Sequence Analysis And Functional Expression Of Cytochrome P450 Genes Of Plutella Xylostella(L.)

The diamondback moth Plutella xylostella(L.)(Lepidoptera:Plutellidae),is one of the most serious pests on the cruciferous vegetables,and is a widely distributed pests in the world.As the control of P.xylostella is mainly dependent on the pesticides for a long time,it has developed resistance to most insecticides.Cytochrome P450 monooxygenases are the most important and common biocatalysts in nature.It is not only involved in the synthesis and degradation of endogenous substances,but also plays an important role in the metabolism of exogenous compounds(pesticides,etc.).Thus,Study on the cytochrome P450 oxidase,there will be conducive to obtaining the understanding of the action modes and roles of P450s,which can help to reveal the metabolism resistance mechanism mediated bu P450s.Therefore,our study was aimed at the diamondback moth P.xylostella,The main research contents include the following two aspects:Cloning and sequence analysis of cytochrome P450 oxidase gene from the diamondback moth;Functional expression of cytochrome P450 gene in the diamondback moth.1.Molecular cloning and sequence analysis of cytochrome P450 genes in diamondback mothThe research of cytochrome P450s in diamondback moth(Px-P450s)was relatively less than other Lepidopterons.The research of gene sequences and functions of Px-P450s became easier after the assembly of diamondback moth genome.So in this study,we fouced on 8 metabolic resistance-related P450 genes:CYP6BG1,CYP6BG4,CYP6BG5,CYP6BFlv4,CYP6AN14,CYP9G2,CYP9G4 and CYP9G11 based on reported results and the information from diamondback moth genome database.We cloned and analyzed the full opening reading frames(ORFs)of eight cytochrome P450 genes.The ORFs of these genes were between 1,397 bp-1,563bp and encoded 464-521 amino acid residues.Furthermore,the bioinfromatic softwares such as ProParam,TMHMM and SignalP3.0 were used to analyze the components of ohysicochemical cahracteristics,transmembrane structure and singal peptide.The results showed the 8 P450 proteins were typical microsomal P450s because the conserved P450 motifs,including the heme binding domain,Helix C,Helix I,Helix K and the meander region could be found in the amino acide sequences of these P450s.The sequence alingments showed the identities of both nucleotide sequences among 8 P.xylostella were between 40.71-95.45%and similarities of amino acid were between 19.14-96.53%.CYP6BG4 and CYP6BG5,which belonged to the same subfamily,shared 96.53%identity of amino acid sequence.This study cloned and analyzed mulitiple metabolic resistant-related Px-P450 genes in CYP6 and CYP9 family and these resultes will make a contribition to the research of P450 gene function and resistant mechanisms of diamondback moth.2.Functional expression of cytochrome P450 gene in the P.xylostellaTo date,the study of metabolic resistances within insecticide resistance research in P.xylostella were still stay in the relationships between gene expression levels and resistance patterns.The direct evidences were lacked.So in this study,we functionally expressed eight cloned P450 genes and co-expressed with P.xylostella cytochrome P450 reductase(Px-CPR)in Sf9 cells via Bac-to-Bac baculovirus expression system.SDS-PAGE analysis showed that 8 samples had specific bands comparing with the non-insertion control(BAC1)and the molecular weight of recombinant P450s were all between 50-60kDa.The molecular weight of CYP6BG1 and CYP6BG5 were slightly lower than predicted data.The reduced CO-difference spectrum of recombinant CYPs in P.xylostella:CYP6BG1,CYP6BG4 and CYP9G4 had an obvious absorption peak at 450 nm.Other 5 P450s and the non-insertion control(BAC1)only had an absorption peak at 420 nm.This result revealed that CYP6BG1,CYP6BG4 and CYP9G4 were successfully expressed in the Sf9 cells and the other 5 P450 did not folded correctly.The enzyme activities to model substrates of CYP6BG1,CYP6BG4 and CYP9G4 were determined(PNOD,BROD,ECOD).It was found that CYP6BG1 had two different substrates(PNOD and ECOD)activity at 0.8 nmol/min/pmol P450 and 0.084 nmol/min/pmol P450 respectively;CYP6BG4 had PNOD activity at 1.44 pmol/min/pmol P450.CYP9G4 also had PNOD activity at a rate of 1.66 pmol/min/pmol P450.The metabolic capabilities of 3 recombinant P.xylostella P450s was detected by in-vitro metabolism.Two hydroxy-pemethrin:trans-hydroxy-permethrin and cis-hydroxy-pemethrin,the oxidative metabolite of permethrin,were identified by UPLC-QTOF at the first time.The MRM methrod for the metabolite quantification was also been builted.The result showed that CYP6BG1 had limited metabolic ability to esfenvalerate,but in the contrary to reported resutes,none of them could metabolise permethrin under the conditions of this study.These results provided direct evidences for the research of pyrethroid resistance of P.xylostella.They also provided new.methods and insights for the studies of pyrethroid resistance in other insects.