The Studies Of Cytochrome P450 Gene Of Bombyx Mandarina

Cytochrome P450 monooxygenase which is composed of various kinds of components is an oxidase system widely exists in aerobic organisms. A lot of evidences indicated that insect cytochrome P450 monooxygenase mediated resistance is the main mechanism of the development of insecticide resistance and adaptability to host plants. Fluoride-induced midguts of Bombyx mandarina were used as experimental materials. Specific primers were designed based on CYP305B1 sequence of Bombyx mori. Two segments of CYP305B1V1 cDNAs were cloned with RT-PCR, and complete CYP305B1 gene was obtained through the juncture of them. Sequencing results showed that the ORF of CYP305B1V1 was 1464 nucleotides long and encodes 487 amino acids which possesses a common characteristic of Cytochrome P450 proteins. Homologous analysis showed that the homology of CYP305B1V1 of Bombyx mandarina and CYP305B1 of Bombyx mori reaches 99%, and both of their deduced sequences of amino acids are exactly the same. It was predicted that CYP305B1V1 gene structure of Bombyx mandarina was possessed of at least 7 introns and all the introns obey the GT-AG rule by searching silkworm genome database in NCBI. The CYP305B1V1 cDNA was cloned into pET28a(+) vector and transformed into E. coli BL21 to express using IPTG as an inducement. SDS-PAGE and Western blotting analyses showed that the molecular weight of recombinant CYP305B1 protein with His6 tag is about 57kDa the same as theoretical value. This result lays the foundatiori for purifying Cytochrome P450 protein and constructs in vitro reaction system in the future. After extracting total RNAs of various' kinds of Bombyx mandarina tissues and producing DIG-tagged random primers of CYP305B1V1 cDNA of Bombyx mandarina, we tried to research the tissue expression specificity of CYP305B1V1. Thepreliminary result showed that CYP305B1V1 transcribes specifically inmidgut, fat body, malpighian tubules, and body wall.