Xenobiotic Metabolism And Gene Expression Regulation Of The CYP6AE Gene Cluster Of Helicoverpa Armigera

Insects and plants have interacted on our planet for more than 400 million years.In the millions of years of coevolution,plants have developed effective defense systems to protect them from insect herbivores with an array of detrimental plant secondary compounds,and herbivorous insects have accordingly evolved diverse enzymes for detoxifying plant toxins,in which insect cytochrome P450 sits at the heart of detoxification enzymes.A large amount of P450 clusters were formed under the pressure of evolution.The presence of multiple,closely related CYP genes in the genomes of pest insects presents a challenge to the functional identification of the genes that are important in adaptation to plant chemicals and detoxification of insecticides.Analysis from 114 P450 genes of Helicoverpa armigera(Hubner)shows that there are 11 subfamilies possessing three or more genes,of which nine form tight head to tail clusters.CYP6AE cluster contains nine genes,which are arranged on the chromosome 16 of H.armigera.Previous studies have indicated that several CYP6AE genes of H.armigera are inducible by xenobiotics suggesting their potential role in detoxification and a controversy existed about whether CYP6AE14 detoxify gossypol.In this study,The CRISPR/Cas9-based reverse genetics approach in conjunction with in vitro metabolism were used to identify the contributions of CYP6AE cluster in xenobiotic detoxification and provide a new perspective to clarify the relationship of CYP6AE14 and gossypol detoxification.Transcriptional regulation of insect cytochrome P450 plays a key role in the stress of xenobiotics.In this study,inducible expression of CYP6AE genes by xenobiotics and response region of CYP6AE19 to xanthotoxin were executed to explore the mechanism of transcriptional regulation of insect P450.Besides,little effort has been made to exploit the transcription factors for xenobiotics response.In this study,four transcription factors for xenobiotics response were tried to be knocked out by CRISPR/Cas9,laying the foundation how transcription factors regulate P450 expression.1.CYP6AE cluster contributes to the detoxification of phytochemicals and insecticidesManagement of H.armigera in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic insecticides.The CRISPR/Cas9 genome editing technology was used to knock out CYP6AE cluster of H.armigera.Bioassays show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals(xanthotoxin and 2-tridecanone)and two classes of insecticides(indoxacarb and esfenvalerate),but not increases the susceptibility to gossypol.In vitro metabolism with each one of the heterologously expressed P450s showed that only CYP6AE19 is capable of metabolizing xanthotoxin,three P450s(CYP6AE11,CYP6AE14 and CYP6AE19)can metabolize 2-tridecanone,and CYP6AE17 and CYP6AE18 are capable of metabolizing indoxacarb.In conclusion,the CRISPR/Cas9-based reverse genetics approach in conjunction with in vitro metabolism not only demonstrate that the CYP6AE cluster contributes to detoxification of xenobiotics,but also show that multiple CYP6AE genes have synergetic effect in defensing xenobiotics.2.The expression pattern of CYP6AE gene under xenobiotics stress and the identification of responsive region to xanthotoxin in the promoter of CYP6AE19.Inducibility of cytochrome P450 is a key mechanism of detoxifying phytochemicals and insecticides metabolic resistance.In this study,Three plant allelochemicals(gossypol,xanthotoxin and 2-tridecanone)and an insecticide(esfenvalerate)were used as inducer to explore the expression pattern of CYP6AE.The results show that the CYP6AE19,which could metabolize xanthotoxin,was very strongly induced by xanthotoxin in midgut and fatbody of H.armigera.Besides,CYP6AE20 was also strongly induced by xanthotoxin,and CYP6AE14 was induced 40-fold by gossypol in the midgut of H.armigera,although CYP6AE20 and CYP6AE14 can't metabolize xanthotoxin and gossypol.Then the induction effect of the stepwise deletion promoter of recombinant plasmid of CYP6AE19 by xanthotoxin were tested through dual-luciferase activity.The results suggested that the response region to xanthotoxin was distributed between-396 and-201 bp in the promoter of CYP6AE19.In conclusion,this study demonstrated that the inducibility of cytochrome P450 is an important mechanism of detoxifying xanthotoxin in H.armigera,presented the inconsistency between the metabolism capability and inducibility,and the inducibility of CYP6AE19 under xanthotoxin was regulated by cis-acting element in its promoter.3.The expression of CYP6AEs and effects of growth and development after HaHR96 and HaAhR knockout by CRISPR/Cas9Exposure to xenobiotics triggers detoxification enzymes to relieve stress.Although transcription factors for xenobiotics response have been studied in vertebrates,little effort has been made in that of insects.In this study,four transcription factors(HaHR96?HaAhR?HaNrf2 and HaKeapl)for xenobiotics response were tried to be knocked out by CRISPR/Cas9.The result shows that the knockout strain for HaNrf2 and HaKeapl can't be obtained,suggesting their key role in growth and development.The knockout of HaHR96 would significantly upregulate the expression of CYP6AE12 and CYP6AE18,and downregulate the expression of CYP6AE11 and CYP6AE14 in midgut of H.armigera,but not affects CYP6AEs in fatbody.The knockout of HaAhR would reduce the pupa weight,affect the development of antennae and wing of adult,and then influence the mating and survival of moth.However the knockout of HaAhR would not affect the expression of CYP6AEs in midgut and fatbody.This study takes the lead in knocking out transcription factors for xenobiotics response in non-model insect H.armigera to explore the transcriptional regulation of P450s and effects of growth and development,and provide several potential target genes for integrated management to H.armigera.