Cloning And Expression Analysis Of Cytochrome P450CYP4G25Gene From The Chinese Oak Silkworm, Antheraea Pernyi

Cytochrome P450monooxygenase enzymes (cytochrome P450monooxygenases, P450s) constitute a multigenic superfamily of enzymes which is widely distributed in the organism and is one of the oldest and largest genesuperfamilies. Cytochrome P450s evolved from a common ancestor which has exisited for more than3.5billion vears. Cytochrome P450s involved in many important life processes of animals, plants, fungi and bacteria. In insects, the cytochrome P450plays an important role:mainly related to growth, development, feeding and other processes. P450s are involved in the metabolism of a wide range of both endogenous and exogenous compounds, which takes part in not only the regulation of endogenous compounds such as hormone. fatty acid and steroid, but also the metabotism of exogenous compounds such as drug insecticide and phytotoxin with important biological and genetic significance.Antheraea pernyi is a kind of developing wild ilk-producing insect, which blongs to Lepidoptera Saturniidae Antheraea. Antheraea pernyi has good officinal and edible economical values except for utilizing in spinning industry as raw marerial. Our results provide preliminary insights into the evolution and biological function, The major results are summarized as follows:1. Cloning and sequence analysis of CYP4G25cDNAA new cytochrome P450gene, CYP4G25(GenBank accession No:GU205081), was isolated from the Antheraea pernyi use RT-PCR and RACE-PCR. The cDNA sequence of CYP4G25,2112bp in length, contains a111-base5-untranslated sequence(5’UTR), an open reading frame of1674nucleotides encoding a putative protein of557amino acid residues with predicted molecular weight of63.6kD and isoelectric point of8.6and a326bp3’-untranslated region (3’UTR). Based on the entire amino acid sequence, the Heme-blinding region, I-helix domain, K-helix domain, C-helix domain, and N-terminal transmembrane anchoring signal were found using the ExPASy Proteomics tools. Phylogenetic analysis indicated that A. pernyi CYP4G25gene had the highest identity with A. yamamai CYP4G25.2. Expressing analysis of CYP4G25Meanwhile, the total RNA of A. pernyi was extracted to analyse the expression of vitellogenin in different developmental stages and tissues, then reversely transcribed and synthesized the first strand cDNA by M-MLV. Take the actin of A. pernyi as a reference gene, we carried the semi-quantitative PCR. The result showed that CYP4G25of A.pernyi ubiquitously expressed in all examined tissues. It was expressed in fat body, integument, midintestine, hemocytes, silkgland, antennae, testis and ovary with no obvious quantitative difference.The ORF was amplified by RT-PCR, ligated to pET-28a(+) expression vector and transformed into Escherichia coli BL21(DE3), induced by IPTG under different concentrations. Through SDS-PAGE electrophoresis and Western blot analysis, the induced fusion protein was successfully expressed. This will provide the foundation for further study on eukaryotic expression and function of CYP4G25gene.