Molecular Cloning, Sequence Analysis And Expression Of Cytochrome P450 Gene In Tribolium Castaneum (Herbst)

P450s(encoded by CYP genes) constitute a multigenic superfamily of enzymes,known for their ability to metabolize a wide range of endogenous and exogenous compounds thus contributing to numerous functions including growth,development,nutrition,and xenobiotic detoxification.P450 enzymes are found in virtually all insect tissues,where they are involved in processes that are vital for insect growth,development and reproduction,including ecdysteroid and juvenile hormone biosynthesis and aspects of ecdysteroid inactivation,as well as metabolism and detoxification of xenobiotics.The first insect P450 gene(CYP6A1) was isolated from an insecticide-resistant strain of housefly,Musca domestica.Subsequently,more and more P450 genes were cloned.To date,more than 200 P450 genes belong to CYP4,CYP6,CYP9,CYP12,CYP15,CYP18,CYP28,CYP48,and CYP305 have been cloned and sequenced。The red flour beetle,Tribolium castaneum(Herbst)(Coleoptera:Tenebrionidae),is a cosmopolitan and destructive pest of raw and processed cereal grains.Further,it is a member of the most species-rich eukaryotic order,a powerful model organism for the study of generalized insect development,and an important pest of stored agricultural products.In 2008,as a model beetle,the genome of T.castaneum has been sequenced.To date,however,studies on Tribolium P450 is lack and premature.Therefore,this study was undertaken and partially supported by the National Natural Science Foundation(30570231) to identify and characterize the P450 genes from T.castaneum. Furthermore,Prokaryotic expression and Real-time quantitative PCR were implemented to investigate the expression character of CYP6BK17 and CYP6BQ13v2 to provide a base of gene expression data for future experiments on the regulation of this gene and its product.The major results are summarized as follows:1.Identification and Sequence characterizationA new cytochrome P450 gene,CYP6BK17(GenBank accession No:EU266589),was isolated from the red flour beetle use RT-PCR.The cDNA sequence of CYP6BK17,1816 bp in length, contains an open reading frame of 1515 nucleotides encoding a putative protein of 505 amino acid residues,contains the classic heme-binding sequence motif F××G×××C×G conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s.Besides,3' sequence of a new cytochrome P450 gene(F06)(GenBank accession No:EU924172),was isolated from the red flour beetle use RT-PCR,614 bp in length.The amino acid residues sequence of F06 contains the classic heme-binding sequence motif F××G×××C×G conserved among all P450 enzymes.An allele of CYP6BQ13 from the Tribolium castaneum genome project,named CYP6BQ13v2 (GenBank accession no:FJ209361),was isolated from the red flour beetle.The cDNA sequence of CYP6BQ13v2,1563 bp in length,contains an open reading frame of 1554 nucleotides encoding a putative protein of 518 amino acid residues,contains the classic heme-binding sequence motif F××G×××C×G as well as other characteristic motifs of all cytochrome P450s.2.Bioinformatic analysisThe predicted molecular weight and theoretical pI of the CYP6BK17 is 58.77 kDa and 9.26, respectively.3D Model of the CYP6BK17 was constructed according to CYP3A4 gene by using SWISS-MODEL.The predicted molecular weight and theoretical pI of the CYP6BQ13v2 is 59.92 kDa and 7.60, respectively.CYP6BQ13v2 was blasted against genomic sequences of T.castaneum in GenBank.It was found that this gene consisted three exons and two introns.The splicing signals are exon/GT-intron-AG/exon.The cDNA sequence had high similarity to only one contig (CtgAAJJ01000808),suggesting that the CYP6BQ13V2 gene was a single-copy gene in the genome.3.Phylogenetic analysisPhylogenetic analysis indicated that the closer phylogenetic relationship of CYP6BK17 with CYP6BK10 from T.castaneum and other insects CYP6 members than other P450 families' members from mammals.Phylogenetic analysis revealed that there was closer phylogenetic relationship of CYP6BQ13V2 with CYP302A1(B.mori) and CYP307A1(D.melanogaster) mediating synthesis of the insect molting hormone,distant relationship with CYP6B1(P.polyxenes) metabolize plant allelochemicals,CYP6D1(M.domestica) linking to pyrethroid resistance and other members of CYP6 family.4.Expression investigantionThe recombinant of pDest-17-CYP6BK17 was obtained to express the recombinant protein of CYP6BK17.The recombinant plasmid was transfected into BL21(DE3).SDS-PAGE showed that CYP6BK17 was successfully expressed in BL21(DE3).To compare the expression levels of CYP6BQ13v2 gene in the different developmental stages of T.castaneum,Q-RT-PCR analysis was performed.The developmental expression in the adults and immature stages revealed that CYP6BQ13v2 was expressed at all life stages investigated.The mRNA expression levels in the 1st instar was 14.9 and 3.86 folds higher than those in pupae and adults,respectively.The CYP6BQ13v2 expression appeared in the order of 1st instar larvae＞4th instar larvae＞7th instar larvae＞adult＞pupae.