CN114480316B - Cytochrome P450 and application thereof in degradation of pesticide residues - Google Patents

Cytochrome P450 and application thereof in degradation of pesticide residues Download PDF

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CN114480316B
CN114480316B CN202210339970.9A CN202210339970A CN114480316B CN 114480316 B CN114480316 B CN 114480316B CN 202210339970 A CN202210339970 A CN 202210339970A CN 114480316 B CN114480316 B CN 114480316B
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organophosphorus
gene
tetranychus urticae
cytochrome
resistance
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王少丽
徐丹丹
张友军
谢文
吴青君
郭兆将
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Abstract

The invention discloses a cytochrome P450, the amino acid sequence of which is shown as SEQ ID NO: 2, the P450 gene has an over-expression phenomenon in populations which generate low-level to medium-level resistance to organophosphorus in fields, can be used as a molecular marker to assist in realizing early detection and early warning on whether the field tetranychus urticae populations generate drug resistance to organophosphorus insecticides, and has a simple and rapid detection process. The gene can metabolize chlorpyrifos and pirimiphos-methyl in vitro and is expected to metabolize other organophosphorus insecticides, so that engineering bacteria expressed by the gene can degrade fruits, vegetables and the like with organophosphorus insecticide pesticide residues, pretreat wastewater and sewage, treat soil polluted by pesticides and the like, and have industrial development and application potential. The cloned P450 gene involved in drug resistance can also be used as a target for specifically designing an inhibitor of a specific detoxification enzyme and providing a thought for the development of a novel medicament in the future.

Description

Cytochrome P450 and application thereof in degradation of pesticide residues
Technical Field
The invention belongs to the fields of biotechnology and environmental protection, and particularly relates to cytochrome P450 and application thereof in degrading pesticide residues.
Background
Tetranychus urticae KochTetranychus urticaeBelongs to the order of true mites of the Arachnida, and is a major agricultural pest mite distributed worldwide. Tetranychus urticae is a complex food, and can eat over 1100 host plants including vegetables, fruit trees, flowers, peanuts and the like. Tetranychus urticae is liked to be clustered on the back of plant leaves, plant juice is absorbed by the Tetranychus urticae in three development stages of adult mites, young mites and nymphs for killing, white spots are formed around main veins of leaves firstly, further the leaves are whitened, plant photosynthesis is influenced, the leaves are yellow and withered in severe cases, even the whole plant is withered, and the yield and the quality of crops are greatly influenced. At present, chemical agents are important measures for preventing and controlling the tetranychus urticae koch in China, but because of unreasonable and unscientific use, single and frequent use of a certain chemical agent or a certain class of chemical agents and the like, the tetranychus urticae koch generates different degrees of drug resistance to various chemical agents, so that the chemical prevention and control of the tetranychus urticae koch in the field face greater difficulty and challenge.
According to literature reports, the tetranychus urticae has developed resistance to at least 93 chemical active ingredients at present, but the resistance degree is inconsistent, and the tetranychus urticae ranks in an international pest resistance database because the tetranychus urticae has many reported cases of resistance to insecticide acaricide at home and abroad. Khajehali et al (Khajehali J, Van Leeuwen T, Grispou M, Morou E, Alout H, Weill M, Tirry L, Vontas J, Tsagkaraku A. Acetylcholinesterase points in European strains ofTetranychus urticae(Acari: Tetranyhidae) resistant to organophosphophosphates. Pest Management Science: for example Pesticide Science, 2010, 66(2): 220-; the earlier stage research results also show that China has the characteristics of high efficiency, high safety and low costTetranychus urticae has developed resistance to avermectins, organophosphates and pyrethroid insecticides (Wang L, Zhang YJ, Xie W, Wu QJ, Wang SL. A bioassay for evaluation of the resistanceTetranychus urticae (Acari: Tetranychidae) to selected acaricides. Systematic and Applied Acarology, 2015, 20(6): 579-590；Xu DD, He YY, Zhang YJ, Xie W, Wu QJ, Wang SL. Status of pesticide resistance and associated mutations in the two-spotted spider mite, Tetranychus urticaeIn China, Pesticide Biochemistry and Physiology, 2018, 150: 89-96.). Organophosphorus pesticides are widely used as chemical agents in farmlands, most varieties of the pesticides have the effect of killing mites, so the pesticides are also called as insecticidal and acaricidal agents, and in recent years, some varieties of the pesticides are replacing due to the toxicity problem of the pesticides, but the pesticides still have certain selective pressure on pest mites of farmlands.
The research on the resistance mechanism of tetranychus urticae to pesticides mainly relates to target resistance and metabolic resistance. After the tetranychus urticae is resistant to organophosphorus insecticides, the mutation of acetylcholinesterase gene is gradually found to cause organophosphorus resistance. Khajehali et al (Khajehali J, Van Leeuwen T, Grispou M, Morou E, Alout H, Weill M, Tirry L, Vontas J, Tsagkaraku A. Acetylcholinesterase points in European strains ofTetranychus urticae(Acari: Tetranyhidae) resistant to organophosphosphates. Pest Management Science 2010, 66(2): 220: 228.) three mutations A201S, T280A and F331W were found to play an important role in the resistance of Tetranychus urticae to organophosphorus insecticides, contemporaneous Kwon et al (Kwon DH, Im JS, Ahn JJ, Lee JH, Clark JM, Lee SH. Acetylchlolines enzyme node properties with monomeric resistance in the two-shot peptide mite, Pest Biochemistry and Physiogy 2010, 96(1): 36-42) found that G119S plays an important role in the resistance of Pennychus urticae; later, it was found that a single mutation of F331W and G328A resulted in moderate resistance to monocrotophos, while The additive effect of these two mutations with A280T or G119S resulted in higher resistance (Kwon DH, Choi JY, Je YH, Si HL. The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticaeInsect Biochemistry and Molecular Biology, 2012, 42(3): 212-. In earlier researches, the inventor also finds that two mutations of G119S and A201S on Ace participate in the drug resistance of Tetranychus urticae in China, but the mutation frequency and the multiple resistance do not show positive correlation, which indicates that other resistance mechanisms exist in the populations.
Cytochrome P450 is an important superfamily of detoxified metabolic enzymes, which are well-defined to be involved in the detoxified metabolic processes of various agents. After the tetranychus urticae has resistance to organophosphorus insecticides, P450 genes participating in detoxification metabolism and having definite physiological functions are rarely disclosed, and the lack of key genes participating in resistance formation also hinders the development process of novel medicaments and the formulation of pest resistance control strategies. Based on the above, the invention discovers that one P450 gene CYP392D8 is overexpressed in a resistant tetranychus urticae population, the gene can metabolize organophosphorus insecticides in vitro after heterologous expression and protein purification, a P450 gene related report that the gene can participate in the formation of organophosphorus insecticide resistance of tetranychus urticae at home and abroad is not yet seen, and the resistance gene can be used for detecting the resistance genes of tetranychus urticae to organophosphorus insecticides such as chlorpyrifos, pirimiphos-methyl and the like, and simultaneously provides a theoretical basis for designing sites of new pesticide targets.
Disclosure of Invention
The invention obtains a P450 gene CYP392D8 participating in the drug resistance of the two-spotted spider mite to the organophosphorus insecticide by cloning in the body of the two-spotted spider mite, and the gene has an over-expression phenomenon in populations generating low-level to medium-level resistance to the organophosphorus in the field, so the gene can be used as a molecular marker to assist in realizing early detection and early warning on whether the two-spotted spider mite populations generate resistance to the organophosphorus insecticide in the field, and the detection process is simple and quick. The gene can metabolize chlorpyrifos and pirimiphos-methyl in vitro, and can also be expected to metabolize other organophosphorus insecticides, so that engineering bacteria expressed by the gene can degrade organophosphorus pesticides remained on the surfaces of fruits, vegetables and the like, can pretreat wastewater and sewage, can treat soil polluted by pesticides and the like, and has industrial development and application potential. The cloned P450 gene involved in drug resistance can also be used as a target for specifically designing an inhibitor of a specific detoxification enzyme and providing a thought for the development of a novel medicament in the future.
Therefore, the invention firstly provides a cytochrome P450, the amino acid sequence of which is shown as SEQ ID NO: 2, respectively.
Further, a nucleic acid encoding the cytochrome P450 is provided. Preferably, the nucleotide sequence is as set forth in SEQ ID NO: 1 is shown.
Further provided are recombinant vectors and recombinant cells containing the nucleic acid.
The invention also provides application of the cytochrome P450 or the coding nucleic acid thereof in detection of whether the tetranychus urticae population generates resistance to organophosphorus insecticides or antibody early warning. Specifically, the sensitive population is used as a control, the expression level of cytochrome P450 genes in the population to be detected is measured, and when the expression level of the population to be detected is higher than that of the sensitive population and reaches a significant difference, the population to be detected is determined to generate resistance to the organophosphorus insecticide.
The invention also provides application of the cytochrome P450 in decomposition of organophosphorus insecticides. Preferably, the organophosphorus insecticide is chlorpyrifos or pirimiphos-methyl. More particularly, the method is used for degrading organophosphorus pesticide residues of fruits and vegetables, pretreating wastewater and sewage polluted by organophosphorus pesticide or treating soil polluted by organophosphorus pesticide.
The invention also provides a method for preparing the tetranychus urticae which is sensitive to the organophosphorus insecticide, and the method is realized by knocking out genes of dsRNA of adult mites eating CYP392D8 genes. This can be used as a control for Tetranychus urticae which is sensitive to organophosphorus insecticides.
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FIG. 1 Tetranychus urticae CYP392D8 gene amplification. Wherein, M: DNA Marker III (from bottom to top, 200, 500, 800, 1200, 2000, 3000, 4500 bp); lanes 1-6: tetranychus urticae resistant population.
FIG. 2 is a graph showing the relative expression levels of the CYP392D8 gene in the sensitive and resistant population of Tetranychus urticae. Wherein, LAB-SS is an indoor sensitive population of tetranychus urticae; MY, CP and SG are respectively Beijing dense cloud population, Beijing Changping population and Shandong Shouguang population.
FIG. 3 shows the expression level (A) of the field resistant population of Tetranychus urticae after the CYP392D8 gene is silenced and the mortality rate (B: chlorpyrifos; C: pirimiphos-methyl) of the two organophosphorus insecticides.
FIG. 4 is a metabolic map of Tetranychus urticae CYP392D8 recombinant protein against chlorpyrifos. Wherein, A, CYP392D8 reacts with chlorpyrifos to generate chlorpyrifos oxide, and the positive ion scanning mode (+ NADPH); b: CYP392D8 reacts with chlorpyrifos to generate 3, 5, 6-trichloropyridin-2-ol, negative ion scanning mode (+ NADPH); c: CYP392D8 reacted with chlorpyrifos, positive ion scanning mode (-NADPH); d: CYP392D8 reacts with chlorpyrifos, in negative ion mode (-NADPH).
FIG. 5 is a metabolic map of the Tetranychus urticae CYP392D8 recombinant protein on pirimiphos-methyl. Wherein, A: CYP392D8 reaction with pirimiphos-methyl to measure pirimiphos-methyl oxide and 2-diethylamino-6-hydroxy-4-methylpyrimidine (+ NADPH); b: CYP392D8 reacts with pirimiphos-methyl (-NADPH).
Detailed Description
The invention is further described with reference to specific examples, which are not intended to be limiting.
Materials and methods
Test insects
A laboratory susceptible population (Lab-IPP) of Tetranychus urticae is provided by insect research group of natural enemy of plant protection research institute of Chinese academy of agricultural sciences, collected in field 10 years ago, and then long-term sponge water isolation platform method is adopted to use kidney bean in artificial climate boxPhaseolus vulgarisLinn) leaf feeding has never been exposed to any chemical agent until now. The breeding conditions are as follows: temperature 26 ± 1 ℃, humidity 60 ± 5% and L: d =16 h: photoperiod of 8 h. The leaf used for feeding the test mites is the 'Bifeng' kidney bean leaf.
Three test field populations were collected from eggplant and strawberry hosts, Beijing Changping (CP), Beijing dense cloud (MY), and Shandong Shouguang (SG), respectively. The two-spotted spider mite population collected from the field is placed on a clean and insect-free kidney bean plant in an artificial climate box for breeding (breeding conditions are as above), and is used for bioassay and other experiments after indoor breeding for 2-3 generations.
Primary reagent
The RNA extraction reagent Trizol, a product of Life Technologies Corporation; the cDNA reverse transcription Kit PrimeScript RT reagent Kit (REAL TIME), a product of TaKaRa company of Japan; DNA molecular weight standards, proteinase K, X-Gal and IPTG, Beijing Tiangen Biochemical technology Co., Ltd; EsTaq has a mixed solution of 6 × Loading Buffer, and Beijing kang is a product of century Biotechnology Limited company; top10 competent cells; PCR product purification kit, pEASY-T1 vector and Beijing Quanji products; q5 high-fidelity DNA polymerase, product of Biotech, Inc., Mizhongyi, Beijing; other laboratory reagents (analytical grade) are commercially available. PCR primer synthesis and amplified product sequencing are completed by Beijing Optimalaceae New Biotechnology Co.
Main instrument
SZX7 olympus stereomicroscope (olympus (china) limited); an intelligent artificial climate box (RXZ-380C, Ningbo Jiangnan Instrument plant); fluorescent quantitative PCR instrument (ABI Q3, usa); gradient PCR instruments (Bio-Rad S1000 and Bio-Rad C1000); water bath and electrophoresis tank (six manufacturers in Beijing); table type refrigerating oscillator (THZ-C-1, Taicano City laboratory plant). High speed centrifuge (Sigma 3K15, germany); nucleic acid electrophoresis system and Gel imaging system (Gel Doc EQ) (Bio-Rad, USA); moist heat autoclave (sanyo, japan); an ultrapure water meter (ZMQ 55VOTI Mini Q water meter); micropipettes (Eppendorf, Germany).
Bioassay of Tetranychus urticae
The bioassay method adopts agar leaf soaking method. Adding about 5 mL of 0.1% liquid agar to the bottom of a plastic dish with the diameter of 3.5 cm, and cooling and solidifying for later use. Diluting the insecticide to 5-7 concentrations by using distilled water. Fresh, clean kidney bean leaves were punched into small leaf disks with a 2.5 cm diameter punch. Soaking the leaf disk in the liquid medicine to be measured for 10s, taking out and drying, and spreading the back of the leaf on the agar of a plastic dish. Soaked leaves were set as blank control. Picking 25-30 healthy female adult mites on each leaf disc by using a zero-number writing brush, then covering a cover with superfine pores, and then transferring the healthy female adult mites to an artificial climate box with the temperature of 26 +/-1 ℃, the humidity of 60 +/-5% and the illumination period of 16 h: 8h (L: D) for feeding and observation. After 24 h, the death number and survival number of the tetranychid mites are checked and recorded under a stereomicroscope, the bodies of the tetranychid mites are lightly touched by a brush pen tip, and the tetranychid mites are judged to be dead if only 1 test mite is active or completely inactive.
Extraction and cDNA Synthesis
Extracting the total RNA of the tetranychus urticae by using Trizol reagent. Taking 150 heads of active tetranychus urticae koch, and freezing in liquid nitrogen. The sample is put into a homogenizer filled with Trizol after high-temperature sterilization for sufficient homogenization, and then transferred into a special RNA centrifuge tube with the volume of 1.5 mL, and kept stand for 5 min at room temperature. Adding 200 μ L chloroform, shaking vigorously with a vortex shaker for 30s, standing for 5 min, and centrifuging at 12000rpm for 15 min. Then transferring the supernatant into another clean centrifuge tube, adding equal volume of pre-cooled isopropanol, reversing and mixing evenly, cooling on ice for 10 min to precipitate RNA, then centrifuging at low temperature and high speed for 10 min, carefully sucking out the supernatant, adding 1 mL of 70% absolute ethyl alcohol for rinsing, and centrifuging at low temperature for 5 min. Carefully absorb the ethanol, blow-dry the ethanol in a super clean bench for 5 min, and add a proper amount of precooled DEPC water for dissolution. Determination of OD260/OD280The ratio and the content of the RNA, and detecting the RNA quality by electrophoresis.
The Kit PrimeScript RT reagent Kit of TaKaRa company is adopted to synthesize cDNA, and the synthesis process is carried out according to the Kit instruction. Briefly, to RNA dedicated PCR tubes, gDNA Eraser and its buffer were added, followed by total RNA, RNase Free ddH2O was replenished to 10. mu.L and quickly placed in a PCR apparatus at 42 ℃ for 2 min. Adding 4.0 μ L of 5 XPrimeScript buffer to the PCR tube from which the DNA had been removed; RT Prime Mix 1.0 μ L; PrimeScriptRT Enzyme Mix 1.0 μ L and ddH2O4.0. mu.L, and rapidly placing into a PCR instrument, holding at 37 deg.C for 15 min, and holding at 85 deg.C for 5 s to inactivate reverse transcriptase, to obtain reverse transcription cDNA.
Gene cloning and qPCR thereof
Tetranychus urticae-based cDNAThe primer pair CYP392D8-F upstream primer is adopted: 5'-ATGTTTCTCGATCATTTCAACGTAAG-3'CYP392D8-R downstream primer: 5'- TTAATGCTTCAAAGTGTGAAAATTTGT -3'And cloning to obtain the full-length sequence of the CYP392D8 gene of the tetranychus urticae. The PCR amplification program is pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 62 ℃ for 20s, and extension at 72 for 40s, for 35 cycles; finally, extension is carried out for 2min at 72 ℃. And (3) recovering the PCR amplification result in a gel recovery kit, then connecting and transforming, carrying out colony PCR verification to obtain a positive cloning spot, then sending the positive cloning spot to Beijing engine science new industry company for sequencing, and analyzing the sequencing result.
The CYP392D8 gene expression levels were tested using qRT-PCR with three biological replicates per population. Upstream primer for qRT-PCR reaction: 5'- CTATACTTCGCAATGTTGGTCTA -3'And a downstream primer: 5'- CACTCAATCCACTCAACTCTT -3'The Actin and ribosomal protein 49 genes were used as reference genes. The fluorescent quantitative PCR result is obtained by adopting 2 according to the CT values of the target gene and the reference gene-ΔΔCTThe method performs data analysis. Differences among sample result groups were analyzed for one-way variance using ANOVA in SPSS 19.0 software, examined for significance of differences using Turkey, and plotted using GraphPad Prism 8 software.
RNA interference of tetranychus urticae CYP392D8 gene
Firstly placing healthy female tetranychus urticae into a centrifugal tube for hungry for 24 hours, then placing bean leaf dishes of 1.5 cm into a 60-oven for baking for 3-5 min, then soaking leaves into 1000 mg/mL target gene dsRNA for standing for 3-5 hours, setting clear water control and EGFP two negative controls, picking out the hungry tetranychus urticae on the bean leaf dishes with the dsRNA, freely taking the bean leaf dishes for 48 hours, picking out the living tetranychus urticae for total RNA extraction and fluorescent quantitative detection test of the target gene, and setting Rp49 and Actin as internal reference genes. After the target gene is knocked out through detection, the two different concentrations of abamectin are adopted to treat the two-spotted spider mites surviving after interference, and 4 times of repetition is set.
Expression in High Five cells
Amplifying the full-length cDN of CYP302D8 from tetranychus urticae by using RT-PCR technologyAnd (B) sequence A. The primers used for amplification are the upstream primers: 5'-ATGTTTCTCGATCATTTCAACGTAAG-3', downstream primer: 5'-TTAATGCTTCAAAGTGTGAAAATTTGT-3' are provided. The ORF was then cloned into the pFastBac vector using the double-restriction method. Recombinant baculovirus DNA was transfected into sf9 cells according to the bac-to-bac baculovirus expression system (Invitrogen, USA). High 5 (H5) cell culture to 2X 106cells/mL, for protein expression of the P450 gene, and then virus titers were detected using the plaque assay. CYP450 was co-expressed with CPR at different fold infections. Cells were cultured for 48 hours, and microsomes were collected and stored at-80 ℃ according to the previous literature. Total protein concentration was determined using the Bradford assay. And detecting the content of the expressed P450 protein by adopting a common difference spectrum method.
Metabolism of organophosphorus insecticides by recombinant proteins
The P450 recombinant target protein with activity detected by differential spectroscopy is reacted in PBS (PH 7.4) buffer solution, an NADPH reaction system (A: 10 mu L, B: 2 mu L) of Promega and an 11.4 mM chlorpyrifos/pirimiphos-methyl standard are added, the reaction is carried out for 3 h under the conditions of 1200rpm/min and 30 ℃ in a shaking metal bath, then 800 mu L acetonitrile is added to stop the reaction, the reaction is carried out for 20 min in the shaking metal bath, then 20000 g is centrifuged for 10 min, 200 mu L of supernatant is absorbed into a chromatographic bottle, and the control is treated as the reaction system without the addition of the NADPH. And (4) carrying out HPLC-MS detection on the sample.
Results of the experiment
1) Resistance of Tetranychus urticae field populations to organophosphorus insecticides
Lab-IPP in Table 1 is indoor sensitive population of Tetranychus urticae, SG, CP and MY are Shandong Shouguang population, Beijing Changping population and Beijing Miyun population collected from the field respectively, and the multiple resistance to chlorpyrifos of the three Tetranychus urticae field populations tested is 10.74-58.85 times, and belongs to medium resistance level; the resistance to pirimiphos-methyl was low, ranging from 4.37 to 5.53 fold (Table 1).
TABLE 1 Tetranychus urticae insecticide resistance to two organophosphorus insecticides in different field populations
Figure 547192DEST_PATH_IMAGE001
2) Gene cloning and sequence analysis
Designing a primer according to the genome sequence of the tetranychus urticae, amplifying cDNA of the tetranychus urticae by adopting RT-PCR (reverse transcription-polymerase chain reaction), and obtaining the full-length sequence of the CYP392D8 gene, wherein an electrophoresis pattern of a PCR amplification product is shown in figure 1.
The gene sequence with the length of 1503bp obtained by Open Reading Frame (ORF) analysis is as follows (shown in SEQ ID NO. 1):
ATGTTTCTCGATCATTTCAACGTAAGCCCAAATTTATTCAACATATTTTGTGGCTGTTTAACTGTTTGTTTGATCAAGTATTTGTTTAATTTTATTAATCGATTTCGTAAACTCCCTTCTGGCCCATGGGGCTTACCAATTTTGGGTTACTTGCCATTCATTAAGAAAGATGCTTATATTCAAATGACTGAACTCGCTAAAAAGTATGGACCAGTATTCAGCTTTAAATGTGGCCAGTGCGATGTGGTTGTGATCAACGAGGTGAAAGCCATTGAGGAAGCTTTGAAGAACGATCATTTATTGGCTCGTCCAAAAACAACTTTCATTCCAGGACGTAATCCTTCATTAGCTGAAATGTCGGGTGAAACTTGGAAACAACAGCGACGAGTTGCTCTAACTATACTTCGCAATGTTGGTCTAGGGAAAAGTACTCTAGAAACAAAGATCAAAGAAGAGATTGGCCATTTTATTGATGTTTTAAAATCAACTCATGGCAAAGAAGTTAGTTTCAAAGAGTTGAGTGGATTGAGTGTTGCAAATAATATCTCAATCCTTATGTTTGGGCATCGATTTGAATATGATGATCCCATTGGAAATGAAATGAGACGTAATATGCAGAAAGTTAGTGATAATATTGAATACTTCACTAAATTTGTGTTTATGCCTGCAATATTTTTCTTCGTTTCTCTTGCTGCTAAATTCAGTCCTGAGCTCAGAAACGCGAATAGCAGCCAGAAAGAACTAGAAGAGAAAATCACTGAGGAAGTTACTAAACATAGAGAAAAGAAAGCAACCCATGAAATCGAGGATTATATTGATGGTTATTTGCAAGAAATGGATAAGCTAAAGGATTCGAAAACGAGTTTTAATATAAACACATTGCGAAGAAATGCTGCTGATTTTTACGCAGCTGGGTCAGATACTACAACGCATGTATTGAATTTCATGATGTTATATTTGGTTACTTATCCTGAGCATCAACAGAAAATACGAGATGAAATTAAGCAAACTATTGGATTCGACCGTCAACCAGATTATGCAGACAGAGCTTCAATGCCTTTCACGATGGCTTTTATTTATGAATCTTTACGAATGTCAACATTAGTTCCTCTTAATGTTCTAAGACGTGCTACTCAAGACACAAAGGTAATGAACTATTCTATACCTAAAGATACATTAGTTATTCTCAACTTCTGGGCTGTTCATCGTGATCCAAAACTATGGAATGATCCACATTCATTCAAACCAGAAAGATTCCTCAGTGAGGATGAAACAACAGTAGTAAAATCACCCTATTTGATGCCATTTAGTGGAGGCAAAAGGATATGTCCTGGTGCAACTTTGGCTTTGATAGAGTTGTTTCTTTATTTGGTTTCAATTGTGCAAAATTTTATCGTTTCACCTCCAGAAGGACATAAAATATCTGATGAAATAAACTATATCTTCATTAGAATGGTGAAAAATCCAGTGAAACTCACAAATTTTCACACTTTGAAGCATTAA。
the nucleotide sequence of the CYP392D8 gene in the tetranychus urticae resistant population encodes 501 amino acids, and the predicted molecular weight is 57.73 KDa. The isoelectric point is 8.83, the N end of the gene contains a transmembrane structure and has typical P450 conserved structural regions, such as WxxxR structural domain (located at 370-387), meander structural domain (located at 1231-1257) and heme-binding structural domain (PFxxGxxxCxG located at 1303-1335).
The 500 amino acid sequences encoded by the CYP392D8 gene in the tetranychus urticae resistant population are as follows (SEQ ID NO: 2):
MFLDHFNVSPNLFNIFCGCLTVCLIKYLFNFINRFRKLPSGPWGLPILGYLPFIKKDAYIQMTELAKKYGPVFSFKCGQCDVVVINEVKAIEEALKNDHLLARPKTTFIPGRNPSLAEMSGETWKQQRRVALTILRNVGLGKSTLETKIKEEIGHFIDVLKSTHGKEVSFKELSGLSVANNISILMFGHRFEYDDPIGNEMRRNMQKVSDNIEYFTKFVFMPAIFFFVSLAAKFSPELRNANSSQKELEEKITEEVTKHREKKATHEIEDYIDGYLQEMDKLKDSKTSFNINTLRRNAADFYAAGSDTTTHVLNFMMLYLVTYPEHQQKIRDEIKQTIGFDRQPDYADRASMPFTMAFIYESLRMSTLVPLNVLRRATQDTKVMNYSIPKDTLVILNFWAVHRDPKLWNDPHSFKPERFLSEDETTVVKSPYLMPFSGGKRICPGATLALIELFLYLVSIVQNFIVSPPEGHKISDEINYIFIRMVKNPVKLTNFHTLKH。
expression levels in different field populations
In 3 field populations of two spotted spider mites collected from Beijing dense cloud (MY), Beijing Changping (CP) and Shandong birthday light (SG), the expression level of the CYP392D8 gene is found to be significantly higher than that of the indoor sensitive population (figure 2). The gene shows that the gene is over-expressed in different tested field tetranychus urticae resistant populations, and the expression level is also highest in dense cloud (MY) populations with the highest resistance multiple.
Interference with CYP392D8 to influence the sensitivity of Tetranychus urticae to organophosphorus insecticides
The dsRNA of the CYP392D8 gene is knocked out by female field-resistant two-spotted spider mite, the expression level of the gene is detected by qRT-PCR after 48 hours, and compared with control treatment, the expression level of the CYP392D8 gene is obviously reduced (60.26 percent), which indicates that the P450 gene is effectively silenced. The female tetranychus urticae after gene silencing is subjected to agent sensitivity measurement by adopting chlorpyrifos (500 ppm and 2000 ppm) and pirimiphos-methyl (50 ppm and 200 ppm), the death number and survival number of the tested mites are checked and recorded after the tetranychus urticae is treated for 24 hours by adopting a leaf soaking method, and the data are treated by adopting Poloplus 2.0 software. The results show that the sensitivity of the two-spotted spider mite after gene silencing to chlorpyrifos and pirimiphos-methyl is respectively increased by 24.32%, 15.93% and 13.62%, and 19.05% (figure 3), and the CYP392D8 is probably involved in the resistance formation of the two-spotted spider mite to the organophosphorus insecticide.
In vitro metabolism of two organophosphorus insecticides
After the P450 protein is determined to have activity by the difference spectrum, the metabolizing capacity of the P450 protein to two organophosphorus insecticides is determined. CYP392D8 can metabolize chlorpyrifos to chlorpyrifos oxide and 3, 5, 6-trichloropyridin-2-ol (fig. 4); it is also possible to metabolize pirimiphos-methyl to pirimiphos-methyl oxide and 2-diethylamino-6-hydroxy-4-methylpyrimidine (FIG. 5).
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> cytochrome P450 and application thereof in degrading pesticide residues
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1503
<212> DNA
<213> Tetranychus urticae
<400> 1
ATGTTTCTCGATCATTTCAACGTAAGCCCAAATTTATTCAACATATTTTGTGGCTGTTTAACTGTTTGTTTGATCAAGTATTTGTTTAATTTTATTAATCGATTTCGTAAACTCCCTTCTGGCCCATGGGGCTTACCAATTTTGGGTTACTTGCCATTCATTAAGAAAGATGCTTATATTCAAATGACTGAACTCGCTAAAAAGTATGGACCAGTATTCAGCTTTAAATGTGGCCAGTGCGATGTGGTTGTGATCAACGAGGTGAAAGCCATTGAGGAAGCTTTGAAGAACGATCATTTATTGGCTCGTCCAAAAACAACTTTCATTCCAGGACGTAATCCTTCATTAGCTGAAATGTCGGGTGAAACTTGGAAACAACAGCGACGAGTTGCTCTAACTATACTTCGCAATGTTGGTCTAGGGAAAAGTACTCTAGAAACAAAGATCAAAGAAGAGATTGGCCATTTTATTGATGTTTTAAAATCAACTCATGGCAAAGAAGTTAGTTTCAAAGAGTTGAGTGGATTGAGTGTTGCAAATAATATCTCAATCCTTATGTTTGGGCATCGATTTGAATATGATGATCCCATTGGAAATGAAATGAGACGTAATATGCAGAAAGTTAGTGATAATATTGAATACTTCACTAAATTTGTGTTTATGCCTGCAATATTTTTCTTCGTTTCTCTTGCTGCTAAATTCAGTCCTGAGCTCAGAAACGCGAATAGCAGCCAGAAAGAACTAGAAGAGAAAATCACTGAGGAAGTTACTAAACATAGAGAAAAGAAAGCAACCCATGAAATCGAGGATTATATTGATGGTTATTTGCAAGAAATGGATAAGCTAAAGGATTCGAAAACGAGTTTTAATATAAACACATTGCGAAGAAATGCTGCTGATTTTTACGCAGCTGGGTCAGATACTACAACGCATGTATTGAATTTCATGATGTTATATTTGGTTACTTATCCTGAGCATCAACAGAAAATACGAGATGAAATTAAGCAAACTATTGGATTCGACCGTCAACCAGATTATGCAGACAGAGCTTCAATGCCTTTCACGATGGCTTTTATTTATGAATCTTTACGAATGTCAACATTAGTTCCTCTTAATGTTCTAAGACGTGCTACTCAAGACACAAAGGTAATGAACTATTCTATACCTAAAGATACATTAGTTATTCTCAACTTCTGGGCTGTTCATCGTGATCCAAAACTATGGAATGATCCACATTCATTCAAACCAGAAAGATTCCTCAGTGAGGATGAAACAACAGTAGTAAAATCACCCTATTTGATGCCATTTAGTGGAGGCAAAAGGATATGTCCTGGTGCAACTTTGGCTTTGATAGAGTTGTTTCTTTATTTGGTTTCAATTGTGCAAAATTTTATCGTTTCACCTCCAGAAGGACATAAAATATCTGATGAAATAAACTATATCTTCATTAGAATGGTGAAAAATCCAGTGAAACTCACAAATTTTCACACTTTGAAGCATTAA 1503
<210> 2
<211> 500
<212> DNA
<213> Tetranychus urticae Koch
<400> 2
MFLDHFNVSPNLFNIFCGCLTVCLIKYLFNFINRFRKLPSGPWGLPILGYLPFIKKDAYIQMTELAKKYGPVFSFKCGQCDVVVINEVKAIEEALKNDHLLARPKTTFIPGRNPSLAEMSGETWKQQRRVALTILRNVGLGKSTLETKIKEEIGHFIDVLKSTHGKEVSFKELSGLSVANNISILMFGHRFEYDDPIGNEMRRNMQKVSDNIEYFTKFVFMPAIFFFVSLAAKFSPELRNANSSQKELEEKITEEVTKHREKKATHEIEDYIDGYLQEMDKLKDSKTSFNINTLRRNAADFYAAGSDTTTHVLNFMMLYLVTYPEHQQKIRDEIKQTIGFDRQPDYADRASMPFTMAFIYESLRMSTLVPLNVLRRATQDTKVMNYSIPKDTLVILNFWAVHRDPKLWNDPHSFKPERFLSEDETTVVKSPYLMPFSGGKRICPGATLALIELFLYLVSIVQNFIVSPPEGHKISDEINYIFIRMVKNPVKLTNFHTLKH 500

Claims (5)

1. The application of cytochrome P450 or coding nucleic acid thereof in detection or resistance early warning of whether tetranychus urticae population generates resistance to organophosphorus insecticides, wherein the amino acid sequence of the cytochrome P450 is shown as SEQ ID NO: 2 is shown in the specification;
the organophosphorus insecticide is chlorpyrifos or pirimiphos-methyl;
and adopting the sensitive population as a reference, measuring the expression level of the cytochrome P450 gene in the population to be detected, and determining that the population to be detected has resistance to the organophosphorus insecticide when the expression level of the population to be detected is higher than that of the sensitive population and the significant difference is achieved.
2. The use of claim 1, wherein the nucleotide sequence of said encoding nucleic acid is as set forth in SEQ ID NO: 1 is shown.
3. An application of cytochrome P450 in decomposing organophosphorus pesticide; the organophosphorus insecticide is chlorpyrifos or pirimiphos-methyl;
the amino acid sequence of the cytochrome P450 is shown as SEQ ID NO: 2, respectively.
4. The application of the compound of claim 3, wherein the compound is used for degradation of organophosphorus pesticide residues on fruits and vegetables, pretreatment of wastewater and sewage polluted by organophosphorus pesticide or treatment of soil polluted by organophosphorus pesticide.
5. The method for preparing the tetranychus urticae which is sensitive to the organophosphorus insecticide is characterized in that the method is realized by gene knockout of dsRNA of a coding gene of cytochrome P450 which is eaten by adult tetranychus urticae;
the amino acid sequence of the cytochrome P450 is shown as SEQ ID NO: 2 is shown in the specification;
the organophosphorus insecticide is chlorpyrifos or pirimiphos-methyl.
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