Functional Expression And Pyrethroid Metabolism Of P450 Genes CYP9A12 And CYP9A17 Of Helicoverpa Armigera (Hübner)

The cotton bollworm,Helicoverpa armigera(Hiibner)(Lepidoptera:Noctuidae) is an important cotton pest.The ability of cotton bollworm to thrive on chemically well-defended plants and develop resistance to insecticides was well recognized.Cytochrome P450 is one of the largest gene families in virtually all living organisms.The cotton bollworm P450 enzymes involved in basic physiological processes and xenobiotic metabolism are of great interest.Overexpression of several P450 genes from CYP9 and CYP6 families were reported to be associated with xenobiotic detoxification in H.armigera.In this study,two CYP9A genes of H.armigera were heterologously expressed in yeast(Saccharomyces cerevisiae),and shuttle expression vectors for CYP6B7 and CYP6AE14 of H.armigera were constructed.1.Comparison of mRNA expression levels of CYP9A12 and CYP9A17v2 in H. armigeraUsing RT-PCR,the mRNA expression levels of two cytochrome P450 genes CYP9A12 and CYP9A17v2 were compared in midguts,fatbodies of final instar larvae,pupae (male/female) and adults(male/female) of a field-collected population(Jiangpu) of H. armigera.The mRNA expression levels of cYP9A12 and cYPgA17v2 in midgut were higher than those in the fatbody respectively.The mRNA expression level of CYP9A17v2 was higher than that of CYP9A12 in either midgut or fatbody,cYP9A12 and CYP9A17v2 were not expressed or expressed at extremely low levels in pupae and adults.2.Metabolism of pyrethroids by H.armigera P450 CYP9A12 and CYP9A17v2 in yeastEnhanced oxidative metabolism by cytochrome P450 is a major factor responsible for pyrethroid resistance in the cotton bollworm Helicoverpa armigera,and constitutive overexpression of CYP9A12 and CYP9A17v2 was associated with pyrethroid resistance.To examine the involvement of cytochrome P450 gene cYP9A12 and CYP9A17v2 in pyrethroid metabolism,we measured metabolism of deltamethrin,cyhalothrin, fenpropathrin and bifenthrin by CYP9A12 and CYP9A17v2 expressed in yeast (Saccharomyces cerevisiae).The metabolism rates of heterologously expressed CYP9A12 against deltamethrin,cyhalothrin and fenpropathrin were 8.58 pmol/min/mg protein,5.85 pmol/min/mg protein and 3.94 pmol/min/mg protein,respectively.The metabolism rates of heterologously expressed CYP9A17v2 against deltamethrin,cyhalothrin were 9.06 pmol deltamethrin/min/mg protein,5.61 pmol cyhalothrin/min/mg protein,respectively.No fenpropathrin metabolism was detected in CYP9A12-containing or CYP9A17v2-containing yeast cell lysates.This study showed CYP9A12 and CYP9A17v2 has the capacity to metabolize several pyrethroids,and also provided direct evidence for the involvement of CYP9A12 and CYP9A17v2 in pyrethroid resistance in H.armigera.3.Construction of shuttle vectors for functional expression of CYP6B7 and CYP6AE14 of H.armigera in yeastTwo cytochrome P450 genes(CYP6B7 and CYP6AE14) from CYP6 subfamily were cloned from H.armigera using gene specific primers.CYP6B7 and CYP6AE14 have open reading frame of 1512 and 1578 nucleotides respectively,encoding 504 and 526 amino acids.The identities of deduced amino acids of CYP6B7 and CYP6AE14 to the members which have deposited in the GenBank were 99%.In order to explore the function of CYP6B7 and CYP6AE14 with the yeast expression system,shuttle expression vectors (pYES2-CYP6B7 and pYES2-CYP6AE14) ready for yeast transformation were successfully constructed.