In Vitro Expression Of A Yeast Like Symbiont P450 YLS375360 From Laodelphax Striatellus And Comparative Study On Its Metabolism Of Two Insecticides

Laodelphax striatellus（Fallén）is an important pest on rice.At present,the L.striatellus has developed resistance to various commonly used insecticides,such as nicotinoids and pyrethroids.It has been shown that yeast like symbionts YLS can help host resist insecticides.In order to further explore the molecular mechanism of YLS in resistance to insecticides,based on the previous findings that YLS375360,a P450 gene encoded by YLS,was overexpressed in deltamethrin and imidacloprid resistant L.striatellus,we used overexpression technology,bioassay,P450 enzyme expression in vitro,enzyme activity determination and insecticide metabolism in vitro to explore the molecular mechanism of YLS to help host resist insecticides.The results are summarized as follows.1.Verification of P450 enzyme function by overexpression of transgenic DrosophilaIn this study,we used GAL/UAS expression system to overexpressed 61 P450 gene of L.striatellus and the P450 gene encoded by YLS in L.striatellus.In the sensitivity test of deltamethrin,compared with the da>9755 control strain,the sensitivity of da>YLS375360,da>CYP6ER2,da>CYP301B1,da>CYP6CW1,da>CYP425A1v2,da>CYP6CS2v1,da>CYP4G76,da>CYP4GK1 and da>CYP4FB1 to deltamethrin was significantly reduced,and the resistance multiple of da>YLS375360 and da>CYP6ER2 Drosophila strain was more than 3 times.In the sensitivity test of imidacloprid,compared with the da>9755 control strain,the sensitivity of da>YLS375360 to imidacloprid was significantly reduced,and the resistance multiple is 3.10.2.In vitro expression,enzyme content and enzyme activity determination of recombinant P450 enzymesWe uesd baculovirus expression system to recombined and expressed P450 enzymes YLS375360,CYP6ER2,CYP4CE2 and cytochrome P450 reductase CPR in vitro.The viral titers of P3 generation of pfast Bac HTA,CPR,YLS375360,CYP6ER2 and CYP4CE2 were 5.0×10⁷,5×10⁷,3.5×10⁷,8×10⁷ and 7.5×10⁷（pfu/m L）.The maximum absorption peak of the three P450 enzymes was found at 450 nm after CO binding by CO difference spectroscopy,indicating that the three P450 proteins were successfully folded and stable in vitro.The contents of P450 enzyme YLS375360,CYP6ER2 and CYP4CE2 were calculated as follows:161.4,187.68 and 180.14pmol/mg protein,respectively.The metabolic activities of three P450 enzymes YLS375360,CYP6ER2 and CYP4CE2against model substrates of p-nitroanisole（PNA）,7-ethoxycoumarin（EC）and methylhaloxymethyl（MR）were determined.The results showed that:compared with the control group,recombinant P450 enzymes YLS375360,CYP6ER2 and CYP4CE2 had higher oxygen demethylation activity to PNA,the activity ratio were 3.09,3.06 and 3.78,respectively;and also showed higher oxygen deethylation activity to EC,the activity ratio were 3.68,2.63 and 2.97,respectively;the three P450 enzymes did not show obvious oxygen demethylation activity to MR.3.In vitro metabolism of deltamethrin by recombinant P450 enzymes YLS375360 and CYP6ER2The in vitro metabolic systems of deltamethrin and recombinant P450 enzymes YLS375360 and CYP6ER2 were constructed,the samples without pfast Bac HTA and without NADPH regeneration system were used as negative control,The results were analyzed by UPLC-DAD-ESI-QTOF primary and secondary mass spectrometry that the suspected compounds were indeed from hydroxydeltamethrin produced by deltamethrin metabolism.Subsequent time-dependent experiments showed that both P450 enzymes metabolized deltamethrin were time-dependent and reached the plateau after 180 min.The K_m values of deltamethrin metabolized by recombinant P450 enzymes YLS375360 and CYP6ER2 were80.45±3.51?M and 97.82±4.30?M respectively,and the K_cat/K_m values were 0.001843381and 0.001753220 respectively.The results showed that YLS375360,a P450 enzyme encoded by YLS,could metabolize deltamethrin,and its activity was similar to CYP6ER2,a P450gene of L.striatellus.4.In vitro metabolism of imidacloprid by recombinant P450 enzymes YLS375360 and CYP4CE2Two recombinant P450 enzymes YLS375360 and CYP4CE2 were incubated with imidacloprid respectively,the samples without pfast Bac HTA and NADPH regeneration system were negative control.The production of 5’-hydroxyimidacloprid was detected by UPLC-Xevo TQ-S micro,and the metabolic activity of recombinant P450 enzyme was characterized by the metabolites;The results showed that the formation rates of 5’-hydroxyimidacloprid from YLS375360 and CYP4CE2 were 0.0075±0.00012 and0.0193±0.0011 pmol/min/pmol P450,respectively.Furthermore,the kinetic parameters K_mof recombinant P450 enzymes YLS375360 and CYP4CE2 were 80.73±1.09?M and96.19±1.02?M respectively,and the intrinsic clearance rate(V_max/K_m)of imidacloprid metabolized by P450 were 0.000093089 and 0.000201165 respectively.The results showed that YLS375360,a P450 enzyme encoded by YLS,could metabolize imidacloprid,and its activity was slightly weaker than CYP4CE2,a P450 gene of L.striatellus.In conclusion,the results of this study confirmed that the P450 enzyme YLS375360encoded by YLS,which was overexpressed in the resistant strain of L.striatellus,could metabolize deltamethrin and imidacloprid,which provided direct evidence for YLS to help the host L.striatellus resist insecticides.