Insecticidal compositions against b. tabaci

Abstract

The present invention discloses a kind of insecticide composition for whitefly, comprising: inhibitor and imidacloprid, inhibitor is letrozole or 1-benzimidazole. The potential of inhibitor as insecticide synergist is further studied by experimental group, and their effects on imidacloprid-resistant adult whitefly are tested. When letrozole and 1-benzimidazole are used with imidacloprid respectively, the mortality of imidacloprid-resistant adult whitefly increases to 91% and 96.3% respectively, while imidacloprid alone is 68%.

Description

[0001] (This application is a divisional application of application number 202310028986.2, which pertains to a method for screening insect P450 enzyme substrates and inhibitors based on yeast.) Technical Field

[0002] This invention belongs to the field of recombinant expression enzyme activity technology, specifically relating to the application of a compound in inhibiting pesticide resistance. Background Technology

[0003] Cytochrome P450 enzymes represent a family of autooxidative heme proteins, also known as cytochrome P450 monooxygenases, and are widely involved in the metabolism of endogenous substances and exogenous substances, including drugs, pesticides, and other compounds. Originating from various organisms from animals to insects, cytochrome P450 enzymes contain heme as a cofactor and exhibit strong absorption at 450 nm. Studies have shown that P450 is a key enzyme in human drug metabolism and also a major metabolic enzyme contributing to pesticide resistance in insects. Currently, two common methods are microsome preparation and whole-cell biotransformation to study P450-mediated drug metabolism. Microsome preparation requires a lengthy centrifugation process, which leads to decreased enzyme activity, and microsomes inevitably contain other P450 enzymes, resulting in low specificity. In whole-cell biotransformation, the substrates or inhibitors required for the reaction inevitably need to cross biological barriers, such as cell walls and cell membranes, significantly reducing the efficiency of drug metabolism and failing to accurately reflect the enzyme-substrate relationship. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide the application of recombinant yeast strain LXP450 expressing insect P450 enzymes in screening substrates for insecticides metabolized by insect P450 enzymes or inhibitors of insect P450 enzymes.

[0005] Another object of the present invention is to provide a method for constructing the above-mentioned yeast strain LXP450.

[0006] Another object of the present invention is to provide the yeast strain LXP450 obtained by the above construction method.

[0007] Another object of the present invention is to provide an enzyme package prepared using yeast LXP450.

[0008] Another object of the present invention is to provide a method for screening luciferin substrates as insect P450 enzyme probes.

[0009] Another object of the present invention is to provide a method for screening insecticide substrates metabolized by insect P450 enzymes using luciferin substrates as probes.

[0010] Another object of the present invention is to provide a method for screening insect P450 enzyme inhibitors using luciferin substrates as probes.

[0011] Another object of the present invention is to provide letrozole / 1-benzylimidazole as an inhibitor in mitigating the detoxification of agricultural pests by pymetrozine / imidacloprid.

[0012] Another object of the present invention is to provide 1-benzylimidazole as an inhibitor for the application of mitigating the detoxification of agricultural pests by pesticides.

[0013] Another object of the present invention is to provide letrozole as an inhibitor for mitigating the detoxification of agricultural pests by pesticides.

[0014] The objective of this invention is achieved through the following technical solution.

[0015] Application of a recombinant yeast strain LXP450 expressing insect P450 enzyme in screening substrates for insecticides metabolized by insect P450 enzyme or inhibitors of insect P450 enzyme.

[0016] A method for constructing a yeast strain LXP450 that recombinantly expresses insect P450 enzymes, comprising the following steps:

[0017] S1. Using the insect P450 gene as a template, PCR amplification and purification were performed to obtain the amplified and purified PCR product; the PCR product and the first vector were used to construct a vector responsible for expressing insect P450.

[0018] In S1, the insect is a Lepidoptera, Homoptera, or Coleoptera insect.

[0019] In the above technical solutions, the Homoptera insects are brown planthoppers (nilaparvatalugens), whiteflies (Bemisia tabaci), black spiny whiteflies (Aleurocanthus spiniferus), gray whiteflies (Laodelphax striatellus), mulberry silkworms (Bombyx mandarina), peach aphids (Myzus persicae), cabbage aphids (Lipaphis erysimi), and cabbage aphids (Brevicoryne brassicae (Linnaeus)). The Lepidoptera insects are fall armyworms (Spodoptera frugiperda), soybean loopers (C. includens), codling moths (Cydia pomonella), beet armyworms (Spodoptera littoralis), cotton bollworms (Helicoverpa zea), sugarcane borers (Sugarcane borer), tobacco bud borers (Heliothis virescens), and diamondback moths (Plutella xylostella). xylostella), beet armyworm (Spodoptera exigua), cabbage armyworm (Mamestra configurata), kaleidoscope armyworm (Mamestra brassicae), black rootworm (Agrotis ipsilon), western rootworm (Agrotis orthogonia), and grain armyworm (A).The following insects are listed: subterranea, kapok beetle (Alabama argillacea), powdery armyworm (Trichoplusia ni), soybean armyworm (Pseudoplusia includens), bean armyworm (Anticarsiagemmatalis), alfalfa green armyworm (Hypena scabra), one-spotted armyworm (Pseudaletia unipuncta), rough-skinned armyworm (Athetis mindara), dark-edged cutworm (Euxoa messoria), Egyptian diamondback moth (Earias insulana), green diamondback moth (Earias vittella), grain armyworm (Heliothis zea), striped caterpillar (Melanchra picta), or citrus armyworm (Egira(Xylomyges)curialis); Coleoptera insects include yellow striped flea beetle (Phyllotreta striolata), Northeast large black scarab beetle (Holotrichia diomphalia), and black velvet scarab beetle (Serica orientalis). The species include the green-brown scarab beetle (Anomala corpulenta Motschulsky), the large black scarab beetle (Holotrichia obilita Falderma on), the potato beetle (Colorado potatobeetle), the sweet potato weevil (Cylas formicarius), and the grain beetle (Trogoderma granarium).

[0020] S2. The vector responsible for expressing insect P450 was introduced into yeast cells expressing cytochrome P450 oxidoreductase, and cultured to obtain recombinant yeast LXP450 that co-expresses cytochrome P450 oxidoreductase and insect P450.

[0021] In the above technical solution, insect P450 includes the CYP3 group, CYP4 group, mitochondrial CYP group, and CYP2 group. The CYP3 group includes: CYP6, CYP9, CYP28 family, CYP308-310 family, CYP317 family, CYP321 family, CYP324 family, CYP329 family, CYP332 family, CYP336-338 family, CYP345-348 family, CYP354 family, CYP357-358 family, CYP365 family, CYP395-400 family, CYP408 family, and CYP413 family. The CYP4 group includes: CYP4, CYP311-3... 13. The CYP316 family, CYP318 family, CYP325 family, CYP340-341 family, CYP349-352 family, CYP380 family, CYP367 family, CYP405 family, and CYP411-412 family; the mitochondrial CYP group includes: CYP12, CYP49, CYP301-302 family, CYP314-315 family, CYP333-334 family, CYP339 family, CYP353 family, and CYP366 family; the CYP2 group includes: CYP15, CYP18, CYP303-307 family, CYP343 family, CYP359 family, and CYP369 family.

[0022] In S2, the yeast may be Pichia pastoris, Saccharomyces cerevisiae, or fission yeast.

[0023] The yeast strain LXP450 obtained by the above construction method.

[0024] In S2, the method for constructing yeast cells expressing cytochrome P450 oxidoreductase includes the following steps:

[0025] Step 1. Using the cytochrome P450 oxidoreductase gene as a template, perform PCR amplification and purification. Construct a vector responsible for expressing cytochrome P450 oxidoreductase using the PCR product obtained from the cytochrome P450 oxidoreductase gene and a second vector.

[0026] Step 2. Introduce the vector responsible for expressing cytochrome P450 oxidoreductase into yeast cells to obtain yeast cells expressing cytochrome P450 oxidoreductase.

[0027] An enzyme package prepared using yeast LXP450, the preparation method of which includes the following steps.

[0028] Step 1: Pick yeast LXP450 and spread it on the first culture medium. Add leucine and thiamine to the first culture medium and incubate at 25-35℃ for 36-72h. Then transfer the yeast LXP450 to the second culture medium and continue to incubate at 25-35℃ for 36-72h.

[0029] In step 1, leucine at a final concentration of 0.05–0.2 g / L and thiamine at a final concentration of 5–10 μM are added to the first culture medium.

[0030] In the above technical solution, the second culture medium is liquid Edinburgh basal medium, YEPD medium, BMGY medium or YPD medium, the first culture medium is a mixture of agar and culture medium, the culture medium is the same as the second culture medium, and the concentration of agar in the first culture medium is 10-40 g / L.

[0031] Step 2: Take the yeast LXP450 cells obtained in Step 1, collect the cells and resuspend them, permeate them on a shaker for at least one hour, wash the permeated cells multiple times, and obtain the enzyme package.

[0032] In step 2, the resuspension is performed using Tris-KCl buffer, and the cells are washed and permeated with ice-cold NH4HCO3 buffer.

[0033] A method for screening luciferin substrates as probes for screening insect P450 enzymes includes the following steps:

[0034] Each fluorescein substrate to be screened was prepared into a sample group. Each sample group was prepared as follows: the fluorescein substrate was dissolved in phosphate buffer to obtain a fluorescein precursor mixture with a concentration of 100-150 μM. An enzyme packet was added to the fluorescein precursor mixture, and the NADPH regeneration system was added to start the enzyme catalytic reaction. The reaction was carried out at 30-40°C for at least 3 hours. After centrifugation, the mixture was mixed with an equal volume of fluorescein detection reagent and incubated at room temperature (20-25°C) for 15-30 minutes to obtain a sample group. Each sample group included two samples. The enzyme packets for the two samples were prepared by yeast strain LXP450 and yeast strain expressing cytochrome P450 oxidoreductase, respectively. The fluorescence intensity of the two samples in a sample group was tested using an ELISA reader, and a T-test was performed. When p was less than 0.05, the fluorescein substrate of the sample group was considered a selected probe.

[0035] In the above technical solution, an equal volume of NADPH regeneration system is added to initiate the enzyme catalytic reaction.

[0036] In the above technical solution, the number of enzyme packets added to the luciferin precursor mixture corresponding to every 5 mmol of luciferin substrate is at least 5*10. 7 indivual.

[0037] In the above technical solution, when p is less than 0.01, the fluorophore substrate of the sample group is the probe obtained through screening.

[0038] A method for screening insecticide substrates metabolized by insect P450 enzymes using luciferin substrates as probes includes the following steps:

[0039] Each insecticide to be screened was prepared into a sample group. Each sample group consisted of: a pre-reaction system made by mixing fluorescein substrate, potassium phosphate buffer, insecticide aqueous solution, and water, wherein the insecticide aqueous solution was a mixture of insecticide and water; an enzyme packet was added to the pre-reaction system and mixed well; the mixture was pre-incubated at 30-40°C for 10-20 minutes; the NADPH regeneration system was added to start the reaction; the reaction was carried out at 30-40°C for at least three hours; and centrifuged. The supernatant was mixed with an equal volume of fluorescein detection reagent and incubated at room temperature (20-25°C) for 20 minutes to obtain a sample group. Each sample group consisted of two samples, one with an enzyme packet prepared using yeast LXP450 and the other with an enzyme packet prepared using yeast expressing cytochrome P450 oxidoreductase.

[0040] In a sample group, if the fluorescence intensity of the sample obtained by using an enzyme pack prepared with yeast LXP450 is less than 80% of the fluorescence intensity of the sample obtained by using an enzyme pack prepared with yeast expressing cytochrome P450 oxidoreductase, then the insecticide used in that sample group is the substrate of the insecticide metabolized by the screened insect P450 enzyme.

[0041] In the above technical solution, the concentration of the insecticide in the pre-reaction system is 100 μmol / L to 1 mmol / L.

[0042] In the above technical solution, the ratio of fluorescein substrate, potassium phosphate buffer, insecticide aqueous solution and water by volume is (0.5-1.5):(1-2):(6-13):(10-17), and an equal volume of NADPH regeneration system is added to start the reaction.

[0043] In the above technical solution, the number of enzyme packets added to the pre-reaction system corresponding to each 1.5 μL of luciferin substrate is at least 5 * 10. 7 indivual.

[0044] In the above technical solution, the fluorochrome substrate is EE, CEE, FEE, BuE, PE, FBuE or CPE.

[0045] A method for screening insect P450 enzyme inhibitors using a luciferin substrate as a probe includes the following steps:

[0046] Each inhibitor to be screened was prepared into a sample group. Each sample group consisted of: mixing fluorescein substrate, potassium phosphate buffer, inhibitor aqueous solution and water to obtain a pre-reaction system; adding enzyme packet to the pre-reaction system and mixing well; pre-incubating at 30-40℃ for 10-20 minutes; adding NADPH regeneration system to start the reaction; reacting at 30-40℃ for at least three hours; centrifuging; mixing the obtained supernatant with an equal volume of fluorescein detection reagent; and incubating at room temperature (20-25℃) for 20 minutes to obtain the sample group.

[0047] Each sample group includes two samples, one containing an enzyme pack prepared using yeast LXP450 and the other containing an enzyme pack prepared using yeast expressing cytochrome P450 oxidoreductase.

[0048] In a sample group, if the fluorescence intensity of the sample obtained by using an enzyme package prepared with yeast LXP450 is less than 70% of the fluorescence intensity of the sample obtained by using an enzyme package prepared with yeast expressing cytochrome P450 oxidoreductase, then the inhibitor used in that sample group is the insect P450 enzyme inhibitor obtained through screening.

[0049] In the above technical solution, the concentration of the inhibitor in the pre-reaction system is 10–100 μmol / L.

[0050] In the above technical solution, the ratio of fluorescein substrate, potassium phosphate buffer, inhibitor aqueous solution, and water by volume is (0.5-1.5):(1-2):(6-13):(10-17), and an equal volume of NADPH regeneration system is added to start the reaction.

[0051] In the above technical solution, the number of enzyme packets added to the pre-reaction system corresponding to each 1.5 μL of luciferin substrate is at least 5 * 10. 7 indivual.

[0052] In the above technical solution, the fluorochrome substrate is EE, CEE, FEE, BuE, PE, FBuE or CPE.

[0053] The method for preparing the enzyme package from the yeast expressing cytochrome P450 oxidoreductase includes the following steps:

[0054] Step 1: Select yeast strains expressing cytochrome P450 oxidoreductase and spread them on the first culture medium. Add leucine to the first culture medium and incubate at 25-35°C for 36-72 hours. Then transfer them to the second culture medium and continue to incubate at 25-35°C for 36-72 hours.

[0055] In step 1, leucine is added to the first culture medium at a final concentration of 0.05–0.2 g / L.

[0056] Step 2: Take the yeast cells obtained in Step 1, collect the cells and resuspend them, permeate them on a shaker for at least one hour, wash the permeated cells multiple times, and obtain the enzyme package.

[0057] In step 2, resuspension was performed using Tris-KCl buffer, and after washing and permeation, the cells were washed with ice-cold NH4HCO3 buffer.

[0058] 1-Benzylimidazol is used as an inhibitor in mitigating the detoxification of agricultural pests by pesticides.

[0059] Letrozole is used as an inhibitor in mitigating the detoxification of pesticides by agricultural pests.

[0060] In the above technical solution, the agricultural pests are leaf-eating pests, piercing-sucking pests, borer pests, or underground pests.

[0061] In the above technical solution, the leaf-eating pests are lepidopteran leaf-eating pests, coleopteran leaf-eating pests, or hymenopteran leaf-eating pests. The lepidopteran leaf-eating pests are bagworms, tussock moths, silkworm moths, geometrid moths, pyralid moths, dead leaf moths, boat moths, fall webworms, Chinese scholar tree geometrid moths, or swallowtail butterflies. The coleopteran leaf-eating pests are leaf beetles, and the hymenopteran leaf-eating pests are sawflies.

[0062] Piercing-sucking pests include aphids, scale insects, whiteflies, psyllids, leafhoppers, stink bugs, thrips, or spider mites;

[0063] Boring pests include Lepidoptera (wood-boring moths, clearwing moths), Coleoptera (longhorn beetles, bark beetles, jewel beetles, weevils), Hymenoptera (horn beetles), or Isoptera (termites).

[0064] Underground pests include mole crickets and crickets (Orthoptera), tigers (Lepidoptera), grubs and wireworms (Coleoptera), or seed flies (Diptera).

[0065] In the above technical solution, the insecticide is a pyridine insecticide, a triazine insecticide, or a neonicotinoid insecticide.

[0066] The application of 1-benzylimidazol / letrozol as an inhibitor in mitigating the detoxification of imidacloprid by agricultural pests.

[0067] In the above technical solution, the method of using 1-benzylimidazole / letrozole as an inhibitor is as follows: the inhibitor and imidacloprid are mixed and used together, wherein the ratio of the inhibitor to imidacloprid is 1:(1-10) by molar amount.

[0068] The application of 1-benzylimidazol / letrozol as an inhibitor in mitigating the detoxification of pymetrozine by agricultural pests.

[0069] In the above technical solution, the method of using 1-benzylimidazole / letrozole as an inhibitor is as follows: the inhibitor and pymetrozine are mixed and used together, wherein the ratio of the inhibitor to pymetrozine is 1:(1-10) by molar amount.

[0070] An insecticide composition for whiteflies includes: an inhibitor and imidacloprid, wherein the inhibitor is letrozole or 1-benzylimidazole.

[0071] In the above technical solution, the ratio of inhibitor to imidacloprid in the insecticide composition for whiteflies is 1:1, based on the amount of substances.

[0072] This invention utilizes recombinant expression of P450 membrane proteins in yeast, leveraging the efficient electron-transferring ability of cytochrome P450 oxidoreductase to assist in the functional expression of insect p450. Through the penetration of a detergent (Triton X-100), several pores are formed on the cell membrane. Luciferin substrates, insecticides, and inhibitors enter the cell through these pores and are metabolized by P450. The luciferin metabolites mix with a luciferin detection reagent to produce fluorescence. The presence or absence of insect-specific substrates and inhibitors is determined by whether candidate compounds significantly reduce the cold light produced by the luciferin substrate.

[0073] This enzyme pack system helps luciferin substrates, pesticides, and inhibitors overcome biological barriers and freely enter the cell to interact with recombinantly expressed proteins in vivo. This avoids the lengthy fractionation process and more realistically simulates the natural state of enzymes in vivo and their interaction patterns with small molecules compared to existing technologies. It also has higher specificity. With the help of luciferin substrates and luciferin detection reagents, enzyme activity can be characterized in 20 minutes, avoiding the lengthy centrifugation process. This makes the entire system more efficient and faster than traditional methods for screening specific insect P450 metabolic substrates and inhibitors.

[0074] Through screening, this invention discovered that letrozole and 1-benzylimidazole are potent insect P450 gene inhibitors that can be used to slow down insecticide detoxification and reduce insect resistance. Attached Figure Description

[0075] Figure 1 The fluorescence of different fluorophore substrates in Example 5;

[0076] Figure 2 The activities of (a) different insecticides, (b) different concentrations of pymetrozine aqueous solution and (c) different concentrations of imidacloprid aqueous solution in Example 6;

[0077] Figure 3The activities of (a) different inhibitors in Example 7, (b) different concentrations of letrozole aqueous solution in Example 7, (c) different concentrations of 1-benzylimidazole aqueous solution in Example 7, (d) different insecticides and inhibitors in Example 7, and (e) the mortality rate of adult whiteflies in Example 8;

[0078] Figure 4 The glass tube is from Example 8. Detailed Implementation

[0079] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0080] The human cytochrome P450 oxidoreductase gene (Cytochrome P450 oxidoreductase gene) shown in SEQ ID NO.1 and the whitefly CYP6CM1 gene (insect P450 gene) shown in SEQ ID NO.2 were both purchased from Anhui General Biotechnology Co., Ltd. (http: / / www.generalbiol.com / ).

[0081] Plasmid pCAD1 (second vector) was obtained from the AddGene Collection (https: / / www.addgene.org) with number 18941. Plasmid pINT was prepared according to the literature. CA,Zearo S,Hannemann F,Bernhardt R,BureikM.Efficient conversion of 11-deoxycortisol to cortisol(hydrocortisone)byrecombinant fission yeast Schizosaccharomyces pombe.FEMS Yeast Res.2005Apr;5(6-7):621-5.doi:10.1016 / j.femsyr.2004.12.001.PMID:15780661. Obtained by transformation;

[0082] The plasmid pREP1 (first vector) was obtained by modifying plasmid pUC119 (number 50010) from the Addgene Collection Center (https: / / www.addgene.org) according to the literature Maundrell K. Thiamine-repressible expression vector spREP and pRIP for fission yeast. Gene. 1993 Jan 15; 123(1):127-30. doi:10.1016 / 0378-1119(93)90551-d.PMID:8422996.

[0083] The fission yeast strain NCYC2036 (yeast) was purchased from the NCYC Collection Center with the number 2036 fission yeast strain.

[0084] Example 1

[0085] A fission yeast CAD62 (a yeast expressing cytochrome P450 oxidoreductase) is constructed by the following steps:

[0086] Step 1. Design the forward primer as shown in SEQ ID NO.3 and the reverse primer as shown in SEQ ID NO.4. Using the human cytochrome P450 oxidoreductase gene as a template, perform PCR amplification to obtain the PCR amplification product. Purify the PCR amplification product by agarose gel electrophoresis to obtain the amplified and purified PCR product. Establish an enzyme digestion system and double-digest the amplified and purified PCR product and plasmid pCAD1 (enzyme digestion sites BamHI / NdeI). Purify and recover the product by agarose gel electrophoresis. Use the Gibson Seamless Assembly Kit (purchased from TransGen Biotech, Beijing) to ligate the amplified gene fragments to plasmid pCAD1 to obtain the vector pCAD1-CPR responsible for expressing human cytochrome P450 oxidoreductase.

[0087] The PCR amplification reaction system included: 10 μL of 2×SuperStar HiFi PCR premix (purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.), 1 μL of the forward primer shown in SEQ ID NO.3, 1 μL of the reverse primer shown in SEQ ID NO.4, 0.5 μL of human cytochrome P450 oxidoreductase gene, and 7.5 μL of double-distilled water. The PCR program was as follows: 95℃ pre-denaturation for 2 min; followed by 30 cycles, with each cycle consisting of 95℃ denaturation for 20 s, 65℃ annealing for 20 s, 72℃ extension for 2 min, and 72℃ extension for 5 min.

[0088] Step 2. Thaw the frozen fission yeast NCYC2036 (genotype h-ura4-D18) parental strain cells in a 40°C water bath for two minutes. Quickly add 1 μL of NotI-digested vector pCAD1-CPR, 5 μL of vector DNA (purchased from Beijing Solarbio Science & Technology Co., Ltd.), and 145 μL of 50% PEG-4000 (purchased from Beijing Solarbio Science & Technology Co., Ltd.) sequentially to 100 μL of a solution containing 10% NotI. 8 Cell suspensions of the parental strains were incubated at 43°C for 15 min to obtain cell suspensions. The cell suspensions were then mixed with an equal volume of TE buffer (10 mmol Tris-HCl, 1 mmol EDTA, pH 7.5) and plated. The mixtures were incubated at 30°C for 3 days to obtain fission yeast CAD62 with the genotype h-ura4-D.18leu1::pCAD1-CPR.

[0089] Example 2

[0090] A fissification yeast LX6CM1 (recombinant yeast LXP450 expressing insect P450 enzyme), the construction method of fissification yeast LX6CM1 includes the following steps:

[0091] S1. Design forward primers as shown in SEQ ID NO.5 and reverse primers as shown in SEQ ID NO.6. Using the whitefly CYP6CM1 gene (purchased from Anhui General Biotechnology Co., Ltd.) as a template, perform PCR amplification to obtain PCR amplification products. Purify the PCR amplification products by agarose gel electrophoresis to obtain amplified and purified PCR products. Establish an enzyme digestion system and double-digest the amplified and purified PCR products and plasmid pREP1 (restriction sites BamHI / NdeI). Purify and recover the products by agarose gel electrophoresis. Use the Gibson Seamless Assembly Kit (purchased from Beijing TransGen Biotech Co., Ltd.) to ligate at 50℃ for 60 minutes to ligate the amplified gene fragments to plasmid pREP1 to obtain vectors responsible for expressing whitefly CYP6CM1.

[0092] Machine sequencing revealed that the vector is responsible for expressing the sequence of the whitefly CYP6CM1, as shown in SEQ ID NO.7.

[0093] The PCR amplification reaction system included: 10 μL of 2×SuperStar HiFi PCR premix (purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.), 1 μL of the forward primer shown in SEQ ID NO.5, 1 μL of the reverse primer shown in SEQ ID NO.6, 0.5 μL of the whitefly CYP6CM1 gene, and 7.5 μL of double-distilled water. The PCR amplification program was as follows: 95℃ pre-denaturation for 2 min; followed by 30 cycles, with the following parameters for each cycle: 95℃ denaturation for 20 s, 65℃ annealing for 20 s, 72℃ extension for 2 min, and 72℃ extension for 5 min.

[0094] S2. Thaw the frozen fissillar yeast CAD62 cells from Example 1 in a 40°C water bath for two minutes. Sequentially add 1 μL of the vector expressing whitefly CYP6CM1, 5 μL of vector DNA (purchased from Beijing Solarbio Science & Technology Co., Ltd.), and 145 μL of 50% PEG-4000 (purchased from Beijing Solarbio Science & Technology Co., Ltd.) to 100 μL of a solution containing 10... 8 The cells of *Schizosaccharomyces CAD62* were mixed in a cell suspension and incubated at 43°C for 15 min to obtain a cell suspension. The cell suspension was then mixed with an equal volume of TE buffer (10 mmol Tris-HCl, 1 mmol EDTA, pH 7.5) and plated. The mixture was incubated at 30°C for 3 days to obtain a recombinant *Schizosaccharomyces cerevisiae* LX6CM1 with the genotype h-ura4-D.18leu1::pCAD1-CPR / pREP1-Bt CYP6CM1, co-expressing human cytochrome P450 oxidoreductase and whitefly CYP6CM1.

[0095] Example 3

[0096] An enzyme package, the preparation method of which includes the following steps:

[0097] Step 1: Select the fission yeast LX6CM1 from Example 2 and spread it on solid Edinburgh basal medium. Add leucine to the solid Edinburgh basal medium to a final concentration of 0.1 g / L and thiamine to a final concentration of 5 μM. Add thiamine to improve the growth of yeast. Incubate at 30°C for 3 days. Then transfer the fission yeast LX6CM1 to liquid Edinburgh basal medium and continue to incubate at 30°C for 36 h at a rotation speed of 230 rpm.

[0098] Step 2, take 5*10 of the fission yeast LX6CM1 obtained in Step 1. 7Cells were collected by centrifugation at 5000 g for 5 minutes and resuspended in 1 mL of 0.3% Triton X-100 Tris-KCl buffer (100 mM Tris-HCl, 200 mM KCl, pH 7.8). The cells were incubated on a shaker at 230 rpm and 30 °C for one hour. The incubated cells were washed three times with 1 mL of 50 mM ice-cold NH4HCO3 (0–4 °C, pH 7.8) buffer to remove residual detergent, yielding the enzyme package.

[0099] Example 4 (Comparative)

[0100] An enzyme package, the preparation method of which includes the following steps:

[0101] Step 1: Select the fission yeast CAD62 from Example 1 and spread it on solid Edinburgh basal medium. Add leucine to the solid Edinburgh basal medium to a final concentration of 0.1 g / L and incubate at 30°C for 3 days. Then transfer the fission yeast CAD62 to liquid Edinburgh basal medium and continue to incubate at 30°C for 36 hours at a speed of 230 rpm.

[0102] Step 2, take 5*10 of fissile yeast CAD62 7 Cells were collected by centrifugation at 5000 g for 5 minutes and resuspended in 1 mL of 0.3% Triton X-100 Tris-KCl buffer (100 mM Tris-HCl, 200 mM KCl, pH 7.8). The cells were incubated on a shaker at 230 rpm and 30 °C for one hour. The incubated cells were washed three times with 1 mL of 50 mM ice-cold NH4HCO3 (0–4 °C, pH 7.8) buffer to remove residual detergent, yielding the enzyme package.

[0103] Liquid Edinburgh Basal Medium:

[0104]

[0105]

[0106] Solid Edinburgh basal medium:

[0107] Potassium hydrogen phthalate 3.00 <![CDATA[Na2HPO4]]> 2.20 <![CDATA[NH4Cl]]> 5.00 <![CDATA[MgCl2.6H2O]]> 1.05 <![CDATA[CaCl2.2H2O]]> 0.0147 KCl 1.00 Sodium sulfate 0.04 pantothenic acid 0.001 niacin 0.01 Inositol 0.01 Biotin 0.001 <![CDATA[H3BO3]]> 0.0005 <![CDATA[MnSO4]]> 0.0004 <![CDATA[ZnSO4.7H2O]]> 0.0004 <![CDATA[FeCl3,6H2O]]> 0.0002 molybdic acid 40mcg KI 0.0001 <![CDATA[CuSO4.5H2O]]> 40mcg Citric acid 0.001 Agar 25

[0108] Example 5

[0109] A method for screening luciferin substrates as insect P450 enzyme probes includes the following steps: Each luciferin substrate to be screened is prepared into a sample group, and each sample group is as follows:

[0110] 5 mmol of luciferin substrate was dissolved in phosphate buffer (80.2 mL of a mixture of 1 M K₂HPO₄ and 19.8 mL of 1 M KH₂PO₄, pH 7.4) to obtain a luciferin precursor mixture with a luciferin substrate concentration of 150 μM. 5 × 10⁻⁶ mmol of luciferin substrate was added to the luciferin precursor mixture. 7 One enzyme packet was used, and an equal volume of NADPH regeneration system (1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, 0.40 U / ml glucose-6-phosphate dehydrogenase) was added to initiate the enzyme-catalyzed reaction. The reaction mixture was reacted at 37°C with shaking at 1000 rpm for 3 hours. The reaction could be terminated by centrifugation at 16000g for 1 minute. An equal volume of fluorescein detection reagent (purchased from Promega, USA) was mixed and incubated at room temperature (20–25°C) for 20 minutes to obtain the sample group.

[0111] Each sample group includes two samples. The enzyme packs for the two samples are prepared using the enzyme pack prepared by *Schizosaccharomyces cerevisiae* LX6CM1 in Example 3 and the enzyme pack prepared by *Schizosaccharomyces cerevisiae* CAD62 in Example 4, respectively (i.e., there are two samples in one sample group, and the only difference between the two samples is the enzyme pack, one of which is prepared by *Schizosaccharomyces cerevisiae* LX6CM1 and the other by *Schizosaccharomyces cerevisiae* CAD62). The fluorescence intensity of the two samples in one sample group is tested using an ELISA reader, and a T-test is performed. When P is less than 0.05, the fluorophore substrate of that sample group is the probe obtained through screening.

[0112] After preparing sample groups using different fluorochrome substrates as shown in Table 1, the fluorescence intensity of two samples in the sample group was tested using an ELISA reader. Figure 1 As shown, Figure 1 The black bars represent data obtained using the enzyme packet from Example 3 and a microplate reader. Figure 1 The gray bars represent data obtained using the enzyme labeler described in Example 4. Figure 1 Samples marked with "***" have a p-value less than 0.001; samples marked with "**" and "***" have a p-value less than 0.01; and samples marked with "*", "**", and "***" have a p-value less than 0.05. Figure 1 The fluorescein substrates used in the sample groups marked with “***”, “**” and “*” can all be used as screening probes for insect P450 enzymes.

[0113] All of these luciferin substrates are protected by an ether bond attached to the 6-carbon position and modified to become the original luciferin substrates. Among them, Luciferin-2FBEME ( Figure 1 2FBEME), Luciferin-3FBEME Figure 13FBEME), Luciferin-TFM2FEME ( Figure 1 TFM2FEME and Luciferin-3TEME Figure 1 The luciferin substrates (3TEME) have carboxylic acid esters instead of carboxyl groups, requiring an additional activation step via deesterification catalyzed by LDR esterases. Catalyzed by human cytochrome P450 oxidoreductases, the O-dealkylation of these luciferin substrates releases a free hydroxyl group at the 6'-carbon, which reacts with luciferase to produce luminescence. Interestingly, all four luciferin substrates showed no significant activity differences in LX6CM1 and CAD62, while the other eight showed significant differences. Among them, Luciferin-FEE ( Figure 1 The signal and selectivity of Luciferin-FEE in LX6CM1 were approximately 8-fold higher than in CAD62, indicating that it is a potential substrate for the CYP6CM1 gene in the whitefly. Therefore, Luciferin-FEE was selected as the optimal probe for the following experiments.

[0114] Table 1

[0115]

[0116]

[0117] Example 6

[0118] Example 6-1

[0119] A method for screening insecticide substrates metabolized by insect P450 enzymes using luciferin substrates as probes includes the following steps:

[0120] Each insecticide to be screened was prepared into a separate sample group. Each sample group consisted of: 1.5 μL Luciferin-FEE, 1 μL potassium phosphate buffer, 12.5 μL insecticide aqueous solution, and 10 μL water, forming a pre-reaction system. The insecticide aqueous solution was a mixture of insecticide and water, and the concentration of the insecticide in the pre-reaction system was 500 μmol / L. 5 × 10⁻⁶ ppm of Luciferin-FEE was added to the pre-reaction system. 7The enzyme packets were mixed thoroughly and pre-incubated at 37°C and 1500 rpm for 10 minutes. An equal volume of NADPH regeneration system was added to start the reaction, which was carried out at 37°C and 1500 rpm for three hours. The reaction was then terminated by centrifugation at 16000g for 1 minute. The resulting supernatant was mixed with an equal volume of luciferin detection reagent (purchased from Promega, USA) and incubated at room temperature (20-25°C) for 20 minutes to obtain the sample group. Each sample group included two samples, with the enzyme packets prepared using *Schizosaccharomyces cerevisiae* LX6CM1 in Example 3 and *Schizosaccharomyces cerevisiae* CAD62 in Example 4, respectively.

[0121] In a sample group, when the fluorescence intensity of the sample obtained using the enzyme pack prepared by *Schizosaccharomyces cerevisiae* LX6CM1 divided by the fluorescence intensity of the sample obtained using the enzyme pack prepared by *Schizosaccharomyces cerevisiae* CAD62 (defined as the activity value) is less than 80%, the insecticide used in that sample group is the substrate of the insecticide metabolized by the screened insect P450 enzyme.

[0122] Activity values ​​of sample groups obtained using different insecticides, such as Figure 2 As shown in a, the insecticide is chlorantraniliprole, deltamethrin, λ-cyhalothrin, spirodiclofen, fipronil, dinotefuran, imidacloprid, or pymetrozine. Figure 2 As shown in a, among the eight insecticides tested, imidacloprid and pymetrozine reduced the luminescence signal by approximately 2 and 3 times, respectively, while the other insecticides did not show significant effects.

[0123] Example 6-2

[0124] A pre-reaction system was prepared by mixing 12.5 μL of imidacloprid aqueous solution with 1.5 μL of Luciferin-FEE, 1 μL of potassium phosphate buffer, and 10 μL of water. The concentrations of imidacloprid in the pre-reaction system were 0.05, 0.1, 0.2, 0.4, 0.6, 1, 2, 4, 6, and 10 mmol / L. 5 × 10⁵ mmol / L of imidacloprid was added to the pre-reaction system. 7The enzyme packets from Example 3 or Example 4 were mixed and pre-incubated at 37°C and 1500 rpm for 10 minutes. An equal volume of NADPH regeneration system was added to start the reaction, which was carried out at 37°C and 1500 rpm for three hours. The reaction was then terminated by centrifugation at 16000g for 1 minute. The resulting supernatant was mixed with an equal volume of luciferin detection reagent (purchased from Promega, USA) and incubated at room temperature (20–25°C) for 20 minutes. The enzyme readings were then recorded using a microplate reader. When the concentration of imidacloprid in the pre-reaction system was fixed, the microplate reader reading obtained from the enzyme packet in Example 3 was divided by the microplate reader reading obtained from the enzyme packet in Example 4 (the result was defined as the activity value). The activity values ​​of imidacloprid at different concentrations in the pre-reaction system were as follows: Figure 2 As shown in c.

[0125] Example 6-3

[0126] 12.5 μL of pymetrozine aqueous solution was mixed with 1.5 μL of Luciferin-FEE, 1 μL of potassium phosphate buffer, and 10 μL of water to obtain pre-reaction systems. The concentrations of pymetrozine in the pre-reaction systems were 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 1, 2, 4, 6, and 10 mmol / L. 5 × 10⁵ mmol / L of pymetrozine was added to the pre-reaction systems. 7 The enzyme packets from Example 3 or Example 4 were mixed and pre-incubated at 37°C and 1500 rpm for 10 minutes. An equal volume of NADPH regeneration system was added to initiate the reaction. The reaction was carried out at 37°C and 1500 rpm for three hours, followed by centrifugation at 16000g for one minute to terminate the reaction. The resulting supernatant was mixed with an equal volume of luciferin detection reagent (purchased from Promega, USA) and incubated at room temperature (20–25°C) for 20 minutes. The enzyme readings were then recorded using a microplate reader. When the concentration of pymetrozine in the pre-reaction system was fixed, the microplate reader reading obtained from the enzyme packet in Example 3 was divided by the microplate reader reading obtained from the enzyme packet in Example 4 (the result was defined as the activity value). The activity values ​​at different concentrations of pymetrozine in the pre-reaction system were as follows: Figure 2 As shown in b.

[0127] Depend on Figure 2 b and Figure 2 As shown in c, the maximum half-maximum inhibitory concentrations (IC50) of pymetrozine and imidacloprid are 248.8 μM and 395.9 μM, respectively, indicating that the CYP6CM1 gene of the whitefly has a high selectivity for pymetrozine.

[0128] Example 7

[0129] Example 7-1

[0130] A method for screening insect P450 enzyme inhibitors using luciferin substrates as probes includes the following steps:

[0131] Each inhibitor to be screened was prepared as a sample group. Each sample group consisted of: 1.5 μL Luciferin-FEE, 1 μL potassium phosphate buffer, 12.5 μL inhibitor aqueous solution, and 10 μL water, mixed to obtain a pre-reaction system. The inhibitor aqueous solution was a mixture of inhibitor and water, and the concentration of the inhibitor in the pre-reaction system was 20 μmol / L. 5 × 10⁻⁶ ppm of Luciferin-FEE was added to the pre-reaction system. 7 The enzyme packets were mixed thoroughly and pre-incubated at 37°C and 1500 rpm for 10 minutes. An equal volume of NADPH regeneration system was added to start the reaction, which was carried out at 37°C and 1500 rpm for three hours. The reaction was then terminated by centrifugation at 16000g for 1 minute. The resulting supernatant was mixed with an equal volume of luciferin detection reagent (purchased from Promega, USA) and incubated at room temperature (20-25°C) for 20 minutes to obtain the sample group. Each sample group included two samples, with the enzyme packets prepared using *Schizosaccharomyces cerevisiae* LX6CM1 in Example 3 and *Schizosaccharomyces cerevisiae* CAD62 in Example 4, respectively.

[0132] In a sample group, when the fluorescence intensity of the sample obtained by using an enzyme reader with enzyme package prepared by fissile yeast LX6CM1 is divided by the fluorescence intensity of the sample obtained by using enzyme package prepared by fissile yeast CAD62 (defined as activity value) and is less than 70%, the inhibitor used in that sample group is the insect P450 enzyme inhibitor obtained through screening.

[0133] Sample groups were prepared using different inhibitors, including synergist ether (PBO), 1-amino-benzotriazole, ketoconazole, clotrimazole, econazolenitrate, miconazole, letrozole, or 1-benzylimidazole. The activity values ​​of the sample groups prepared with different inhibitors are shown below. Figure 3 As shown in a, as Figure 3 As shown in a, among these inhibitors, letrozole and 1-benzylimidazole showed inhibitory effects of 36% and 80% on the luminescence signal (inhibition effect = 1 - activity value), respectively. However, at a final concentration of 20 μM in the pre-reaction system, the other inhibitors did not show significant inhibitory effects on the luminescence signal.

[0134] The structural formula of letrozole is as follows:

[0135]

[0136] The structural formula of 1-benzylimidazole is as follows:

[0137]

[0138] Example 7-2

[0139] A pre-reaction system was prepared by mixing 12.5 μL of 1-benzylimidazole aqueous solution, 1.5 μL of Luciferin-FEE, 1 μL of potassium phosphate buffer, and 10 μL of water. The 1-benzylimidazole aqueous solution was a mixture of 1-benzylimidazole and water. The concentrations of 1-benzylimidazole in the pre-reaction system were 0.005, 0.02, 0.5, 2, 8, 20, 40, 200, and 800 μM. 5 × 10⁵ μM of 1-benzylimidazole was added to the pre-reaction system. 7 The enzyme packets from Example 3 or Example 4 were mixed and pre-incubated at 1500 rpm for 10 minutes at 37°C. An equal volume of NADPH regeneration system was added to start the reaction, which was carried out at 1500 rpm for 3 hours at 37°C. The reaction was then terminated by centrifugation at 16000g for 1 minute. The resulting supernatant was mixed with an equal volume of luciferin detection reagent (purchased from Promega, USA) and incubated at 20–25°C for 20 minutes. The enzyme readings were then recorded using a microplate reader. When the concentration of 1-benzylimidazole in the pre-reaction system was fixed, the microplate reader reading obtained from the enzyme packet in Example 3 was divided by the microplate reader reading obtained from the enzyme packet in Example 4 (the result was defined as the activity value). The activity values ​​obtained at different concentrations of 1-benzylimidazole in the pre-reaction system are shown below. Figure 3 As shown in c.

[0140] Example 7-3

[0141] A pre-reaction system was prepared by mixing 12.5 μL of letrozole aqueous solution, 1.5 μL of Luciferin-FEE, 1 μL of potassium phosphate buffer, and 10 μL of water. The letrozole aqueous solution was a mixture of letrozole and water. The concentrations of letrozole in the pre-reaction system were 4, 12, 40, 80, 160, 400, and 800 μM. 5 × 10⁵ μM of letrozole was added to the pre-reaction system. 7 The enzyme packets from Example 3 or Example 4 were mixed and pre-incubated at 37°C and 1500 rpm for 10 minutes. An equal volume of NADPH regeneration system was added to start the reaction, which was carried out at 37°C and 1500 rpm for three hours. The reaction was then terminated by centrifugation at 16000g for 1 minute. The resulting supernatant was mixed with an equal volume of luciferin detection reagent (purchased from Promega, USA) and incubated at room temperature (20–25°C) for 20 minutes. The enzyme readings were then recorded using a microplate reader. When the letrozole concentration in the pre-reaction system was fixed, the microplate reader reading obtained from the enzyme packet in Example 3 was divided by the microplate reader reading obtained from the enzyme packet in Example 4 (the result was defined as the activity value). The activity values ​​obtained at different letrozole concentrations in the pre-reaction system are shown below. Figure 3 As shown in b.

[0142] Depend on Figure 3 b and Figure 3 As can be seen from c, the IC50 values ​​of letrozole and 1-benzylazole are 23.74 μM and 1.30 μM, respectively.

[0143] Further verification was conducted on their effects on the metabolism of imidacloprid and pymetrozine.

[0144] A mixture of equal volumes of 2 mM insecticide aqueous solution and 80 μM inhibitor aqueous solution was prepared to obtain a mixture in which the insecticide was imidacloprid (IM) or pymetrozine (PM), and the inhibitor was letrozole or 1-benzylimidazole. 12.5 μL of this mixture was then mixed with 1.5 μL Luciferin-FEE, 1 μL potassium phosphate buffer, and 10 μL water to obtain a pre-reaction system. 5 × 10⁻⁶ ppm of the pre-reaction system was added to the mixture. 7 Mix the enzyme packets from Example 3 or Example 4 thoroughly, pre-incubate at 1500 rpm for 10 minutes at 37°C, add an equal volume of NADPH regeneration system to start the reaction, react at 1500 rpm for 3 hours at 37°C, centrifuge at 16000g for 1 minute to terminate the reaction; mix the resulting supernatant with an equal volume of luciferin detection reagent (purchased from Promega, USA), incubate at room temperature (20-25°C) for 20 minutes, and read the enzyme reading using a microplate reader. The activity value is defined as the microplate reader reading obtained using the enzyme packet from Example 3 divided by the microplate reader reading obtained using the enzyme packet from Example 4. The activity value is as follows: Figure 3 As shown in figure d, letrozole reduced the luminescence signal by 8% and 4% compared to pymetrozine and imidacloprid, respectively; while 1-benzylimidazole reduced the signal by 31% and 37%, respectively, indicating that 1-benzylimidazole is a strong inhibitor of the CYP6CM1 gene in whiteflies and can be used to slow down the detoxification of pymetrozine and imidacloprid. Figure 3 The method represented by "IM" in d is basically the same as that in Example 6-1, except that in this example, "the insecticide is only imidacloprid, and the concentration of the insecticide in the pre-reaction system is 500 μM". Figure 3 The method represented by "PM" in d is basically the same as that in Example 6-1, the only difference being that in this example, "the insecticide is only pymetrozine, and the concentration of the insecticide in the pre-reaction system is 250 μM".

[0145] Example 8

[0146] All bioassays were performed on adult imidacloprid-resistant whiteflies (Mediterranean, "Q" type, captured on the surface of chili crops in Yangling, Shaanxi).

[0147] Control group: Sucrose (Guangdong Xilong Science Co., Ltd., China), yeast extract (Thermo Scientific, MA, USA), imidacloprid (Shanghai Yuanye Co., Ltd., China) and water were mixed to obtain the control group feed solution. The concentration of sucrose in the control group feed solution was 30 g / L, the concentration of yeast extract in the control group feed solution was 5 g / L, and the concentration of imidacloprid in the control group feed solution was 50 mM.

[0148] Experimental group: The feed solution in the experimental group was basically the same as that in the control group. The only difference was that the feed solution in the experimental group contained an inhibitor. The concentration of the inhibitor in the feed solution in the experimental group was 50 mM. The inhibitor was letrozole, 1-benzylimidazole or synergist.

[0149] like Figure 4 As shown, a transparent glass tube with a length of 50 mm and an inner diameter of 20 mm was prepared. A sealing film was placed on the right end of the glass tube. 100 μL of feed solution (either the experimental or control group feed solution) was placed on the side of the sealing film closest to the glass tube. Another sealing film was placed over the feed solution, minimizing air bubbles, to form a double-sealed pouch containing the feed solution for the whiteflies to feed on. The left end of the glass tube was placed on a leaf infested with whiteflies, and the leaf was gently tapped to allow 25 adult whiteflies to fly into the glass tube. Another sealing film was then stretched and used to seal the left end of the glass tube, with several small holes for ventilation. A black light-blocking sleeve was wrapped around the curved surface of the glass tube. The tube was placed in an incubator with a light source at the right end. The incubation conditions were 25°C, 14 hours of light per day (the light source was turned off at other times), and 80% humidity (higher humidity prevents nutrient solution evaporation). After 48 hours, the mortality rate was calculated by counting the surviving insects.

[0150] All experiments were repeated three times, and the average was calculated. The mortality rate of adult whiteflies fed the control group's feed solution was as follows: Figure 3 As indicated by "IM" in "e", the mortality rate of adult whiteflies in the experimental group when the inhibitor was letrozole was as follows: Figure 3 As shown in “IM+letrozole” in the e, the mortality rate of adult whiteflies in the experimental group when the inhibitor was 1-benzylimidazole was as follows: Figure 3 As shown in "IM+1-benzylimidazole" in the e, the mortality rate of adult whiteflies in the experimental group when the inhibitor was a synergistic ether was as follows: Figure 3The "IM+PBO" designation in section e is shown. Further investigation into the potential of inhibitors as insecticide synergists was conducted using experimental groups, testing their effects on imidacloprid-resistant adult whiteflies. When letrozole and 1-benzylimidazole were used in combination with imidacloprid, the mortality rates of imidacloprid-resistant adult whiteflies increased to 91% and 96.3%, respectively, compared to 68% when imidacloprid was used alone. In contrast, PBO did not significantly alter the mortality rate.

[0151] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. An insecticide composition for whiteflies, characterized in that, include: Inhibitors include imidacloprid, with letrozole or 1-benzylimidazole being the inhibitors.

2. The insecticide composition according to claim 1, characterized in that, The ratio of inhibitor to imidacloprid in the insecticide composition for whiteflies is 1:1, based on the amount of substances involved.

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