In Vitro Expression Of Five Cytochrome P450s And Their Metabolism Studies On Imidacloprid In Laodelphax Striatellus

The small brown planthopper(SBPH),Laodelphax striatellus(Fallen),is an important agricultural pest.The application of insecticides is the main measure to control small brown planthopper(SBPH).At present,SBPH has developed resistance to imidacloprid and other commonly used insecticides.It was found that the increased activitity of cytochrome P450 enzyme was the main mechanism of the resistance to imidacloprid.Previous studies have shown that CYP6AY3v2 and CYP353D1v2 which were overexpressed in imidaclopridresistant strains of SBPH can metabolize imidacloprid.Because of the evolutionary plasticity of cytochrome P450 enzymes,there must be other P450 enzymes in SBPH that can metabolize imidacloprid.In order to identify these P450 enzymes,transgenic Drosophila melanogaster and bioassay studies were carried out in previously studies.CYP4DC1,CYP15G1,CYP427A1,CYP307A1 and CYP6ER2 were screened from 63 SBPH P450s.In this study,the functions of these five enzymes were verified by in vitro expression,enzyme activity determination and in vitro metabolism studies.The main results are summarized as follows:1.Expression and enzyme activity assays of five SBPH P450sCYP4DC1,CYP15G1,CYP427A1,CYP307A1,CYP6ER2 and cytochrome reductase CPR were expressed in vitro by using baculovirus expression system.The content and spectral characteristics of P450 enzymes were determined by CO difference spectroscopy.The results showed that all the five P450 enzymes had a maximum absorption peak at 450 nm after binding to CO,which indicating that the five recombinant proteaseswere stable and had the characteristics of P450 enzymes.The contents of the five P450 enzymes were 211.86 pmol/mg protein,295.03 pmol/mg protein,96.58 pmol/mg protein,286.66 pmol/mg protein and 207.23 pmol/mg protein,respectively.The metabolic activities of recombinant CYP4DC1,CYP15G1,CYP427A1,CYP307A1 and CYP6ER2 to p-nitroanisole(PNA),7-ethoxycoumarin(EC),7-benzyl-4trifluoromethyl coumarin(BFC)and 7-methyl halogen(MR)were determined respectively.Comparing to the control,CYP427A1 and CYP6ER2 had higher oxygen deethylation activities to EC,the specific activity values were 3.46 and 2.74,respectively;CYP307A1 and CYP6ER2 showed high oxygen demethylation activityies to PNA,and the specific activity values of were 2.40 and 2.25,respectively.The metabolic activities of the five P450 enzymes to the substrate BFC and MR were not detected.2.Metabolism of imidacloprid of five SBPH P450 enzymes in vitroThe enzyme of CYP4DC1,CYP15G1,CYP427A1,CYP307A1 and CYP6ER2 were incubated with imidacloprid respectively,and the protein extract of no-load pFastBacHTA was used as negative control.The metabolic activity of P450 enzyme to imidacloprid was characterized by the formation rate of 5’-hydroxy-imidacloprid.The results showed that the rates of formation of 5’-hydroxy-imidacloprid from CYP4DC1,CYP15G1,CYP427A1,CYP307A1 and CYP6ER2 were 0.0060 ± 0.0008 pmol/min/pmol P450,0,0037 ± 0.0003 pmol/min/pmol P450,0.0291 ± 0.0014 pmol/min/pmol P450,0.0039±0.0003 pmol/min/pmol P450 and 0.0199±0.0017 pmol/min/pmol P450,respectively.The rates of formation of 5’-hydroxy-imidacloprid by CYP427A1 and CYP6ER2 were higher,and the ability of metabolizing imidacloprid was stronger.While,the rates of formation of 5hydroxyimidacloprid by CYP4DC1,CYP15G1 and CYP307A1 was higher than that of the negative control,but the activity was low.Further enzyme kinetic studies showed that the formation of 5’-hydroxy-imidacloprid was time-dependent when imidacloprid was metabolized by CYP427A1 and CYP6ER2,and the kinetic parameters Km of CYP427A1 and CYP6ER2 were 38.93 ±4.18 μM and 92.63±2.90 μM,respectively,and the intrinsic clearance rates(Vmax/Km)were 0.00075 and 0.00021,respectively.In summary,the results of this study confirmed the metabolic effects of CYP4DC1,CYP15G1,CYP427A1,CYP307A1 and CYP6ER2 on imidacloprid.CYP4DC1,CYP15G1 and CYP307A1 have weak effect on imidacloprid metabolism,while CYP427A1 and CYP6ER2 have strong effect on imidacloprid metabolism.These two enzymes are potential P450 enzymes that may be involved in the metabolic resistance of SBPH to imidacloprid.