Phylogenetic Analysis Of Cytochrome P450 Genes And Functional Characterization Of Xenobiotic Metabolism Related P450 Genes In Helicoverpa Armigera

The cotton bollworm,Helicoverpa armigera(Hiibner),is one of the most commonly reported pests all over the world.It created resistance against almost all kinds of insecticides because of polyphagy,high fecundity and migration capacities,and facuitative diapause.Pyrethroid resistance was an early reported and one of the most serious resistant patterns in H.armigera.It was reported that P450-mediated involvement of detoxification was related to pyrethroid resistance in H.armigera from many parts of the world.But most pieces of evidences were only focus on the level of enhanced detoxification or several overexpressed P450 genes.As an important and complicated insect enzyme,cytochrome P450 has numerous genes and diversified functions.But systematic study and direct evidence were still lacked for the causal relationship between most cytochrome P450 and resistant pattern.In this study,108 cytochrome P450 genes from reported H.armigera genome data were organized by phylogenetic analysis.The evolution of cytochrome P450 genes in Clan2 and ClanM was relatively conserved.Most genes in these two groups from H.armigera and Bombyx mori showed 1:1 orthologies.Cytochrome P450 genes in Clan3 and Clan4 were massively amplified and might be linked with the detoxification of xenobiotics.Twenty one P450 genes from three pyrethroid resistance-related H.armigera P450 subfamilies,CYP6B,CYP9A and CYP6AE,were functionally expressed in an insect cell line by baculovirus expression system(Bac-to-Bac).In addition,substrate recognition sites were predicted by the alignments of their primary and secondary structures and exchanged between several CYP6AE P450s which had high sequence similarities but great difference on metabolic capacities.The resultes of functional expression showed that the metabolic capacities of CYP6AE P450s were influenced by SRS1 and SRS6.This research provided direct evidences for the relationships between cytochrome P450s in CYP6B and CYP9A subfamilies and fenvalerate resistance in H.armigera.We firstly identified the metabolic capabilities of CYP6AE to multiple insecticides,and the protein stabilities and metabolic abilities to esfenvalerate of CYP6AE P450s might be influenced by SRS.These results made a contribition to the research of P450-mediated pyrethroid resistance mechanism in H.armigera and structure-function interaction of insect cytochrome P450 genes.1.Phylogenetic analysis of H.armigera P450 genesWe obtained 114 cytochrome P450 genes from H.armigera genome data and 105 correct genes were selected after checking.Physical map of whole H.armigera P450s was drawed according to their absolute position on genome.Phylogenetic analysis was performed with 108 P450s(105 selected genes and three un-annotated genes:CYP9A12,CYP9A18 and CYP337B3)and 83 B.mori P450s.These results showed that H.armigera P450s distributed in 21 chromosomes and divided to four Clans(2,3,4,M),28 families and 55 subfamilies.There were 8 genes in Clan2;10 genes in ClanM;48 genes in Clan3 and 42 genes in Clan4.The evolution of genes in Clan2 and ClanM was relatively conserved and most of them showed 1:1 orthology in H.armgiera and B.mori.Genes in Clan3 and Clan4 were massively amplified and formed ten subfamilies with over 3 genes in multiple chromosomes and probably related to xenobiotic detoxification.These results comprehensively showed the distribution and classification of H.armigera P450 genes and laid the foundation for the future work on the systematic function and evolution study of H.armigera P450s.2.Functional expression of P450 genes in CYP6B and CYP9A subfamiliesAccording to the recent researches,a number of H.armigera P450 genes in Clan3 and Clan4 were constitutively overexpressed in many pyrethroid resistance strains or induced by multiple chemical compounds including pyrethorids.Most of them were located in CYP6B and CYP9A subfamilies,but only CYP6B6,CYP6B7,CYP9A12 and CYP9A14 were demonstrated having metabolic capacities with esfenvalerate in vitro.Twelve P450s were found in CYP6B and CYP9A subfamilies in H.armigera genome.Do high sequence similarities indicate similar functions?So we cloned eleven P450 genes in these two subfamilies and heterologously expressed them in insect cells by Bac-to-Bac system.The final results showed that all P450s in CYP6B and CYP9A subfamilies can metabolise esfenvalerate except CYP6B43.In the same conditions,CYP6B6 had the highest metabolic rate(V=0.46 pmol 4'-OH product/min/pmol P450)and CYP9A18 was the second one V=0.3 pmol 4'-OH product/min/pmol P450).CYP6B2 showed the lowest rate(V=0.01 pmol 4'-OH product/min/pmol P450).2'-hydroxyesfenvalerate was spotted in some samples for the first time and the main metabolite of CYP9A12 was 2'-hydroxyesfenvalerate.These results showed the metabolic capabilities of CYP6B and CYP9A subfamilies to esfenvalerate.These lines of evidences demonstrated the protential roles of H.armigera CYP6B and CYP9A P450s in fenvalerate resistance for the first time.3.Phylogenetic analysis,tissue expression pattern and functional characterization of P450 genes in H.armigera CYP6AE subfamilyThere are ten P450 genes in H.armigera CYP6AE subfamily share at highest 91.8%amino acid sequence similarity,and nine of them form an end-to-end gene cluster on chromosome 16.The results of current studies showed over half of H.armigera CYP6AE subfamily members could be induced by many kinds of pesticides and plant allelochemicals including pyrethroid and gossypol.They were probably related to the detoxification of these compounds.Only the functions of CYP6AE14 were identified,but the results from in-vitro tests were inconsistent with in-vivo assays.In this study,the evolution pattern showed conservative synteny and close phylogenetic relationships of CYP6AE genes from H.armigera,Helicoverpa zea and B.mori.CYP6AE14 and CYP6AE18 were diverged from recent gene duplication events and CYP6AE19/20 clade was the early diverged genes in H.armigera CYP6AE subfamily.CYP6AE12,CYP6AE14 and CYP6AE19 showed tissue-specific expression pattern:CYP6AE12 and CYP6AE19 mainly expressed in fatbody and CYP6AE14 mainly expressed in midgut.CYP6AE11 showed the most abundent expression level and it was hard to detect expression of CYP6AE20 in both midgut and fatbody.The in-vitro metabolism tests showed CYP6AE P450s have high metabolic efficiencies to esfenvalerate except CYP6AE20.CYP6AE11 had the highest metabolic rate(Vmax=2.3 pmol/min/pmol P450)and CYP6AE14 had the highest metabolic efficiency(Vmax/Km=0.51).Similarly,nine CYP6AE P450S can metabolise aldrin except CYP6AE20.CYP6AE12 showed highest metabolic rate of aldrin(V=0.45 pmol/min/pmol P450).Seven P450s can metabolise imidacloprid but even the highest activity was 1/10 of the positive control,CYP6CMlvQ.Gossypol could not be metabolised by all CYP6AE P450s.These results demonstrated that P450s in H.armigera CYP6AE subfamiy can metabolise multiple insecticides but can not decrease gossypol in-vitro for the first time.The CYP6AE subfamily might involved in the resistance pattern mainly by induction and gossypol might play a limited role in the evolution of CYP6AE genes.4.Effects of substrate recognition sites SRS1 and SRS6 on H.armigera CYP6AE P450s in the metabolism of esfenvalerateGreat difference on metabolic capacities can be found in CYP6AE subfamily P450s despite the high sequence similarities between them.Acorrding to the difference of metabolic capacities and phylogenetic relationships,we analyzed the primary sequences and secondary structrues of two pairs of CYP6AE P450s,one pair was CYP6AE17/18 in the same clade with over 90%sequence identity and over 7-fold difference on metabolic effeciency to esfenvalerate.The other pair was CYP6AE11 from late diverged clade with high metabolic ability and CYP6AE20 which represented ancient clade that lost metabolic capabilities to three kinds of pesticides.SRS1 and SRS6 had the lowest identities among six substrate recognition sites(SRS).So we exchanged the SRS1 of CYP6AE11-CYP6AE20 and SRS1 or SRS6 between CYP6AE17 and CYP6AE18.Six mutants were functionally expressed and the results showed great variations on the stability of protein and metabolic ability.The mutant of CYP6AE11 could not fold correctly in High Five cells but keep slight metabolic capabilities and same affinity to esfenvalerate(Km=6.6±1.7?M).The absorption peak of CYP6AE20-11 SRS 1 in reduced CO difference spectrum assay shifted to 457nm but obtained relatively low metabolic capacity to esfenvalerate(Vmax/Km=0.05).The metabolic abilities of two mutants of CYP6AE18 had been promoted greatly(Vmax/Km=0.25 and 0.4)and even higher than wild CYP6AE17(Vmax/Km=0.27).The content of P420 was high in recombinant mutants of CYP6AE17 and the metabolic capacities of these two mutants fall heavily with a 2-fold and 13-fold decreases.These results demonstrated the important roles of SRS1 and SRS6 regions of CYP6AE P450s in protein stability and esfenvalerate metabolism,and the polymorphisms in these two regions promoted the functional distribution of CYP6AE subfamily.It also made a contribution to the research of function-structure relationships of insect cytochrome P450s.