Relationship Between GST And UGT Of Spdoptera Exigua And Insecticide Resistance

Glutathione-s-transferase(GST)and uridine diphosphate glycosyltransferase(UGT)are widely distributed phase II detoxifying metabolic enzymes in vivo and play an important role in the evolution of insect resistance.However,there are few reports on the relationship between GST,UGT and insecticide detoxification metabolism and drug resistance in Spodoptera exigua,and its role in resistance is still unclear.In this study,the field population of S.exigua in Huizhou,Guangdong Province,which had developed high levels of resistance to chlorpyrifos and cypermethrin,was used to explore the relationship between the phase II detoxification enzymes and insecticide resistance of S.exigua.The resistance level of S exigua to chlorpyrifos was assayed by leaf dip method,the difference of UGT gene expression between resistant and susceptible strains was analyzed by quantitative PCR,UGT enzyme activity was measured by using α-naphthol as a substrate and UDP-glucose as a sugar donor,and the effect of UGT enzyme inhibitors on the toxicities of insecticides were determined to explore the relationship between UGT and chlorpyrifos resistance.Also,the mechanism of GSTs metabolic resistance by in vitro expression of GSTs and in vitro metabolism of recombinant GSTs were analyzed.Furthermore,upstream sequence of GST genes with higher expression in resistance population were cloned and the transcription faction binding sites were predicted.And the promoter transcription activities were analyzed by fluorescent reporter plasmid analysis to demonstrate mechanisms for the overexpression of multiple GSTs.1.Relationship between glutathione-S-transferase and insecticide resistance in S.exiguaThe leaf dip method was applied to determine the resistance level of the field populations to six commonly used insecticides.This population had developed very high resistanct to chlorpyrifos and cypermethrin,the RF was 1155-fold and 931-fold,respectively.The glutathione-S-transferase inhibitor,DEM,significantly increased the toxicities of chlorpyrifos and cypermethrin against this resistant population,with the synergistic ratios of 5.5 and 4.1,respectively.The assay of GST enzyme activity also demonstrated a 2.9-fold higher enzymatic activity in resistant population than that in susceptible one,suggesting that elevated GST enzyme activity played an important role in chlorpyrifos and cypermethrin resistance.The gene expression level analysis presented that six(SeGSTd3,SeGSTe1,SeGSTe6,SeGSTe9,SeGSTo2 and SeGSTs1)among the 31 analyzed GST genes were significantly overexpressed in the HZ16 population compared with the susceptible population.The GST genes with the highest over-expression in the HZ16 population were SeGSTo2,SeGSTe6 and SeGSTd3,which were up-regulated by 109.1-fold,60.7-fold and 34.8-fold,respectively.These up-regulated GST genes may be associated with chlorpyrifos and cypermethrin resistance.The prokaryotic expression of three GST genes with the most up-regulated expression were made,and purified GST protein were obtain,the kinetic parameters of these three recombinant GST proteins were determined using the substrates CDNB and GSH,the maximum reaction rates(Vmax)of SeGSTo2,SeGSTe6 and SeGSTd3 were 1.57μmol/min/mg,4.98 μmol/min/mg and 2.77 μmol/min/mg,respectively.Enzyme activity inhibition assay showed that chlorpyrifos and cypermethrin inhibit the metabolism of GST substrate CDNB by recombinant GST enzymes,the inhibition rates of chlorpyrifos on CDNB metabolism were 68.0%for GSTe6,61.8%for GSTo2,and 45.8%for SeGSTd3;the inhibition rates of cypermethrin were 73.7%for GSTo2,65.5%for GSTe6,and 54.1%for GSTd3,suggesting a direct interaction between SeGSTs and insecticides.In vitro metabolism demonstrated that the clearance abilities of GSTd3 over chlorpyrifos,cypermethrin,carbaryl,triazophos,chlorantraniliprole,indoxacarb and hexaflumuron,the clearance rates of these 7 insecticides by GSTd3 were 55.3%,37.7%,29.1%,10.1%,8.8%,1.7%,and 29.3%,respectively;the clearance rates by GSTe6 were 27.3%,35.4%,14.1%,5.0%,19.9%,61.1%and 15.2%,respectively,and the clearance rates by GSTo2 were 45.1%,33.3%,17.9%,14.5%,18.0%,3.3%and 4.1%,respectively.In general,chlorpyrifos and cypermethrin can be significantly metabolized by these recombinant GST enzymes,suggesting SeGSTs participate in insecticide resistance in S.exigua.Then,the upstream sequences of SeGSTd3,SeGSTe6 and SeGSTo2 genes were conled by genome walking.Although the HZ 16 and resistant populations of S.exigua were different sources and had different genetic backgrounds,there was no significant difference in the upstream sequence of the SeGST gene between the two populations.The transcription factor binding sites in these upstream sequences were predicted using the online tools JASPAR and ALGGEN.The binding sites for Dfd,BR-C and CncC/Maf were observed in the upstream sequence of SeGSTd3,binding site for AhR/ARNT,HSF,CncC/Maf and PXR/RXR were found in SeGSTe6 and the upstream sequence of SeGSTo2 contains HSF,AhR/ARNT,BR-C and CncC/Maf binding sites.The upsrtream sequences of these three GST genes all harbor the CncC/Maf binding sequence.This suggests that the expression of SeGSTd3,SeGSTe6 and SeGSTo2 in S.exigua are under the control of the same cis-regulatory elements and transcription factors CncC and Maf.By quantitative PCR.Therefore,the relative expression levels of transcription factors were compared between the resistant and susceptible population of S.exigua.the mRNA levels of CncC,Maf and AhR in HZ16 population were significantly up-regulated by 8.5-fold,2.8-fold and 1.6-fold,respectively.Therefore,we hypothesized that upregulation of transcription factors with binding sites in the upstream sequence of the SeGST gene enhances transcription of the corresponding GST gene in the HZ 16 population.Then fluorescent reporter plasmids were constructed for the upstream sequences of these three GST genes,these three reporter plasmids have strong constitutive fluorescence reporter activity,and the the reporter plasmid with upstream sequence of SeGSTe6 showed the highest fluorescence activity,therefore,was selected for further research.Co-transfection of the CncC and/or Maf expression construct vector with the SeGSTe6 reporter plasmid significantly enhanced promoter activity,indicating that up-regulated expression of CncC or Maf enhances the transcription level of its downstream target gene.Similarly,due to the presence of the AhR/ARNT binding site in the upstream sequence of the SeGSTe6 gene,co-transfection of the construct expressing AhR and/or ARNT with the reporter plasmid also significantly increased the fluorescence reporter activity.After mutating these two loci,co-transfection of the CncC/Maf or AhR/ARNT expression construct with the mutant reporter plasmid no longer increased the transcriptional activity,indicating that the binding sites of the two transcription factors are transcribed in the gene.It plays an important role.Since the upstream sequences of the HZ16 population SeGSTd3,SeGSTe6 and SeGSTo2 genes are not significantly different from those of the sensitive population,the transcription factors CncC,Maf and AhR are constitutively overexpressed in the HZ 16 population,and the up-regulated expression of these transcription factors results in related target genes.Up-regulation of constitutive expression,including up-regulation of multiple GST genes,may be the molecular mechanism by which multiple GST genes are up-regulated and lead to drug resistance.2.Effect of UGTs on toxicity of chlorpyrifosWe have found that the field population of S.exigua in Huizhou,Guangdong Province,has produced a very high level of resistance to chlorpyrifos.The resistant population not only had higher GST enzyme activity,but also significantly higher than the sensitive population.The resistant larvae UGT enzyme activity was three-fold that of the sensitive strain.Comparison of 32 UGT gene expression levels in resistant and susceptible populations by real-time PCR,in the resistant populations,we found that 17 of the 32 UGT genes were up-regulated by more than 3 fold and were significantly different from the sensitive populations,Among them,UGT33J3 had the highest expression(42 folds),followed by UGT33F7(37 folds)and UGT33F5(34 folds),indicating that the resistance of resistant populations may be related to the up-regulation of UGT gene expression.Using UGT expression inducer(phenobarbital)to treat resistant larvae,we found that phenobarbital and chlorpyrifos alone can increase the expression levels of UGT33F5,UGT33F7 and UGT33J3 genes,of which UGT33F5 is the most obvious,but phenobarbital Proper use with chlorpyrifos did not further increase gene expression levels;at the same time,UGT gene activity of larvae of resistant population increased significantly after UGT gene treatment for 24 h in exogenous phenobarbital,treatment of larvae with chlorpyrifos(LC30)also produces an induction effect similar to phenobarbital,the UGT enzyme activity was further increased significantly after the mixture of chlorpyrifos and phenobarbital,this indicates that the increase in UGT enzyme activity should be due to the up-regulated expression of the UGT gene.When treated with the LC50 concentration of phenobarbital and chlorpyrifos,relative to the control,the mortality rates of sensitive and resistant populations decreased by 36.6%and 23.3%,respectively.This indicates that the up-regulated expression of UGT increases the detoxification and metabolism ability of the stressed larvae to chlorpyrifos.UGT inhibitors were used to inhibit the UGT activity of larvae,the inhibitory concentrations of 5-nitrouracil and benzenesulfazolone against UGT enzyme activities of S exigua larvae were 348.7 μM/L and 489.8 μM/L,respectively;these two inhibitors can be used to inhibit UGT enzyme activity of S exigua.When the resistant and sensitive larvae were treated with two strains of chlorpyrifos LC50,the mortality rate was 46.7%;when mixed with the UGT enzyme inhibitor 5-nitrouracil or sulfinpyrazone,the mortality rate of sensitive strain larvae increased to 73.3%and 83.3%,respectively,two inhibitors increased the virulence of chlorpyrifos by 57%and 78%,respectively,the mortality rate of resistant larvae increased to 76.6%and 67.7%,respectively,increasing the virulence of chlorpyrifos by 64%and 45%respectively,compared with chlorpyrifos alone,the mortality rate is significantly improved.It is indicated that the UGT enzyme inhibitors in resistant and sensitive populations can significantly increase the virulence of chlorpyrifos.In summary,the phase Ⅱ detoxification metabolic enzymes GST and UGT in S.exigua play a certain role in insecticide resistance,which increases the understanding of the mechanism of resistance to beet armyworm and it will contribute to the resistance control of S.exigua and provide theoretical guidance for exploring resistance management techniques.