The Transcription Regulation Mechanism Of Cytochrome P450 And Glutathione-S-Transferase Genes In Spodoptera Exigua

Cytochrome P450 and Glutathione-S-transferase can metabolize different structural insecticides.P450-and GST-mediated metabolic resistance mechanism is prevalent in insecticide resistance.Previous studies indicated that insecticide resistance was related to the enhanced detoxification ability of P450 and GST,but mainly focused on the synergistic and enzyme activity experiment,and the molecular mechanism research was not understood.Overexpression of detoxifying enzyme genes is often the cause of increased resistance level.Therefore,exploring the transcriptional regulation mechanism of detoxification enzyme genes is crucial for studying the cause of resistance,however,there is no relevant study in Spodoptera exigua.In this study,we identified the P450 and GST family genes by PCR and bioinformatic analysis was performed.On the one hand,the fat body cell line of Spodoptera exigua was induced by the insecticides,and the P450 and GST genes whose expression levels were changed after exposure to insecticides were studyed,and the fluorescent reporter plasmid system was further used to explore the regulation mechanism of co-induced expression of the GST genes.On the other hand,we analyzed the expression level of P450 family genes in resistant and susceptible strain.The P450 genes with high constitutive expression in resistant populations were identified,and the four P450 genes with the highest expression level were studyed for insecticide resistance by constructing transgenic Drosophila and and functional expression of P450s.The transcriptional regulation mechanism of constitutive up-regulation of P450 gene was studied.1?Most of Spodoptera exigua P450 genes can be induced by insecticidesIn order to study the mechanism for detoxification of insecticides in S.exigua,the cytochrome P450 genes from this insect are cloned and analyzed.68 P450 expressed in larvae are cloned,and the phylogenetic tree combined with CYPs from Bombyx mori,Drosophila melanogaster and Tribolium castaneum are constructed.The 68 CYPs from S.exigua belong to 25 families and 40 subfamilies.CYP2 clan and mitochondrial clan have few members,while CYP4 clan and CYP3 clan has multiple P450 genes which imply the extensive gene expansions during evolution.Heat map cluster analysis showed that the P450 gene showed a specific tissue expression pattern.In order to understand the expression response of S.exigua on insecticide exposures,the expression levels of these P450 genes are analyzed with fat body cells exposed by indoxacarb,metaflumizone,chlorantraniliprole,lambda-cyhalothrin and abamectin separately.After treatment with five insecticides,the expression levels of five P450 genes showed significant effects.The expression levels of CYP6AE47,CYP6AB31,CYP9A9 and CYP9A10 in these five P450 genes were significantly up-regulated after treatment with these five insecticides.CYP321A8 decreased in expression level after avermectin treatment,while other four insecticides were up-regulated after treatment.The expression levels of CYP321A9,CYP6AE10 and CYP321A16 were significantly increased after treatment with indoxacarb,cyanofluorfen,chlorantraniliprole and lambda-cyhalothrin,while avermectin had no significant effect on the expression of these three P450s.In the analyzed insecticides the gene expression response in S.exigua to abamectin is obviously different from the response to indoxacarb,metaflumizone,chlorantraniliprole and lambda-cyhalothrin exposures.The P450 expression responses to indoxacarb,metaflumizone,chlorantraniliprole and lambda-cyhalothrin exposures are very similar.2?Metabolic resistance to insecticide in Spodoptera exigua relies on multiple P450 enzymesThe P450-mediated metabolic resistance mechanism is prevalent in insect resistance to various insecticides.Previous studies have shown that the resistance of beet armyworm to various insecticides is related to the enhanced ability of P450,but mainly focus on synergistic and enzyme activity experiments.In this study,the inhibitor PBO significantly increased the toxicity of chlorpyrifos in resistant strain with a synergistic ratio of 8.09,which also increased the toxicity of pyrethroid insecticides.The P450 enzyme activity in the resistant strain was 2.93 times that of the susceptible strain.We also analyzed the expression levels of P450 in resistant and susceptible strains by qRT-PCR_Compared to susceptible strain,21 P450s(30%of P450 genes)were up-regulated more than 2-fold in resistant strain(p-value<0.05).These 21 overexpressed P450s are mainly derived from the CYP6,CYP9 and CYP321 families,suggesting that multiple P450s may be involved in chlorpyrifos and pyrethroid insecticides resistance.The transgenic Drosophila bioassay results showed that the over-expressed transgenic Drosophila strain actin5 C>CYP321A8,actin5C>CYP321A16,actin5C>CYP6AE70 and actin5C>CYP332A1 can not only improve the resistance to chlorpyrifos,but also improve resistance to the pyrethroid insecticides cypermethrin and deltamethrin.In vitro recombinant protein metabolism experiments also confirmed that recombinant CYP321A8,CYP321A16,CYP6AE70,CYP332A1 and CYP321B1 proteins can metabolize chlorpyrifos,as well as pyrethroid insecticides cypermethrin and deltamethrin.The results of the kinetic parameters of these five recombinant proteins on chlorpyrifos showed that these five P450s have certain affinity and metabolic rate for chlorpyrifos.We characterized the difference in enzyme metabolic rate by intrinsic clearance(Vmax/Km)and found that CYP321A8 and CYP321B1 have the highest metabolic rate and CYP6AE70 has the lowest metabolic rate.These results indicate that multiple P450s of Spodoptera exigua are involved in the resistance of chlorpyrifos,cypermethrin and deltamethrin.3?Cis-acting elements and tra ns-acting factors coordinate ly regulate the expression of CYP321A8 and CYP321B1 conferring resistance to insecticides in lepidopteran S.exiguaPrevious studies have found that CYP321A8 and CYP321B1 were involved in chlorpyrifos and pyrethroid insecticide resistance,however the regulation mechanism is not cleared.In the present study,we cloned the upstream promoter sequences of CYP321A8 and CYP321B1 and found multiple transcription factor binding sites on these two upstream sequences.We further analyzed the relative expression levels of the relevant transcription factors in the resistant and susceptible strains and found that the CncC and Maf mRNA levels in the resistant strain were up-regulated by 5.2-fold and 2.8-fold,respectively.Fluorescence reporter assays confirmed that overexpressed CncC and Maf can regulate the constitutive high expression of CYP321A8 and CYP321B1 in resistant strain,and also found that there are two CncC/Maf binding sites in the proximal and distal CYP321A8 upstream sequence,and CYP321B1 only have an CncC/Mafbinding site in the proximal sequence.The promoter sequence analysis of CYP321A8 and CYP321B1 in resistant and susceptible strains was also analyzed,and it was found that there were many differences in the CYP321A8 and CYP321B1 resistant and susceptible promoter regions.The fluorescence reporter assay indicated that the CYP321A8 promoter activity in the resistant strain was 10.4 times that of the susceptible strain,and the CYP321B1 promoter activity in the resistant strain was 1.95 times that of the susceptible strain.The deletional segmentation study showed that the difference in CYP321A8 promoter activity was between-385 and-142,and the difference in CYP321B1 promoter activity was between-258 and-210.The mutation test showed that the site mutation at M5 between-385 and-142 is responsible for the high activity of the resistant CYP321A8 promoter.The site mutation at M2 between-258 and-210 is responsible for the high activity of the resistant CYP321B1 promoter.Further predictions revealed that the mutation at M5 between-385 and-142 of CYP321A8 was located in the cis-acting element of the transcription factor Knirps.Co-transfection showed that overexpressed Knirps significantly up-regulated the resistance plasmid P(-385/-1)-R fluorescence activity,but had no significant effect on the susceptible plasmid P(-3 85/-1)-S fluorescence activity.These results indicate that mutations at the resistance promoter M5 recruited the transcription factor Knirps,enhancing the overexpression of CYP321A8 in resistant strain.This suggests that the high promoter activity caused by mutations in the upstream promoter of the resistant strain is also responsible for the high expression of CYP321A8 and CYP321B1 in resistant strain.The final co-transfection assay confirmed that the overexpressed CncC/Maf and cis-acting element mutations coordinately regulate constitutive overexpression of CYP321A8 and CYP321B1.4?Insecticides induce the co-expression of glutathione S-transferases through ROS-activated CncC/Maf pathway in S.exiguaGlutathione S-transferases(GSTs)are a family of multifunctional enzymes that are involved in detoxification of electrophilic toxic compounds.Although the co-induced expression of GST genes by insecticides in insects has been documented in recent years,the underlying regulatory mechanisms are not understood.In this study,a total of thirty-one cytosolic S.exigua GSTs(SeGSTs)was cloned and identified.The bioinformatics and gene expression patterns were also analyzed.Out of them,SeGSTe9,SeGSTs6,SeGSTel,SeGSTe6,SeGSTe8,SeGSTe14,and SeGSTd1 were significantly co-expressed following exposure to three insecticides(lambda-cyhalothrin,chlorpyrifos and chlorantraniliprole).The analysis of upstream sequences revealed that all of these seven SeGSTs harbored CncC/Maf binding site.The luciferase reporter assay showed that the pGL3-SeGST promoter construct exhibited a significant increase in luciferase activities after exposure to insecticides,and mutation of CncC/Maf binding site diminish the induction effect.These data indicate that CncC/Maf pathway regulates the co-expression of GST genes in response to different insecticides in S.exigua.Insecticides significantly enhanced the ROS content and treatment with the ROS inhibitor N-acetylcysteine(NAC)decreased the insecticide-induced luciferase activities of the PGL3-GSTe6 promoter construct,but not the CncC-mutated construct.These results indicate that ROS mediates GST gene expression after exposure to insecticides through CncC/Maf pathway.Overall,these data show that insecticides induce the co-expression of glutathione S-transferases through ROS-activated CncC/Maf pathway in S.exigua.