Screening And Preliminary Functional Verification Of P450 Enzymes Metabolizing Neonicotinoid Insecticides In Laodelphax Striatellus

The Laodelphax striatellus（Fallén）,small brown planthopper（SBPH）,is an important agricultural pest.The long-term application of chemical insecticides has caused the resistence of small brown planthopper to a variety of insecticides.Cytochrome P450 is an important detoxification enzyme in organism.It is found that the increase of its activity is the main mechanism to insecticides.Neonicotinoids are important insecticides after organophosphorus,carbamate and pyrethroids.They are often used to control rice planthopper.The previous studies have shown that rice planthoppers have resistence and cross resistence to imidacloprid,acetamiprid,thiamethoxam and dinotefuran.However,the mechanism of cross resistance between these neonicotinoid insecticides is still unclear.In order to answer the above questions,we used overexpression technology in transgenic Drosophila melanogaster and bioassay to select from 62 cytochrome P450s of Laodelphax striatellus.By expressing them with Bac-to-Bac system in vitro,we can verify the functions of the genes that we have chosen.The main results are summarized as follows.1.Bioassay of acetamiprid,thiamethoxam and dinotefuran in 62 transgenic D.melanogaster with P450 gene of L.striatellus.The acetamiprid,thiamethoxam and dinotefuran were used to bioassay 62 D.melanogaster strains with P450 gene of small brown planthopper.The experimental strains were the hybrids of male（female）of pure transgenic D.melanogaster strains and female（male）of da-Gal4 D.melanogaster strains.The control strains were the hybrids of male（female）of da>9755 D.melanogaster strains and female（male）of da-Gal4 D.melanogaster strains.The results showed that the sensitivity of da>CYP6ER2 to acetamiprid,thiamethoxam and dinotefuran was significantly reduced than da>9755.Compared with the da>9755,the sensitivity of da>CYP6CS2v1 and da>CYP15G1 to acetamiprid and dinotefuran was significantly reduced,the sensitivity of da>CYP3115A1 and da>CYP6AX to acetamiprid and thiamethoxam was significantly reduced.The results of bioassay showed that CYP6CS2v1,CYP6ER2,CYP15G1,CYP3115A1 and CYP6AX were involved in the formation of cross resistance to neonicotinoid insecticides.2.In vitro expression of CYP6CS2v1,CYP6ER2 and CYP15G1 in L.striatellusCYP6CS2v1,CYP6ER2 and CYP15G1 were expressed by baculovirus expression system.The content and spectral characteristics of these three P450 enzymes were determined by arbon monoxide difference spectrum analysis.The results showed that the three P450 enzymes all had a maximum absorption peak at 450 nm after CO binding,indicating that the three P450 proteins were stable and possessed the characteristics of P450enzymes.According to the absorbance difference between 450nm and 490nm and molar absorptivity,the contents of the three P450 enzymes were 284.11 pmol/mg protein,219.78pmol/mg protein and 137.36 pmol/mg protein respectively.The metabolic activities of recombinant CYP6CS2v1,CYP6ER2 and CYP15G1 to p-nitroanisole（PNA）,7-ethoxycoumarin（EC）and 7-methylhaloxymethyl（MR）were determined.The results showed that CYP6CS2v1 and CYP6ER2 had higher oxygen demethylation activity to PNA,the specific activity values were 2.27 and 2.06 respectively;the three P450 enzymes showed higher oxygen demethylation activity to EC,the specific activity values were 2.34,2.54 and2.10 respectively;but they had no metabolic activity to substrate MR.3.Preliminary study on metabolism of acetamiprid,thiamethoxam and dinotefuran in vitro by CYP6CS2v1,CYP6ER2 and CYP15G1 of L.striatellusThe recombinant CYP6ER2 and CPR were mixed and incubated with acetamiprid for 3h.The recombinant p Fast Bac HTA was a negative control.The metabolite of acetamiprid was N-dimethyl-acetamiprid detected by UPLC-MS with a mass spectrum peak of 209.05m/z.No mass spectrum peak of N-dimethyl-acetamiprid was found in the control group and the other two recombinant P450 enzymes.Therefore,CYP6ER2 could metabolize acetamiprid to N-dimethyl-acetamiprid.In the tests of metabolizing thiamethoxam by CYP6CS2v1,CYP6ER2 and CYP15G1,CYP6ER2 can metabolize thiamethoxam to thiamethoxam.The rates of generating clothianidin by CYP6ER2 was 0.0102±0.0010 pmol/min/pmol P450,which was significantly higher than negative control p Fast Bac HTA.The results of metabolic kinetics showed that when CYP6ER2 metabolized thiamethoxam,the production of thiamethoxam was time-dependent;The Michaelis constant（K_m）of CYP6ER2 was 94.56±5.74μM.The clearance rate(V_max/K_m)was 0.00011.The metabolic rates of CYP6CS2v1and CYP15G1 to thiamethoxam were similar to that of the negative control,so CYP6CS2v1and CYP15G1 had no metabolic activity to thiamethoxam.In the three experimental groups,the substance that might be the metabolite of dinotefuran was not detected.Therefore,the results showed that CYP6ER2 had metabolic activity to acetamiprid and thiamethoxam,and it was a P450 enzyme that mediated the cross resistance of L.striatellus to neonicotinoid insecticides.