The Cloning, Expression Of CYP4G2 Gene And Induction Of P450s By Fenvalerate From Xylotechus Rusticus L.

The cytochrome P450-dependent monooxygenases are an extremely important metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. Insecticide resistance has proven to be correlated with Monooxygenase-mediated metabolism. Cytochrome P450 is a hemoprotein which actsas the terminal oxidase in monooxygenase systems to oxidize widely diverse substrates.Xylotrechus rusticus Linnaeus is a kind of the stem pest that endangers various broad-leaved trees of poplar,the partial region resulted in the serious bane in the our country northeast.In order to know more about the structures and functions of individual P450s in Xylotechus rusticus L.,efforts were given to identify the individual P450 as well as cloning,array analysis and expressing it in expression systems, and discusses the induction characteristic of fenvalerate on cytochrome P450 gene.The main results are as followed.A pair of primers were designed,coding for conservative amino acid regions in all kinds of insects CYP4 protein family.CYP4G2 of Xylotechus rusticus L.was successfully cloned from their midguts by RT-PCR with GenBank accession numbers EF429250.Sequence analysis showed that the full length of CYP4G2 open reading frame(ORF) was 387 bp, encoding 129 amino acid residuum,the predicted MW and pI were 16.9kD and 5.75, respectively.The deduced amino acid sequence showed a high identity to the reported sequences of CYP4 family amino acid from other insects(63%～86%).The comparison of CYP4G2 amino acid sequence with that of nearly all the other members of CYP4 family showed that they shared highest identity to CYP4 from Leptinotarsa decemlineata and Tribolium castaneum.The CYP4G2 gene was cloned into prokaryotic expression pET-28a(+),transformed into E coli.JM109,and then expressed by the induction of 0.3mmol/L IPTG.SDS-PAGE analysis indicated that CYP4G2 of Xylotechus rusticus L.showed a specific approximately 22 kD band compared with the control groups of empty JM109 and JM109 transformed by empty pET-28a(+).The molecular weight was the same as the prediction of fusion protein,So we predicted that CYP4G2 protein of Xylotechus rusticus L.was successfully expressed.Scribbling 2mg/g Fenvalerate solution in adult body well,then dissecting the check and sample and mensuating the quality of P450 in body well,midgut as well as fat body,the results indicate that Fenvalerate had complex induction mechanism for different induction function on different components of P450 enzyme department.The induction of P450 by Fenvalerate was observed in midgut.The successful clone and expressions of CYP4G2 proteins from Xylotechus rusticus L.lay the foundation for constructing in vitro recombinant enzymatic systems to study CYP4G2-mediated xenobiotics metabolism and finally confirm their functions.