Cloning, Sequence Analysis, And Prokaryotic Expression Of CYP6B29 Genes From Bombyx Mori And Bombyx Mandarina

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 acts as the terminal oxidase in monooxygenase systems to oxidize widely diverse substrates. According to the predicted P450 sequences from the genome of Bombyx mori, a pair of primers were designed, and Bombyx mori CYP6B29 and Bombyx mandarina CYP6B29 were successfully cloned from their midguts by RT-PCR with GenBank accession numbers DQ252324 and DQ252325 respectively. Sequence analysis results showed that they both contain a 1 518 bp open reading frame, which encode 505 amino acid residues (m.w. 58 kD) with pI 8.29 and 8.49 respectively. The comparison of CYP6B29 amino acid sequence with that of nearly all the other members of 6B subfamily showed that they shared highest identity (60.5%) to CYP6B27 from Helicoverpa zea. Because many members of CYP6B subfamily are related with insecticide and plant toxin resistences, we predict that CYP6B29 from Bombyx mori and Bombyx mandarina might be related with insect resistence.The CYP6B29 genes were cloned into prokaryotic expression pET-28a respectively, transformed into E coli. BL21, and then expressed by the induction of 1 mmol/L IPTG. SDS-PAGE analysis indicated that Bombyx mori and Bombyx mandarina both showed a specific approximately 62 kD band compared with the control groups of empty BL21 and BL21 transformed by empty pET-28a. So we predicted that CYP6B29 proteins from Bombyx mori and Bombyx mandarina were...