In Vitro Expression Of Several Laodelphax Striatellus P450 Genes And Metabolic Assays To Imidacloprid

The small brown planthopper(SBPH),Laodelphax stratellus(Fallén),as an important pest on rice,has developed resistance to many common insecticides such as imidacloprid.Studies have shown that the enhancement of cytochrome P450 enzyme activity is an important mechanism for the imidacloprid resistance of Laodelphax striatellus.In this study,we identified that CYP4FB1,CYP6FK1 and CYP6ER2 may be involved in the metabolism of imidacloprid based on the pre-existing transgenic Drosophila overexpression technique.Therefore,the baculovirus expression system was used to express CYP4FB1,CYP6FK1 and CYP6ER2,respectively.In vitro metabolism experiments of imidacloprid by CYP6ER2 were carried out.The research results are summarized as follows:1.Expression of CYP4FB1,CYP6FK1 and CYP6ER2 in vitro.The baculovirus expression system was used to express CYP4FB1,CYP6FK1,CYP6ER2 and the phytochrome reductase of Laodelphax striatellus.The results showed that the recombinant CYP4FB1,CYP6FK1 and CYP6ER2 could catalyze the conversion of pnitroanisole(PNA)to p-nitrophenol(PNP)with a Vmax value of 21.06.±0.24 mOD/min/mg protein-55.37±6.94 mOD/min/mg protein,the enzyme activity ratio was between 1.8 and 4.7 times compared with the negative control group.Content and spectral characteristics of CYP4FB1,CYP6FK1 and CYP6ER2 were determined by CO difference spectroscopy.The results showed that P450 bound with CO had obvious absorption peak at 450nm,indicating that the recombinant protein is stable and has the functional structure of P450 enzyme.The conten of the P450 enzyme between 10 pmol/mg protein to 20 pmol/mg protein.That laid the foundation for the next functional verification.2.Study on the metabolism of imidacloprid by CYP6ER2Recombinant CYP6ER2 protein incubated with imidacloprid after mixed with the whitefly CPR.The sample without the NADPH regeneration system was used as the control.Suspected hydroxylated imidacloprid product was detected by LC-MS and determined by LC-MS/MS to make sure that suspected compound is indeed derived from the hydroxylated imidacloprid produced by imidacloprid metabolism.In addition,we can studied the dynamic process of recombinant CYP6ER2/CPR mixed enzyme solution on imidacloprid metabolism with a kinetic method.The results showed that when CYP6ER2 metabolized imidacloprid,the consumption of imidacloprid and the production of hydroxylated imidacloprid metabolites were time-dependent;and the kinetic parameter Km of CYP6ER2 was 43.96±0.89 μM,the Kcat value was 0.050±0.005 pmol/min/pmol P450.This result further confirmed that the potential metabolic capacity of CYP6ER2 for imidacloprid,providing evidence that CYP6ER2 of SBPH can participate in the formation of metabolic resistance to imidacloprid.