Transcriptional Responses Of All P450 Genes To Xenobiotic Exposure And Functional Expression Of Three Subfamilies Genes In Helicoverpa Armigera

The cotton bollworm Helicoverpa armigera(Hübner)is a typical polyphagous pest with more than 300 species of host plants.At present,it has developed high-level resistance to more than 20 chemical pesticides and causes more than 3 billion dollars in economic losses each year around the world.Therefore,the cotton bollworm is one of the most serious agricultural pests in the world.It was reported that cytochrome P450 oxidase-mediated detoxification of insecticides and plant secondary substances is one of the main reasons for the cotton bollworm`s host shift and insecticide resistance.However,most studies are limited to indirect evidence such as the enhancement of cytochrome P450s’ enzyme activity and transcriptional responses of some P450 s to xenobiotics,lacking direct evidence such as gene function studies.The relationship between P450 transcriptional responses and detoxificationcapacities needs to be systematically studied.In this study,transcriptional responses of 117 cytochrome P450 genes to xanthotoxin and esfenvalerate in H.armigera were detected by RNA-seq.The transcriptional responses and metabolic capacities of the members of CYP9 A,CYP6B,and CYP321 A subfamilies were verified by Real-time Quantitative PCR(RT-q PCR)and baculovirus mediated expression system in Sf9 cells.The results are helpful to clarify the relationship between the inducibilities and detoxification-capacities of P450 and provide more direct evidence for host adaptation and pesticide resistance development in H.armigera.1.Transcriptional responses of H.armigera P450 genes to xanthotoxin and esfenvalerateIt is generally believed that insect cytochrome P450 s can respond to the stress of xenobiotics at the transcriptional level,which is an important reason for insects’ adaptation to host plants and insect resistance.In this study,RNA-seq was used to identify the global transcriptional variation of H.armigera P450 s in the midgut and fatbody after 48 h treatment of xanthotoxin and esfenvalerate.After 1 mg/g xanthotoxin treatment,the transcriptional levels of 11 P450 s were up-regulated in the midgut.The transcriptional levels of CYP6AE19 and CYP6AE20 had the most extreme changes(1366-fold and 109858-fold,respectively).Moreover,the transcriptional levels of 15 P450 s decreased to 2% and 38% of basic expression in the midgut.In the fatbody,the transcriptional levels of 16 P450 s were upregulated 2～132-fold,and the transcriptional levels of 5 P450 s decreased.Five μg/g esfenvalerate increased the expression of 15 P450 s in the fatbody(2-to 21-fold)and only 6P450 s in the midgut(2-to 6-fold).Furthermore,it decreased the expression of 6 P450 s in the fatbody and 8 P450 s in the midgut.Among the 44 P450 s whose transcriptional levels changed significantly after the treatment with two xenobiotics,most belonged to Clan3 and Clan4,and the members from CYP321 A,CYP6AE,CYP6 B,and CYP9 A subfamilies were the majority.RT-q PCR was used to detect the expression change of 13 P450 genes in the three subfamilies of CYP9 A,CYP6B,and CYP321 A further.After treating esfenvalerate,the expression of 6 P450 s,including CYP9A3,CYP9A12,CYP9A15,CYP9A16,CYP9A23,and CYP321A1,increased 1-to 3-fold in the midgut.Moreover,the expression of 3 P450 s increased in the fatbody,including CYP9A3(6-fold),CYP9A12(5-fold),and CYP321A1(3-fold).After the xanthotoxin exposure,the expression of 5 P450 s in the midgut(CYP9A3,CYP9A12,CYP321A1,CYP321A2,and CYP321A5)increased 3-to 14-fold,and the expression of 7 P450 s in the fatbody(CYP9A3,CYP9A12,CYP6B2,CYP6B7,CYP321A1,CYP321A2,and CYP321A5)had 1-to 9-fold increase.In general,more H.armigera P450 s have transcriptional responses to xanthotoxin than to esfenvalerate.The inducibilities of P450 s have specificities in xenobiotics and tissues.Due to different analysis methods,results detected by the two technologies were inconsistent.This study screened P450 s that have transcriptional responses to xanthotoxin and esfenvalerate and may be involved in the metabolism of these xenobiotics.The results could provide important clues for further study of P450s’ metabolic capacities to these compounds.2.Functional expression of P450 genes in CYP6 B,CYP9A,and CYP321 A subfamilies of H.armigeraIt is reported that 2-tridecanone and xanthotoxin,two important plant secondary substances in host plants of H.armigera,and esfenvalerate induce several P450 s extremely.Most of those inducible P450 s belong to CYP6 AE,CYP6B,CYP9 A,and CYP321 A subfamilies.Nevertheless,the metabolic capacities of most members are still unknown.There are four P450 s in CYP6 B,eight P450 s in CYP9 A,and three P450 s in CYP321 A in the H.armigera genome.In this study,three P450 s from the CYP321 A subfamily were cloned and expressed in Sf9.At the same time,those genes of CYP6 B and CYP9 A subfamilies were expressed in Sf9.The results of the in vitro metabolism indicated that CYP6B2,CYP6B6,and CYP321A1 could metabolize xanthotoxin and 2-tridecanone,and others have no metabolic capacities to these plant allelochemicals.CYP321A1 exhibited the fastest metabolism of xanthotoxin with a metabolic rate of 0.91 ± 0.06 pmol/min/pmol P450.CYP6B6 came next,with the metabolic rate of 0.14 ± 0.012 pmol/min/pmol P450,and CYP6B2 metabolized xanthotoxin at the lowest rate(0.053 ± 0.014 pmol/min/pmol P450).For metabolism of 2-tridecanone,CYP6B6 showed the fastest metabolic rate(0.11 ± 0.010pmol/min/pmol P450).CYP6B2(0.041 ± 0.0007 pmol/min/pmol P450)and CYP321A1(0.083 ± 0.004 pmol/min/pmol P450)metabolized 2-tridecanone slowly.In addition,CYP321A5 could metabolize esfenvalerate at a lower rate(0.030 ± 0.001 pmol/min/pmol P450)than reported CYP6 B and CYP9 A P450s.Therefore,these results clarified that multiple P450 s in CYP6 B and CYP321 A subfamilies were involved in the detoxification of xanthotoxin,2-tridecanone,and esfenvalerate.Moreover,they played essential roles in the host adaptation and pesticide resistance evolution of cotton bollworms.Through comprehensive analysis of P450s’ transcriptional responses and metabolic function,we found there is no absolute causal relationship between inducibilities and detoxification-capacities of insect P450 s.