Molecular Cloning And Functional Expression Of Cytochrome P450 Genes Of Helicoverpa Armigera (Hübner)

The cotton bollworm, Helicoverpa armigera (Hubner)(Lepidoptera:Noctuidae) is oneof the most serious pests on cotton and seriously restrict the production of cotton.This pestdistributes widely in space from 50°North latitude to 50°South Latitude. As the control ofH.armigera is mainly dependent on the pesticides for a long time, it has developedresistance to most insecticides especially to pyrethroids. Many studies have indicated thatthe enhanced oxidative metabolism by cytochrome P450 is a major factor responsible forpyrethroid resistance in cotton bollworm from Asia. In this study further experiments wereconducted to identify the role of P450 genes in pyrethroid resistance.1. Molecular cloning and sequence analysis of cytochrome P450 genes from H.armigeraTwo novel cytochrome P450 genes from CYP6 and CYP9 subfamily were cloned fromH. armigera using RACE (Rapid Amplification of cDNA Ends) strategy. These twocytochrome P450 genes were designated as CYP6AE12 and CYP9A18. CYP6AE12 andCYP9A18 have open reading frame of 1569 and 1690 nucleotides respectively encoding523 and 530 amino acids. These two P450 genes have several conserved domains such asthe heine-binding motif of FXXGXRXCXG, the K helix EXXR and a high hydrophobicsignal sequence in amino terminus. The homologous sequence analysis showed the identityof deduced amino acids of CYP6AE12 was 55％to CYP6AE1. The identities of deducedamino acids of CYP9A18 to the CYP9 members were 49％-66％. Phylogenetic tree analysisindicated that insect CYP6, CYP9 and mammalian CYP3 derived from the same ancestor.2. The mRNA expression of cytochrome P450 genes in resistant and susceptiblestrains in H. armigera Using the real-time quantitative PCR, the mRNA expression levels of six cytochromeP450 genes in the YS-FP and YS-FM strains were compared with those in a relativelysensitive strain YS. CYP6AE12, CYP9A12 in midgut of the YS-FP strain had 3.6- and133-fold overexpression respectively when compared with those in YS strain; In fatbodythe expression of CYP6AE12,CYP9A12,CYP9A14 and CYP9A17v2 had 1.3-,81.4-,2- and3.3-fold overexpression respectively compared with those in the YS strain. In the YS-FMstrain, expression levels of six P450 genes in either fatbody or midgut were similar or lesscompared with the YS strain. Constitutive overexpression of several cytochrome P450genes such as CYP9A12, CYP6AE12, CYP9A17v2 and CYP9A14 may be associated withpyrethroid resistance in the YS-FP strain.3. Heterologous expression of CYP9A genes of H. armigera in yeast (Saccharomycescerevisiae)Previous studies indicated that constitutive overexpression of cytochrome P450 genes(CYP9A12, CYP9A14 and CYP9A17v2) was possibly responsible for enchanced oxidativemetabolism of pyrethroid in H. armigera. In this study, a system for the heterologousexpression of three CYP9A genes in S. cerevisiae using the vector pYES2 was developed.Heterologous expression of CYP9A12, CYP9A14 and CYP9A17v2 can be induciblyexpressed in the culture media using galactose as the sole carbon source. The MRODacitivity of the cell lysate prepared from the recombinant yeasts containing CYP9A12,CYP9A14 and CYP9A17v2 were 2.98, 5.41 and 2.80 pmol resorufin/min/mg proteinrespectively, and the PNOD acitivity were 0.59, 0.42 and 0.47nmol p-nitrophenol/min/mgprotein respectively. The rate of esfenvalerate metabolism was 8.18, 4.29 and 1.71 pmolesfenvalerate/min/mg protein respectively. However, no MCOD, ECOD activities can bedetected in enzymes prepared from both recombinant and control yeast cells.