Determination Of The Detoxification Roles Of Cytochrome P450s In Clan 2 And Mitochondria Clan Of Helicoverpa Armigera

Cytochrome P450 enzymes are oxidases widely distributed in almost all organisms on earth.They catalyze the insertion of oxygen atom into the substrate and increase the hydrophilicity to promote subsequent reactions such as hydrolysis,sequestration,and glycosylation.In insects,P450 is an important detoxification enzyme that participates in the chemical defense of insects against plant secondary substances and insecticides,and has important contributions for insect host shift and the evolution of insecticide resistance.Insect P450 is mainly divided into 4 clans,but functional study has been mainly focus in P450 s of Clan 3 and Clan 4,which are widely expanded and considered relating to chemical defense.However,P450 s of Clan 2 and mitochondrial Clan(Clan M)are evolutionarily conserved,but their functions are rarely studied.Generally,it is considered relating to the physiological process of insects,mainly involved in the synthesis or metabolism of insect endogenous hormones and signal compounds.However,a small number of previous studies found that the transcription level of P450 genes in Clan 2 and Clan M could be up-regulated by multiple allelochemicals and several P450 s in Clan M could metabolize pesticides,indicating that the P450 s in these two groups are potential detoxification P450 s involved in metabolism of xenobiotics.In this study,we aimed at all eight Clan 2 and ten Clan M P450 genes in a typical polyphagous pest-Helicoverpa armigera.The potential roles of these 18 P450 s in the detoxification of plant secondary substances and pesticides were evaluated by in vitro functional characterization and exogenous compound induction.It provides important evidence and new perspectives for the research on chemical defense and molecular mechanism of insecticide resistance of cotton bollworm.The results are as follows:1.In vitro expression and metabolic system construction of Clan 2 and Clan M P450sGenomic data shows that there are eight Clan 2 and ten Clan M P450 s in the cotton bollworm.The transcription levels of many members can be induced by exogenous compounds or constitutively up-regulated in insecticide-resistant strains indicating the potential role in detoxification of xenobiotics.In order to verify its function,we cloned these eighteen P450 genes,and expressed in Sf9 cells by insect cell-baculovirus expression system(Bac-to-Bac).Clan 2 P450 s were co-expressed with the cytochrome P450 reductase(CPR)of Helicoverpa armigera.Clan M P450 s were co-expressed with adrenodoxin(Adx)and adrenodoxin reductase(Ad R)of Helicoverpa armigera.Reduced CO difference spectrum showed sixteen P450 s were successfully expressed in vitro except CYP49A1 and CYP428A1.The results of model substrate metabolism showed that several members in both Clan 2 and Clan M P450 s exhibited O-dealkylation or O-debenzylation activity.It implied that both in vitro mitochondrial and microsomal P450 electron transport systems worked well.In this study,the successful expression of sixteen Clan 2 and Clan M P450 s of Helicoverpa armigera and the construction of mitochondrial P450 electron transport system provided an important basis for further study of detoxification roles of Clan 2 and Clan M P450 s in H.armigera.2.In vitro metabolism of xenobiotics by cytochrome P450 s in Clan 2 and Clan MPrevious studies found that insecticides represented by fenvalerate and plant secondary compounds represented by 2-tridecanone could induce multiple P450 genes within Clan 2and Clan M in the cotton bollworm.However,to data,there was no evidence that these P450 s were directly involved in the detoxification of these compounds.In this study,recombinant P450 s were used to detect the metabolism of aldrin,esfenvalerate,2-tridecanone and xanthotoxin in vitro.Results showed that CYP305B1 and CYP18A1 in Clan 2 and mitochondrial P450 CYP333B3 could transfer aldrin to dieldrin,CYP305B1 and CYP339A1 in Clan2 could metabolize esfenvalerate,CYP303A1 in Clan M could efficiently metabolize 2-tridecanone,but all P450 s could not metabolize xanthotoxin.In addition,it was found that the co-expression ratio of P450 and Adx/Ad R would affect the expression and metabolic activity of recombinant mitochondrial P450 s.The P450 protein content and metabolic activity reach the best balance under the MOI ratio = 1:1:1.This study provides important clues for insect chemical defense and the molecular mechanism of insect-host co-evolution,and provides more potential resistance genes for the study of insecticide resistance mechanisms.3.Expression pattern of Clan 2 and Clan M P450 s in H.armigeraThe temporal and spatial expression pattern of cytochrome P450 in insects and the response to xenobiotics are usually related to its functions.In order to further understand the role of Clan 2 and Clan M P450 in the growth,development and detoxification of the cotton bollworm,in this study,the expression patterns of 18 Clan 2 and Clan M P450 s were analyzed by RNA-seq.It was found that CYP333B3,CYP333A1 and CYP18A1 were highly expressed in all tested stages and tissues of the cotton bollworm.CYP303A1 and CYP49A1 expressed in the pupal.CYP307A2 is specifically expressed in the trachea.In addition,the transcriptome results showed that CYP18B1 in Clan 2 was 2.1-fold induced by xanthotoxin.Quantitative Real-time PCR results showed CYP301A1 in Clan M,CYP307A2,CYP15C1 and CYP18A1 in Clan 2 could be induced by esfenvalerate(the fold change were 1.7).In general,the P450 gene in Clan 2 and Clan M have limited responses to two exogenous compounds.This result provides predictive information for the study of P450 functions in Clan 2 and Clan M.Meanwhile,the results of transcriptional induction and functional studies indicated that there is no causal relationship between inducibility and metabolic competence of these P450 s in H.armigera.