Expression Regulation And Evolution Of Cytochrome P450 CYP9A Subfamily Genes In Helicoverpa Armigera (Hübner)

The cotton bollworm Helicoverpa armigera is a serious cotton pest of global importance.This pest has developed high levels of resistance to pyrethroids,which threatens cotton production.Numerous studies show that enhanced detoxification through overexpression of related P450 genes is an import mechanism to pyrethorid resistance in H.armigera.In the current study,genomic organization and 5'-upstream region sequences of three P450 genes(CYP9A12,CYP9A14,and CYP9A17) related to pyrethroid resistance of H.armigera were investigated.These results provide a basis for further work on regulatory mechanism of P450 gene overexpression.1.Intron/extron structure of three P450 CYP9A subfamily genes from H. armigeraAccording to obtained cDNA sequences of CYP9A12,CYP9A14 and cYPgA17v2 a group of specific primers were designed to amplify complete genome sequences of these three P450 genes from a susceptible strain(SCD),a field-collected strain(YG) and a pyrethroid-resistant strain(YGF) of H.armigera.Coding areas of CYP9A12, CYP9A14 and CYPgA17v2 were inserted by 9 introns followed the GT-AG rule without exception.Similarities in the protein sequences and intron positions indicate that these three P450 genes may be derived from a common ancestral gene.The evolugtionay mechanism of the three P450 genes may be gene duplication and subsequently divergence.2.Molecular cloning and sequence analysis of the 5'-UTR region of cytochrome P450 CYP9A subfamily of H.armigeraOverexpression of some cytochrome P450 CYP9A subfamily genes is associated with pyrethroid resistance in H.armigera.Based on the 5'-end nucleotide sequence of the full length cDNA of CYP9A12,CYP9A14 and CYP9A17v2,the 5'-untranslated region(UTR) of these genes in SCD strain were obtained by genome walking technique in order to investigate the regulatory mechanism of overexpression.The 5'-UTR fragments of these genes in YGF and YG strains were cloned using specific primers based on obtained 5'-UTR sequences of these genes in SCD strain.The transcriptional start sites of CYP9A12,CYP9A14 and CYP9A17v2 were predicted by the NNPP software and cis-acting elements were predicted by TFSEARCH 1.3 software.It will provide an important basis for investigating expression regulation of cytochrome P450 genes in pyrethroid-resistant H.armigera.3.Evolution of insect cytochrome P450 CYP9A subfamily genesAccording to sequences of cytochrome P450 CYP9A subfamily genes deposited in GenBank and related P450 databases,a multiple sequence alignment was carried out by clusta1X,and the N-J tree was constructed by MEGA3.1.The results showed that CYP9A subfamily genes may come from a common ancestral gene.The mechanism of evolution may be gene duplication and subsequent divergence,and intron loss may be one of the most important factors for divergence.High levels of similarities between the amino acid sequences of the CYP9A subfamily genes suggest that they may have common mechanisms in metabolizing endogenous substrates (steroid hormones,lipids,etc.) and detoxifying enobiotics(pesticides,allelochemicals, etc.).