CN116809624A - Application of spider mite cytochrome P450 gene in pesticide degradation - Google Patents
Application of spider mite cytochrome P450 gene in pesticide degradation Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to application of a spider mite cytochrome P450 gene in pesticide degradation. The invention discovers thatCYP392A11The gene of the tetranychus urticae cytochrome P450 shows over-expression in abamectin resistant tetranychus urticae population, and abamectin can be metabolized in vitro after heterologous expression. Therefore, the engineering bacteria expressed by the gene can degrade fruits and vegetables and the like with pesticide residues of avermectin, pre-treat wastewater and sewage, treat soil polluted by pesticides and the like, and has the potential of industrialized development and application. Participation of clonesThe P450 gene with drug resistance can also be used as a target for specifically designing an inhibitor of specific detoxification enzyme, and provides a thinking for the development of novel medicaments in the future.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a spider mite cytochrome P450 gene in pesticide degradation.
Background
Tetranychus urticaeTetranychus urticaeBelongs to the genus Tetranychus of the order Europe, the family Tetranychidae, and is a great agricultural pest mites which are widely distributed and cause harm worldwide. Tetranychus urticae host plants are extremely wide-ranging and can be used for the pest of at least 1100 kinds of host plants including capsicum, eggplant, watermelon, melon, strawberry, etc. (Dermauw et al A link between host plant adaptation and pesticide resistance in the polyphagous spider mite)Tetranychus urticaePNAS, 2013, 110 (2): E113-E122). The two-spotted spider mites, young mites and if mites can pierce plant leaves to cause harm, the damaged leaf mainly shows some white small spots from two sides of a main vein of a leaf stalk, the whole leaf becomes grey white or even dark brown along with the serious damage degree, some plants also show rusty red, photosynthesis of the plant leaf is inhibited, the damaged leaf turns yellow, serious people can scorch and even fall off, and the yield and quality of crops are influenced (Ji Xiuzhi, etc. the damage of the two-spotted spider mites of vegetables, chinese fruits and vegetables, 2007, 124 (5): 36). The mites are usually 15% -20% lost economically when control is lost.
In actual production, control of spider mites often relies on chemical agents. Due to the long-term use of the agents, tetranychus urticae have inevitably developed different degrees of resistance to the various agents. Abamectin is a sixteen-membered macrolide compound with insecticidal, acaricidal and nematicidal activities which is first developed by university of North Mitsui, japan, country, etc. and Merck company, america, and is prepared from Streptomyces griseusStreptomyces avermitilisFermentation production ()Zhong Xuan Avermectin research progress and industry review, J.Chinese antibiotics, 2022, 47 (11): 1139-1148). Avermectin as a nerve agent has excellent contact poisoning and stomach poisoning activities, and is widely used for controlling various pests in fields, such as lepidoptera pests, root-knot nematodes, mites and the like. Inevitably, the selective pressure of the agents brought about by long-term use of the agents causes various pests/mites to develop resistance to the agents, so that chemical control of field pests faces greater difficulties and challenges. Among them, the report of resistance to avermectin by Tetranychus urticae is more common, for example, cases where there is a high level of resistance to avermectin by Tetranychus urticae in foreign areas such as Russian, turkey, greek, the United states, canada, australia etc. (Ç a ğ atay et al Identification and characterization of abamectin resistance in)Tetranychusurticae Koch populations from greenhouses in Turkey. Crop Protection, 2018, 112: 112-117; Herron et al. Development of abamectin resistance in Tetranychusurticae in Australian cotton and the establishment of discriminating doses for T. lambiInd Experimental and Applied Acarology, 2021, 83 (3): 325-341), liu Qingjuan et al (Liu Qingjuan, et al, determination of resistance to seven acaricides and study of its mechanism by Tetranychus urticae, applied insect theory, 2012, 49 (2): 376-381), found that egg phase in Tetranychus urticae and the Alternaria glabra population in the source of the Yi-source was reduced in sensitivity to avermectin, and subsequently also reported successively in 2014-2021 that Tetranychus urticae, shanxi, shandong, zhejiang, hainan et al, in China also showed high resistance or extremely high level resistance to avermectin (Tang et al, stage-specific expression of resistance to different acaricides in four field populations of)Tetranychusurticae (Acari: Tetranychidae). Journal of Economic Entomology, 2014, 107(5): 1900-1907; Xu, et al. Status of pesticide resistance and associated mutations in the two-spotted spider mite, TetranychusurticaeIn China. Pesticide Biochemistry and Physiology, 2018, 150:89-96). Therefore, the problem of the resistance of the two-spotted spider mites to the avermectin at home and abroad is becoming more and more evidentSevere.
The research on the drug resistance mechanism of the tetranychus urticae on abamectin mainly relates to target resistance and metabolic resistance. Earlier studies have found that mutation of the G314D site of the GluCl1 gene in the glutamate-gated chloride channel (Gluamate-gated chloride channel, gluCl) and the G326E site in the GluCl3 gene resulted in resistance of Tetranychus urticae to avermectin (Kwon et al A point mutation in a glutamate-gated chloride channel confers abamectin resistance in the two-spotted spider mite,Tetranychus urticae Koch. Insect Molecular Biology, 2010, 19 (4): 583-591; Dermauw et al. The cys-loop ligand-gated ion channel gene family of Tetranychus urticaeimplications for acaricide toxicology and a novel mutation associated with abamectin resistance Insect Biochemistry and Molecular Biology, 2012, 42 (7): 455-465); the I321T site on the GluCl3 gene was found by the researchers to be likely associated with avermectin resistance (Xue et al Untangling a Gordian knot: the role of a GluCl 3I 321T mutation in abamectin resistance inTetranychus urticaePest Manag. Sci., 2021, 77 (4): 1581-1593), but the site mutation was detected only in European avermectin-resistant Tetranychus urticae red-type populations (Simmaet al Acaricide resistance status and identification of resistance mutations in populations of the two-spotted spider mite)Tetranychus urticaefrom ethiopa. Exp. Appl. Acarol 2020, 82 (4): 475-491). The two-spotted field population in China also has different point mutations of the genes, but the mutation and the resistance multiple of the two-spotted spider mites do not show linear correlation, even some populations have no point mutation frequency of the resistance genes, but the resistance multiple of the abamectin is still high (Xu, et al Status of pesticide resistance and associated mutations in the two-spotted spider mite,Tetranychus urticaein China. Pesticide Biochemistry and Physiology, 2018, 150:89-96). In addition, xu et al find that Piperonyl Butoxide (PBO) has remarkable synergy on two resistant populations by performing a synergist test on two field avermectin resistant populations in China, which shows that the P450 detoxification enzyme is used in two spotsTetranychus urticae plays a key role in the development of avermectin resistance (Xu et al Transcriptome profiling and functional analysis suggest that the constitutive overexpression of four cytochrome P450s confers resistance to abamectin in)Tetranychus urticaefrom China, pest Manag, sci, 2021, 77 (3): 1204-1213), but it was elucidated in further studies whether the P450 gene, which is highly expressed in the field resistant population, was involved in resistance to avermectin by Tetranychus urticae, whether avermectin could be metabolized.
Disclosure of Invention
Based on transcriptome data analysis of resistant and sensitive populations of Tetranychus urticae, the present inventors found a P450 geneCYP392A11(GenBank accession number: MT787325.1, the sequence is shown in SEQ ID No. 1) shows over-expression in avermectin resistant Tetranychus urticae population, and after heterologous expression, avermectin can be metabolized in vitro, and related reports that the gene can participate in the formation of avermectin pesticide resistance of Tetranychus urticae are not seen at home and abroad. The resistance gene can be used as a target gene for detecting the resistance of the tetranychus urticae to abamectin, and can also provide a theoretical basis for a new pesticide target design site; meanwhile, the engineering bacteria expressed by the gene can degrade fruits and vegetables with avermectin pesticide residues, pre-treat wastewater and sewage, treat soil polluted by pesticides and the like, and have the potential of industrialized development and application.
The invention firstly provides an application of a spider mite cytochrome P450 gene or a coded protein thereof in degrading abamectin, wherein the spider mite cytochrome P450 gene isCYP392A11And (3) a gene.
Specifically, the encoded protein is obtained by recombinant expression.
In specific embodiments, the encoded protein is applied to fruit and vegetable degrading pesticide residues, or applied to wastewater or soil contaminated with pesticides to degrade pesticides.
Application of recombinant cells expressing spider mite cytochrome P450 gene in degrading abamectin, wherein the spider mite cytochrome P450 gene isCYP392A11And (3) a gene.
Preferably, the recombinant cell is an insect cell.
More preferably, the recombinant cell is an H5 cell.
Specifically, recombinant cells were obtained by introducing a baculovirus expression system containing the spider mite cytochrome P450 gene into insect cells.
In practical implementation, the recombinant cells are applied to fruits and vegetables to degrade pesticide residues, or applied to wastewater and sewage or soil polluted by pesticide to degrade pesticide.
Specifically, the recombinant cells are prepared into powder, water agent, suspending agent and other forms.
The invention also provides an application of the tetranychus urticae cytochrome P450 gene in the targeted design of the inhibitor of specific detoxification enzyme or in the screening of novel medicaments for preventing and treating tetranychus urticae, wherein the tetranychus urticae cytochrome P450 gene isCYP392A11And (3) a gene.
The invention discovers thatCYP392A11The gene of the tetranychus urticae cytochrome P450 shows over-expression in abamectin resistant tetranychus urticae population, and abamectin can be metabolized in vitro after heterologous expression. Therefore, the engineering bacteria expressed by the gene can degrade fruits and vegetables and the like with pesticide residues of avermectin, pre-treat wastewater and sewage, treat soil polluted by pesticides and the like, and has the potential of industrialized development and application. The cloned P450 gene participating in drug resistance can also be used as a target for specifically designing an inhibitor of specific detoxification enzyme, and provides a thought for the development of novel medicaments in the future.
Drawings
FIG. 1, tetranychus urticaeCYP392A11Amplifying the gene fragment. Wherein M: DNAMarker III (200, 500, 800,1,200,2,000,3,000,4,500 bp from bottom to top); 1: the indoor sensitive population Lab-IPP of the tetranychus urticae, 2-6 are field resistant populations of the tetranychus urticae: MY-BJ, FS-GD, SY-HN, WF-SD, LY-HN populations, respectively.
FIG. 2, tetranychus urticae resistant populationCYP392A11Relative expression level of the gene. Wherein the expression level of the sensitive population gene is set to 1. Average ± standard error, n=3, indicated in the figureHas significant difference (P)< 0.001）。
Figure 3, mortality after two concentrations of abamectin treatment (B) of expression level after gene silencing (a) of the spider mite resistant population CYP392a 11.
Figure 4, analysis of avermectin metabolites by the spider mite P450 protein. Wherein, (A) an abamectin chromatogram; (B) CYP392A11 protein and abamectin, and NADPH reaction chromatogram is not added; (C) CYP392A11 protein and abamectin are added with an NADPH reaction chromatogram.
Detailed Description
1. Materials and methods
1.1 Test insects
The indoor sensitive population (Lab-IPP) of Tetranychus urticae is given away by natural enemy insect research group of plant protection institute of China national academy of sciences, and kidney beans are used in artificial climate box by sponge water isolation platform method for a long timePhaseolus vulgarisLinn) leaves were raised until now, without any chemical exposure. The feeding conditions are as follows: temperature 26±1 ℃, humidity 60±5% and L: d=16h: photoperiod of 8 h. The two-spotted spider mite population is raised by using 'Bifeng' bean variety.
Five test field populations used in the study were collected from host plants such as Beijing Micloud (MY-BJ), guangdong Buddha mountain (FS-GD), hainan three-layer (SY-HN), shandong Luoyang (WF-SD) and Henan Luoyang (LY-HN), respectively. The two-spotted spider mite population collected in the field is placed on clean and insect-free kidney bean plants in a climatic chamber for breeding (breeding conditions are as above), and is used for bioassay and other experiments after being subjected to indoor breeding for 2-3 generations.
1.2 Main reagent
RNA extraction reagent Trizol (Invitrogen, carlsbad, calif., USA); cDNA reverse transcription kit PrimeScript ® RT reagent Kit (TaKaRa); q5 ultra-fidelity DNA polymerase (New England Biolabs, NEB); monarch PCR&DNA purification kit (NEB, england); cloning vector pEASY-Blunt Cloning Vector vector (Beijing full gold Biotechnology Co., ltd.); X-Gal and IPTG (Beijing Tiangen Biochemical technologies Co., ltd.); fastFire rapid fluorescent quantitative PCRPremixing the reagent; dsRNA synthesis kit AmpliScribe TM T7- Flash TM Transcription Kit (Lucigen); other laboratory chemicals (analytically pure) are commercially available. PCR primer synthesis and amplification product sequencing were both delegated to be completed by Beijing qingke biotechnology Co.
1.3 Main instrument
Stereoscopic microscope (Olinbas SZX-7, japan); intelligent artificial climate box (RXZ-380C, ningbo Jiangnan Instrument Co.); gradient PCR apparatus (Bio-Rad S1000 and Bio-Rad C1000); nucleic acid electrophoresis systems and Gel imaging systems (Gel Doc EQ) (Bio-Rad, usa); water bath (Beijing Liuyi manufacturing plant); full-automatic sample rapid grinding instrument (Shanghai Jing Xin technology Co., ltd.); high speed centrifuge (Sigma 3K15, germany); ultraviolet spectrophotometer NanoDrop 2000c (Thermo Scientific); fluorescent quantitative PCR apparatus (ABI Quantum studio 3, simer Feishmanic technologies); moist heat autoclave (japan sanyang); ultrapure water meter (ZMQ VOTI Mini Q pure water meter); micropipettes (Eppendorf, germany).
1.4 Biological assay of Tetranychus urticae
The drug resistance of Tetranychus urticae was determined by an optimized agar leaf dipping method (Xu et al Transcriptome profiling and functional analysis suggest that the constitutive overexpression of four cytochrome P450s confers resistance to abamectin in)Tetranychus urticaefrom China, pest Manag, sci., 2021, 77 (3): 1204-1213), the specific steps of operation are as follows: preparing agar with a proper volume and a concentration of 0.1%, dripping the agar melted by a microwave oven on the bottom of a plastic dish by using a suction tube, and cooling to solidify at room temperature. The insecticide was diluted to 5-7 concentrations with distilled water. The bean leaves are beaten into small leaf discs by a puncher with the diameter of 2.5 and cm, the leaf discs are fully immersed in the liquid medicine by forceps, the time is 10s, the bean leaves are taken out and dried, and then the dried leaf discs are spread on plastic dish agar back to back. 4 replicates were set for each agent concentration and leaf discs immersed in distilled water and air dried were placed in the blank. After the work is completed, healthy female adult mites with consistent physiological state are picked up by a zero-number writing brushAnd (3) 25-30 leaf mites are arranged on each leaf dish, then a culture dish upper cover with very small holes is covered, and the leaf mites are transferred into an intelligent artificial climate box for feeding and observation, wherein the temperature is set to be 26+/-1 ℃, the humidity is set to be 60+/-5%, and the illumination period is set to be 16h:8h (L: D). After 24 h is fed, the number of the live and dead spider mites is checked and recorded under a stereoscopic microscope, the spider mites are lightly touched by a writing brush tip, and only 1 person with enough activity or complete immobility is judged to be dead. Note that the spider mite samples that escaped the leaf dish and stuck to the agar did not count up.
1.5 RNA extraction and cDNA Synthesis
1 mL TRIzol and two sterilized steel balls were added to the centrifuge tube containing the spider mite sample, and the mixture was placed in a low temperature tissue mill for sufficient milling, followed by incubation at room temperature for 5 min. 0.2. 0.2 mL chloroform was added, immediately vigorously shaken for 30s, and placed on ice for 5 min. Centrifuging at 12000 rpm at 4deg.C for 15 min; sucking the supernatant solution into a new centrifuge tube, adding precooled isopropanol with the same volume, gently mixing up and down, and standing on ice for 30-60 min. Centrifuge at 12000 rpm for 10 min at 4℃and aspirate the supernatant from the tube with a pipette, then add 1 mL of 75% ethanol for washing the pellet. Centrifuging at 7500 rpm at 4deg.C for 5 min, sucking out ethanol, standing at room temperature for 5 min to volatilize ethanol completely, adding appropriate amount of DEPC water, and dissolving RNA completely. Finally, the concentration and the integrity of RNA are detected by a micro ultraviolet spectrophotometer and electrophoresis.
cDNA synthesis was performed using the TaKaRa Kit PrimeScript Kit, and the synthesis procedure was performed according to the Kit instructions. Briefly, first, the RNase-removed centrifuge tube was added with the genomic DNA remover gDNA Eraser and its buffer, then with total RNA, and finally with the RNase-removed double distilled water to 10. Mu.L, and rapidly placed in a PCR apparatus at 42℃for 2min. Adding 4.0 [ mu ] L of 5 XPimeScript buffer to the PCR tube from which the DNA had been removed; RT Prime Mix 1.0 [ mu ] L; primeScriptRT Enzyme Mix 1.0.0 [ mu ] L and 4.0 [ mu ] L of double distilled water were put into a PCR instrument, kept at 37℃for 15 minutes, and kept at 85℃for 5 s to inactivate reverse transcriptase, thereby obtaining reverse transcribed cDNA.
1.6 CYP392A11Gene clone and its expression quantity detection
Based on the cDNA of Tetranychus urticae, the primer pair CYP392A11-L upstream primer is adopted: 5'-ATGCAAAAAGTTATGTCTTTATTGG-3' (SEQ ID No. 2), CYP392A11-R downstream primer: 5'-TCAGTCAGAATTGGAAATTTTCTC-3' (SEQ ID No. 3), clone obtained two-spotted spider miteCYP392A11The gene CDS is full-length. The PCR amplification procedure was: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 59℃for 20s, extension at 72 for 40s,35 cycles; finally, the extension is carried out at 72 ℃ for 2min. And (3) connecting and converting the PCR amplification result after recovering the PCR amplification result in a gel recovery kit, and carrying out colony PCR verification to obtain positive clone spots, then sending the positive clone spots to Beijing qingke biotechnology company for sequencing, and analyzing the sequencing result.
CYP392A11Gene expression levels were detected using qRT-PCR with three biological replicates per population. The qRT-PCR primer is an upstream primer: 5'-TCCCTGGTGCTGTTGATGAC-3' (SEQ ID No. 4), downstream primer: 5'-GCTCGTACCAAGACCGACAT-3' (SEQ ID No. 5) with the action gene as an internal reference gene. The fluorescent quantitative PCR result is 2 according to CT value of target gene and reference gene -ΔΔCT The method performs data analysis. Differences between sample results groups were analyzed for one-way variance using ANOVA in SPSS 19.0 software, for significance of differences using Turkey's method, and for results plotted using GraphPad Prism 8 software.
1.7 Tetranychus urticaeCYP392A11RNA interference assay of Gene
Firstly, placing healthy two-spotted She Manci mites into a centrifuge tube for starving 24 h, then placing a bean leaf dish of 1.5 cm into a baking oven at 60 ℃ for baking for 2min, then soaking the leaves into 500 ng/ml target gene dsRNA, standing for 3 h, setting two negative controls of clear water control and enhanced green fluorescent protein EGFP at the same time, picking up two-spotted spider mites after starving on the bean leaf dish with dsRNA, taking 48 h of the two-spotted spider mites freely, picking up living two-spotted spider mites for total RNA extraction and fluorescence quantitative detection test of the target gene, and setting action as an internal reference gene. After detection, the target gene is silenced, two different concentrations of abamectin are adopted to treat the two-spotted spider mites which survive after interference, and three repetitions are set.
1.8 CYP392A11 protein heterologous expression and in vitro metabolism
By double enzyme digestionCYP392A11Gene cloning into pFastBac vector recombinant baculovirus DNA was transfected into sf9 cells for subculture according to the bac-to-bac baculovirus expression system (invitrogen, USA) and protein expression was performed using High Five (H5) cells. Adding P3 and CPR P3 virus into H5 cells, suspending and culturing in a shaking table at 27 ℃ and 135 rpm/min, collecting cells after 48 and H, and detecting the content and activity of the expressed P450 protein by adopting a carbon monoxide difference spectrum method.
The recombinant target protein with activity detected by the differential spectrum detection is reacted in PBS (PH 7.4) buffer solution, an NADPH reaction system (A: 10 mu L, B: 2 mu L) of Promega and a 55 mu M avermectin standard substance are added, 3 h is reacted in a vibrating metal bath at 1,200rpm/min and 30 ℃, then 800 mu L of acetonitrile is added to terminate the reaction, the reaction is reacted in the vibrating metal bath for 20 min, then the centrifugation is carried out at 20,000g for 10 min, 200 mu L of supernatant is sucked into a chromatographic bottle, and the reaction system without NADPH is processed by contrast. The samples were subjected to HPLC-MS detection.
2. Results and analysis
2.1 Resistance of Tetranychus urticae field population to avermectin
The spider mite Lab-IPP adopted by the control shows a sensitive state to the population avermectin. The five field populations tested all belonged to extremely high levels of resistance to avermectin, at 5,567-100,000 fold (table 1).
TABLE 1 resistance of different field populations of Tetranychus urticae to avermectin insecticides
2.2 Gene cloning and sequence analysis
Based on the genome data of the tetranychus urticae, the clone is obtainedCYP392A11The CDS full-length sequence of the gene, 1,512 bp (FIG. 1), encodes 503 amino acids, and predicts a protein molecular weight of 57.25 kDa, with typical P450 conserved domains, such as WxxxR domains (positions381-393), the reader domain (at 1,242-1,263), the heme-binding domain (PFxxGxxxCxG at 1,314-1,341) (sequence information shown in SEQ ID NO. 1).
2.3 CYP392A11Expression level of genes in different field populations
Compared with sensitive population, the two-spotted spider mite is found in 5 field populations of two-spotted spider mites collected from Beijing Micloud, guangdong Buddha mountain, hainan three-way, henan Luoyang and Weifang birthday lightCYP392A11Genes exhibited significantly higher expression in these test populations (fig. 2).
2.4 Tetranychus urticaeCYP392A11Post-gene interference sensitivity assay
To test the efficiency of interference, gene silencing was performed on the spider mite resistant population, and fluorescent quantitative PCR was performed on surviving spider mites after dsCYP392a1148 h of feeding. The results indicate that, relative to control treated EGFP,CYP392A11the gene expression level is obviously reduced by 70.58%, which indicates thatCYP392A11The gene was effectively silenced (A in FIG. 3). Then, MY-BJ population is selected for interference, and the sensitivity of the MY-BJ population to avermectin is determined. The results indicate that MY-BJ populationCYP392A11After gene disruption, mortality increased by 13.43% and 14.93% at two concentrations of abamectin 200 and 400 mg/L, respectively (B in fig. 3). The results demonstrate thatCYP392A11The gene is related to the resistance of the tetranychus urticae to abamectin.
2.5 In vitro metabolism of abamectin by CYP392A11 protein
The constructed recombinant protein is determined to have correct folding by a reduction type CO difference spectrum method, and after the activity is checked, the in vitro metabolism condition of CYP392A11 protein on abamectin is determined. The metabolic results are shown in fig. 4, and the results show that the CYP392A11 protein derived from the two-spotted spider mites can successfully metabolize abamectin in vitro under the condition of having NADPH (providing electrons for P450), and a metabolite (hydroxy-abamectin) peak of abamectin is generated (C in fig. 4).
The invention clones and obtains a P450 gene which participates in the drug resistance of the mite to avermectin insecticide from the inside of the tetranychus urticaeCYP392A11The gene generates resistance to avermectin in the fieldThe population has over-expression phenomenon, so the gene can be used as a molecular marker to assist in early detection and early warning of abamectin insecticide by the field tetranychus urticae population. The expressed protein of the gene can successfully metabolize abamectin in vitro, so that the engineering bacteria expressed by the gene can degrade fruits and vegetables and the like with abamectin pesticide residues, perform pretreatment of wastewater and sewage, perform soil treatment polluted by pesticides and the like, and have industrial development and application potential. The cloned P450 gene participating in drug resistance can also be used as a target for specifically designing an inhibitor of specific detoxification enzyme, and provides a thought for the development of novel medicaments in the future.
Claims (10)
1. Application of tetranychus urticae cytochrome P450 gene or coded protein thereof in degrading abamectin is characterized in that the tetranychus urticae cytochrome P450 gene isCYP392A11And (3) a gene.
2. The use according to claim 1, wherein the encoded protein is obtained by recombinant expression.
3. The use according to claim 1, wherein the encoded protein is applied to fruit and vegetable degrading pesticide residues or to wastewater or pesticide contaminated soil to degrade pesticides.
4. Application of recombinant cells expressing tetranychus urticae cytochrome P450 genes in degrading abamectin, wherein the tetranychus urticae cytochrome P450 genes areCYP392A11And (3) a gene.
5. The use of claim 4, wherein the recombinant cell is an insect cell.
6. The use of claim 4, wherein the recombinant cell is an H5 cell.
7. The use according to claim 6, wherein the recombinant cell is obtained by introducing a baculovirus expression system containing the spider mite cytochrome P450 gene into an insect cell.
8. The use according to any one of claims 4 to 7, wherein the recombinant cells are applied to fruit and vegetable degrading pesticide residues or to wastewater or pesticide contaminated soil to degrade pesticides.
9. The use according to claim 8, wherein the recombinant cells are prepared in the form of powders, mistura, suspensions.
10. Application of tetranychus urticae cytochrome P450 gene as target in targeted design of inhibitor of specific detoxification enzyme or in research and development and screening of novel medicament for preventing and treating tetranychus urticae, wherein the tetranychus urticae cytochrome P450 gene isCYP392A11And (3) a gene.
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