Cloning And Expression Of Cytochrome P450 From Helicoverpa Armigera

Cytochrome P450-dependent monooxygenases are a very important enzymatic system, which has been found in all living organisms systems examined. These monooxygenases are able to metabolize a phenomenal number of endogenous and exogenous compounds. It has been regarded as a key object in the biology field because of its diversity in structures and functions. Helicoverpa armigera is an economically important pest around the world. It has caused damage to many different crops. In order to know more about the structures and functions of individual P450s in H.armigera, efforts were given to identify the individual P450 and to express it in heterologous expression systems. The main results are as followed. An EST(Expressed Sequence Tag) encoding a cytochrome P450 was cloned from the midgut cDNA library of the sixth instar larvae of a laboratory strain of the H. armigera, by using mRNA differential display RT-PCR(DDRT-PCR) with upstream primers designed on the conserved heme-binding region of P450 sequence. The fragment is 342bp in length, including F--G---C-G, which is conserved in all cytochrome P450, and 127bp of 3'untranslated region. The 5'terminal region of the EST transcript was determined by using 5'RACE(Rapid Amplification of cDNA Ends) procedure. The 5'RACE fragment is 1605bp in length, including partial or all the 5 ′untranslated region, and the conserved sequence, PFGFGPRNCIG ,which is the same with the EST fragment. The entire cytochrome P450 cDNA sequence was obtained by combining the EST fragment with 5'RACE fragment. The final results showed that the cytochrome P450 is 1891bp in length, including 171bp of 5'untranslated region, 1593bp of coding region and 127bp of 3'untranslated. The coding region encodes a protein of 531 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus signal sequence. Analysis of the NH2-terminal sequence indicated that it is a microsomal P450.The molecular weight of the putative protein is 61kDa, and its predicted isoelectric point is 8.30. The cDNA sequence was deposited in GenBank under the accession number of AY753201 and named CYP9A17 by Dr. Nelson. Comparisons of the deduced amino acid sequence with CYP9A12 from the H.armigera, CYP9A14 from the H.armigera, CYP9A1 from the Heliothis virescens, CYP9E2 from the Blattella germanica, CYP9G2 from the plutella xylostella and CYP9L1 from the Anopheles gambiae show the amino acid identity of 93%,62%,54%,42%,40% and 38%,respectively. The entire coding cDNA of CYP9A17 was amplificed by RT-PCR with a pair of primers designed on the entire coding sequence of CYP9A17 . And this cDNA was cloned into pGEM-T easy vector. The full-length coding cDNA clone was successfully obtained, which can facilitate to study the structure, function and regulation of CYP9A17. We also detected the expression of CYP9A17 in different life stages of H.armigera by using RT-PCR. The results indicated that CYP9A17 was only expressed we examined in the sixth larval stage of H.armigera, but not in the pupae and adults , nor in the eggs. CYP6B7 genomic DNA sequence was obtained from the midgut genomic DNA of the sixth instar larvae of a laboratory strain of the cotton bollworm, H.armigera ,by using a pair of primers specific to CYP6B7 from GenBank. The resultant sequence is 1836bp in length, encoding a protein of 505 amino acid residues, containing 321bp introns in length. In order to heterolously express CYP6B7 we cloned, we used inverse PCR to eliminate the introns from the CYP6B7 and obtained a CYP6B7 allele with 99% of similarity to CYP6B7 (GenBank N0.AF031468 ) .Then we constructed the fusion gene of CYP6B7 and MBP(Maltose Binding Protein)with pMAL-c2x expression vector. After induction of 0.3mM IPTG at 30℃overnight, the fusion gene was successfully expressed in TB1, but we failed to purify the fusion protein.