypti larvae. FDL22 The mRNA level of is negatively correlated with the toxicity of trifluoxetine. Therefore, the mixed application of S-(+)-methoprene and trifluoxetine in Example 3 enhances the toxicity of trifluoxetine to Aedes aegypti larvae, which may also be due to the inhibition of Aedes aegypti FDL22 expression.

[0067] Example 4 Knockdown of the Agricultural Pest Myzus persicae Using dsRNA Interference FDL22 Homologous genes FDL242 expression, enhancing the toxicity of trifluoxetine to peach aphid.

[0068] The amino acid sequence of farnesol dehydrogenase FDL22 from Aedes aegypti was compared with that from the Genbank database. M. persicae The gene encoding the homologous amino acid sequence is numbered LOC111037242 and named FDL242 , which is Myzus persicae farnesol dehydrogenase FDL242, and has an amino acid sequence similarity of 44.90% with that of Aedes aegypti farnesol dehydrogenase FDL22. Figure 7 Schematic diagram of the alignment of the amino acid sequence of farnesol dehydrogenase FDL22 from Aedes aegypti and the amino acid sequence of farnesol dehydrogenase FDL242 from Myzus persicae.

[0069] The amino acid sequence of green peach aphid farnesol dehydrogenase FDL242 is shown in SEQ ID NO. 13, specifically: MIFQIFSYCLDTGKHWSCSFYNIKIYIFKTKMEKWNGKVAVVTGASSGIGEETCRQLVERGMIVVGFARREDKLQVLENDLKGKLGKFYYVKVDLCSEENIMEAFNWVKSTLKSLDVLINNAGVLKQTDLLGSTKDWKQMFDTNVIGLNICSREAIKIMEQVQIKEGHIININSVAGHYQFPFMKDISVYSTTKHSVTIITESLRELLGMKNLPIRVTSISPAGVETEMTVELSKLEGFKMLKSIDIAEAILYALSAPQRVNVAEIIIRPTSENTLDFIKSLV.

[0070] The gene encoding the green peach aphid farnesol dehydrogenase FDL242, also known as the green peach aphid farnesol dehydrogenase FDL242The nucleotide sequence is as shown in SEQ ID NO.14, specifically: 5’-atgatatttcagattttcagttattgtttggacaccggtaagcactggtcgtgtagtttttataatattaaaatatatatttttaaaacgaaaatggaaaagtggaatggaaaagttgctgtagtgacgggtgcttcttctggtatcggagaggagacttgtcgacaactggttgaaaggggaatgattgttgttggttttgctagaagagaagataaacttcaggtgttggaaaacgatttaaaaggaaaactgggtaaattttattacgttaaagttgatttatgttcggaagaaaacattatggaagcttttaattgggtaaagagtacattgaaatcgttagacgtactaattaacaatgctggtgttttgaagcaaactgatttattaggaagcacaaaagattggaaacaaatgtttgacactaatgtcataggacttaatatctgcagcagagaggccataaaaattatggagcaagtacaaatcaaagagggtcacataataaatattaacagtgttgcaggtcattatcaatttccatttatgaaggatatttcagtttatagcactaccaaacatagtgttactataatcacagagagtttaagagaattgctgggcatgaaaaatttacccatcagagtaacgagtatcagtccggccggagtagaaaccgaaatgactgtagagttaagcaaattggaaggatttaaaatgttgaaatctattgacattgctgaagctattttatacgcgttgagtgcaccgcaacgtgttaacgtcgccgaaatcatcattagaccaacaagtgaaaatacattagattttattaagagtcttgtgtag-3’。

[0071] 1. LC of ethoprophos against the fourth instar nymphs of Myzus persicae 50The LC value of trifluoxetine against the fourth instar nymphs of green peach aphid was determined according to the Agricultural Industry Standard of the People's Republic of China NY / T 1154.6-2006 Pesticide Indoor Bioassay Test Guidelines Insecticides Part 6: Insecticide Activity Test Insect Dipping Method. 50 Each experimental group was tested with 30 fourth-instar nymphs of green peach aphid. The results are shown in Table 7.

[0072] Table 7 Mortality of peach aphid nymphs after treatment with different concentrations of trifluoxetine for different periods of time (%)

[0073] Note: 1-3 are 3 biological replicates.

[0074] 2. FDL242 dsRNA synthesis was performed by querying the mRNA sequence of the gene in the Genbank database using the accession number LOC111037242. siRNA prediction was performed using siDirect (https: / / sidirect2.rnai.jp / ), and a region with the highest possible siRNA yield and low off-target efficiency was selected as the dsRNA template. The template sequence, SEQ ID NO. 15, is: 5'-gacaactggttgaaaggggaatgattgttgttggttttgctagaagagaagataaacttcaggtgttggaaaacgatttaaaaggaaaactgggtaaattttattacgttaaagttgatttatgttcggaagaaaacattatggaagcttttaattgggtaaagagtacattgaaatcgttagacgtactaattaacaatgctggtgttttgaagcaaactgatttattaggaagcacaaaagattgg aaacaaatgtttgacactaatgtcataggacttaatatctgcagcagagaggccataaaaattatggagcaagtacaaatcaaagagggtcacataataaatattaacagtgttgcaggtcattatcaatt tccatttatgaaggatatttcagtttatagcactaccaaacatagtgttactataatcacagagagtttaagagaattgctgggcatgaaaaatttacccatcagagtaacgagtatcagtccggcc-3'.

[0075] Based on the dsRNA template sequence, primers with T7 promoter were designed using the primer design online website (https: / / crm.vazyme.com / cetool / simple.html?enzymeLociFlag=Y). The primer sequences are as follows: ds FDL242 F: 5'-taatacgactcactatagggGACAACTGGTTGAAAGGGGA-3' (SEQ ID NO. 16), ds FDL242 R: 5'-taatacgactcactatagggGGCCGGACTGATACTCGTTA-3' (SEQ ID NO. 17).

[0076] RNA was extracted from fourth-instar nymph tissues of peach aphids using the TransZol Up Kit (Full Gold). The extracted RNA was subjected to 1% agarose gel electrophoresis, concentration determination, and quality assessment, confirming its quality. The HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novozymes) was used to reverse transcribe and synthesize cDNA using 1 µg of RNA as a template. The cDNA was used as a template to amplify the ds FDL242 The PCR product was verified by 1% agarose gel electrophoresis in the template region, revealing a single band. The DNA template was then extracted using the phenol / chloroform method and purified using ethanol / sodium acetate precipitation. dsRNA was synthesized using the TranscriptAid T7 High-Yield Transcription Kit (Thermo Fisher Scientific) using 1 µg of DNA as a template. Finally, the dsRNA was extracted using phenol / chloroform and purified using ethanol / sodium acetate precipitation. After electrophoresis on a 1% agarose gel and concentration determined, the dsRNA was aliquoted and stored at -80°C.

[0077] 3. GFP dsRNA (named ds GFP ) is synthesized in the same manner as in Example 1. The dsRNA of the GFP gene is referred to as ds GFP As a control, the sequence refers to SEQ ID NO. 8 in Example 1; MpFDL242 The dsRNA of a gene is called ds FDL242 .

[0078] 4. Delivery of dsRNA GFP and ds FDL242 The following methods were used to introduce dsRNA into peach aphid nymphs.

[0079] dsRNA was delivered by microinjection. GFPand ds FDL242 Dilute to 10000 ng / μL with 0.1% DEPC water; select the fourth-instar nymphs of peach aphids with uniform growth and place them in a 5 mL centrifuge tube for later use; briefly stun the peach aphid nymphs with carbon dioxide and place them on a solidified 1% agarose gel plate, and use the Nanoinject Ⅲ microinjection system to inject ds GFP or ds FDL242 The injection was performed into the body of peach aphid nymphs at the mid-leg coxae fossa. The injection volume was 30 nL per aphid at a rate of 30 nL / s. After the injection, the peach aphids were placed in a culture dish containing fresh radish seedlings for breeding. Samples were taken at 24 h and 48 h for RNA extraction to check the efficiency of RNA interference.

[0080] 5. Interference efficiency test: TransZol Up kit (full gold) was used to test the interference efficiency of ds GFP and ds FDL242 RNA was extracted from treated peach aphid nymphs. The extracted RNA was subjected to 1% agarose gel electrophoresis, concentration determination, and quality assessment. After the RNA was found to be of acceptable quality, 1 µg of RNA was used as a template for reverse transcription and cDNA synthesis using the HiScript III 1st Strand cDNA Synthesis Kit (with gDNA wiper) (Novozymes). The synthesized cDNA was diluted 10-fold and used for fluorescence quantitative PCR to detect the presence of FDL242 Changes in expression levels. Primers used in fluorescent quantitative PCR were designed using an online website (same as 4 in Example 1). The sequence information is as follows: q-FDL242 F: 5'-GGTGCTTCTTCTGGTATCG-3' (SEQ ID NO. 18), q-FDL242 R: 5'-CCCTCTTTGATTTGTACTTGC-3' (SEQ ID NO. 19); quantitative internal reference primers q-GAPDH F: 5'-ACGGACCATCTGGAAAATTATGGA-3' (SEQ ID NO. 20), q-GAPDH R: 5'-GCAACTGGTACTCTGAAAGCC-3' (SEQ ID NO. 21). qPCR reactions were performed using the ChamQ Universal SYBR qPCR Master Mix kit on a Bio-Rad Real-Time PCR system. -ΔΔCt The results are shown in Table 8 and Figure 8 shown. Figure 8 To inject ds FDL242 Significantly reduced the number of green peach aphid nymphs FDL242 Graph showing the results of mRNA expression levels.

[0081] Table 8 ds GFP and ds FDL242 24 and 48 hours after treatment of peach aphid nymphs FDL242 Relative expression

[0082] From Table 8 and Figure 8 Available, microinjection delivery of ds FDL242 In the body of the green peach aphid nymph FDL242 The expression level of ds GFP The control group decreased by about 64% at 24 h and by about 72% at 48 h. FDL242 Can significantly interfere with green peach aphid FDL242 The expression of FDL242 Knockdown, functional analysis and subsequent dsRNA interference in green peach aphid nymphs FDL242 Requirements for the toxicity test of trifluoxetine to green peach aphid nymphs after expression.

[0083] 6. Toxicity test for green peach aphid nymphs Toxicity test was conducted on green peach aphid nymphs in the fourth instar that had not been exposed to trifluanid according to the leaf dipping method. GFP 48 h later larvae and 23 ds-injected FDL242 After 48 hours, the nymphs were immersed in 128.64 mg / L or 230.16 mg / L triflumuron for 10 seconds and then transferred to fresh radish seedlings for feeding. The mortality rate at 24 and 48 hours was counted, and the change in mortality rate was used as the analysis indicator.

[0084] The results are shown in Tables 9-10 and Figures 9 and 10 shown. Figure 9 Farnesol dehydrogenase in green peach aphid nymphs FDL242 The results of the reduction of mRNA expression level on the toxicity enhancement of 128.64 mg / L trifluoxetine solution to green peach aphid nymphs; Figure 10 Farnesol dehydrogenase in green peach aphid nymphs FDL242 The results of the reduction of mRNA expression level on the toxicity enhancement of 230.16 mg / L trifluanid solution to green peach aphid nymphs.

[0085] Table 9 Mortality of green peach aphid nymphs after treatment with 128.64 mg / L trifluoxetine

[0086] Table 10 Mortality of green peach aphid nymphs after treatment with 230.16 mg / L triflumuron

[0087] From Tables 9~10 and Figures 9 and 10 Available, ds FDL242 The mortality of peach aphid nymphs treated with 128.64 mg / L trifluoxetine for 24 and 48 hours was significantly lower than that of ds GFP The number of peach aphid nymphs treated for 48 hours increased by about 2.90% and 11.60%. FDL242 The mortality of peach aphid nymphs treated with 230.16 mg / L trifluoxetine for 24 and 48 hours was significantly lower than that of ds GFP The number of peach aphid nymphs increased by about 8.69% and 13.04% after 48 hours of treatment. FDL242 Under the premise of expression, the toxicity of trifluoxetine to green peach aphid nymphs can be significantly improved, and the expression level of FDL242 in green peach aphids is negatively correlated with the toxicity of trifluoxetine to larvae.

[0088] In summary, the schematic diagram of the present invention for inhibiting the expression of farnesol dehydrogenase to enhance the toxicity of trifluoxetine is as follows: Figure 11 Aedes aegypti larvae ingest ds FDL22 After knocking down the expression of FDL22 in the body, the toxicity of trifluoxetine to Aedes aegypti larvae was significantly enhanced; the green peach aphid nymphs ingested ds FDL242 Knocking down FDL242 expression in the cells significantly enhanced the toxicity of trifluoxetine to green peach aphid nymphs. Furthermore, the mixed application of S-(+)-methoprene and trifluoxetine also significantly enhanced the toxicity of trifluoxetine to larvae.

[0089] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of farnesol dehydrogenase inhibition in enhancing the toxicity of trifluoxetine.

2. The application according to claim 1, characterized in that The farnesol dehydrogenase includes any one of the following (1) to (3): (1) The amino acid sequence of Aedes aegypti farnesol dehydrogenase FDL22 is shown in SEQ ID NO. 1; the nucleotide sequence encoding the Aedes aegypti farnesol dehydrogenase FDL22 protein is shown in SEQ ID NO. 2; (2) The amino acid sequence of Myzus persicae farnesol dehydrogenase FDL242 is shown in SEQ ID NO. 13; the nucleotide sequence encoding the Myzus persicae farnesol dehydrogenase FDL242 protein is shown in SEQ ID NO. 14; (3) Homologous genes of (1) or (2) in pests other than Aedes aegypti and Myzus persicae.

3. The application according to claim 1, characterized in that Methods for inhibiting the expression of farnesol dehydrogenase include knocking down farnesol dehydrogenase by dsRNA and / or exogenously administering juvenile hormone analogs to inhibit the expression of farnesol dehydrogenase.

4. The application according to claim 3, characterized in that The juvenile hormone analogs include S-(+)-methoprene.

5. A farnesol dehydrogenase from Aedes aegypti FDL22 The dsRNA is characterized in that The nucleotide sequence of the dsRNA is shown in SEQ ID NO.

3.

6. A Myzus persicae farnesol dehydrogenase FDL242 The dsRNA is characterized in that The nucleotide sequence of the dsRNA is shown in SEQ ID NO.

15.

7. A synergistic insecticide, characterized in that: These include dsRNA that inhibits the expression of farnesol dehydrogenase and trifluoxetine.

8. The synergistic insecticide according to claim 7, characterized in that: dsRNA that inhibits the expression of farnesol dehydrogenase includes Aedes aegypti farnesol dehydrogenase FDL22 Myzus persicae farnesol dehydrogenase FDL242 of dsRNA.

9. Use of the dsRNA according to claim 5 or 6 or the synergistic insecticide according to claim 7 or 8 in controlling pests.

10. A method for controlling pests using the synergistic insecticide according to claim 7 or 8, characterized in that: include: When the synergistic insecticide comprises a dsRNA that inhibits the expression of farnesol dehydrogenase, the dsRNA is delivered to the pest, which is then exposed to trifluoxetine.

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