Study On The Function And Expression Pattern Of Detoxification Related Genes In Ectropis Oblique

Ectropis oblique（Eo）is one of the most common and most harmful leaf-eating pests in tea gardens.It’s mainly distributed in tea areas of Guangdong,Guangxi,Sichuan,Jiangxi and Zhejiang.At present,the main control method of the Ectropis oblique is spraying with chemical Insecticide,so some genes in the Ectropis oblique may play a role in pesticide metabolism.In this study,four genes with increased expression were screened out from the transcriptome analysis of the theophylline after treatment with deltamethrin:EoGST534（glutathione s thiotransferase）,EoGST968,Eo UGT438（UDP glycosyltransferase）and Eo UGT678.Recombinant EoGST534,EoGST968,Eo UGT438 and Eo UGT678 were expressed in E.coli BL21,and the optimal temperature,p H and enzyme activity of the fusion proteins EoGST534 and EoGST968 were determined.The q RT-PCR technology was used to detect the gene changes of the four genes after chlorpyrifos,fenvalerate,and deltamethrin stimulation.The RNAi technology was used to interfere with EoGST534,EoGST968,Eo UGT438and Eo UGT678 respectively to preliminary study on their role in the insecticide metabolism of Ectropis oblique.The specific results are as follows:1.Bioinformatics analysis of EoGST534,EoGST968,Eo UGT438 andEo UGT678Some proteins Bioinformatics software and website were used to analyze the physical and chemical characteristics of the four proteins.EoGST534’s molecular weight is 24.669 kDa and theoretical isoelectric point is 5.42.The N-terminus contains a signal peptide consisting of 21 aminos.It contains two conserved domains,the N-terminus Domain and C-terminal Domain;EoGST968’s molecular weight is24.274 kDa,theoretical isoelectric point is 5.44,no signal peptide;contains two conserved domains:N-terminal domain and C-terminal Domain;They both have the highest homology to GST1 of Cydia pomonella.Eo UGT438’s molecular weight is59.369 kDa.It’s theoretical isoelectric point is 8.19,the N-terminus contains a signal peptide consisting of 20 aminos,it’s contains a glycosyltransferase domain;The molecular weight of Eo UGT678 is 57.635 kDa.The theoretical isoelectric point of Eo UGT678 is 9.17,the N-terminus contains a signal peptide consisting of 20 aminos,it’s contains a glycosyltransferase domain.2.EoGST534,EoGST968,Eo UGT438 and Eo UGT678 prokaryotic expressionand enzyme activity measurementThe molecular weight of recombinant EoGST534 is about 40kDa,the optimal temperature is 50℃,the optimal p H is 10,and the enzyme activity is 7.32 U/mg under the optimal conditions;The molecular weight of recombinant EoGST968 is about 40 kDa,the optimal temperature is 50℃,and the optimal p H is 9.5,and the enzyme activity was 1.064 U/mg under the optimal conditions.The molecular weight of recombinant Eo UGT438 is about 68 kDa,and the molecular weight of recombinant Eo UGT678 is about 69 kDa.Both proteins were expressed in E.coli BL21（DE3）after using the p ET-32a vector,and then they expressed in form of inclusion bodies.3.Real-time PCR to detect gene expressionAfter the 2^nd,3^rd,and 4^th instar larvae of Ectropis oblique were stimulated by deltamethrin,the expressions of EoGST534 and EoGST968 were significantly reduced within 12 h,and the expressions of Eo UGT438 and Eo UGT678 were significantly increased.At 24h,the expressions of EoGST534 and EoGST968 were significantly increased,and Eo UGT438 and Eo UGT678 expression gradually decreased.This results show that GSTs and UGTs may play different roles in the metabolism of deltamethrin in Theophyllotus esculenta.After 24h of treatment with chlorpyrifos and methrinthrin in the larvae,the expression of the four genes increased to varying degrees.These Genes also play a role in chlorpyrifos and fenvalerate metabolism.4.RNAiThe ds RNA of EoGST534,EoGST968,Eo UGT438,and Eo UGT678 were synthesized chemically in vitro.Electrophoresis detection shows high purity and concentration,their concentrations are 570.2 ng/μL,352.3 ng/μL,681.0 ng/μL,696.4ng/μL,868.2 ng/μL respectively.Four ds RNA injections compared with EGFP ds RNA showed that EoGST534 ds RNA and EoGST968 ds RNA suppressed efficiency more than 90%,and Eo UGT438 ds RNA suppressed efficiency about 70%,and Eo UGT678 ds RNA suppressed efficiency about 80%.After ds RNA injection,chlorpyrifos,fenpropathrin,and deltamethrin were used to stimulate the larvae.The expression levels of the four genes were compared with the control group.The expression levels of the four genes were reduced to different extents,indicating that the four ds RNAs have effects on the expression of EoGST534,EoGST968,Eo UGT438and Eo UGT678.After RNAi,the virulence of chlorpyrifos,fenpropathrin and deltamethrin to the 4^thinstar larvae of Ectropis oblique was determining again.It was found that the sensitivity of the EoGST534 ds RNA-treated group to fenpropathrin and deltamethrin was increased,and the EoGST968 ds RNA-treated group Increased sensitivity to chlorpyrifos and deltamethrin,no change in the Eo UGT438 ds RNA treatment group,and enhanced sensitivity to chlorpyrifos and deltamethrin in the Eo UGT678 ds RNA treatment group,further evidence that EoGST534,EoGST968,and Eo UGT678 are involved in the Theatrical process of Ectropis oblique.