朱砂叶螨TcGSTM7的体外表达及其功能  

In Vitro Expression of TcGSTM7 of Tetranychus cinnabarinus and Its Function

作　　者：申光茂[1] 李恒 梁金辉 何林[1] SHEN GuangMao, LI Heng, LIANG JinHui, HE Lin(College of Plant Protection, Southwest University, Chongqing 40071)

机构地区：[1]西南大学植物保护学院,重庆400715

出　　处：《中国农业科学》2018年第5期905-913,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金青年基金(31401748)

摘　　要：【目的】朱砂叶螨(Tetranychus cinnabarinus)是一种重要的农业害螨,长期化学防治导致其对多种药剂产生了抗性,因此明确该螨对化学药剂的解毒代谢机制对开展有效的抗性治理具有重要意义。谷胱甘肽S-转移酶(GST)是节肢动物主要的解毒代谢酶之一,笔者前期研究中筛选出了朱砂叶螨体内高表达的一条GST基因TcGSTM7,该基因的表达在药剂处理下具有明显的可诱导性。因此,本研究以TcGSTM7为研究对象,进一步利用异源表达技术分析TcGSTM7重组蛋白和甲氰菊酯、丁氟螨酯的互作效应,以及利用RNAi沉默该基因的表达后,检测朱砂叶螨对这两种药剂的敏感性变化,为明确TcGSTM7在药剂代谢中的功能打下基础。【方法】利用NCBI数据库对TcGSTM7编码蛋白的序列特征进行注释,并在SWISS MODEL构建的蛋白三维结构中对相关结构域、结合位点以及活性中心等进行展示。利用异源表达系统在大肠杆菌(Escherichia coli)中表达TcGSTM7重组蛋白,对重组蛋白进行分离纯化后使用特异性底物检测其GST催化活性,并通过检测杀螨剂对该蛋白活性的抑制效果分析其与甲氰菊酯和丁氟螨酯的互作效应。在此基础上,为明确TcGSTM7基因表达水平对朱砂叶螨药剂敏感性的影响,参照该基因的序列设计并合成特异dsRNA,利用叶碟饲喂法将其导入朱砂叶螨体内,经qPCR检测TcGSTM7表达水平显著下调后,进行生物测定分析基因沉默后朱砂叶螨对不同剂量甲氰菊酯和丁氟螨酯药剂敏感性的变化。【结果】TcGSTM7的氨基酸序列分析结果表明,其N端具有一个Mu家族GST特有的"thioredoxin-like"结构域,以及一个GSH的结合位点(G-site),在C端则具有催化底物结合域(H-site)。该蛋白具有9个α螺旋和4个β折叠结构,呈经典的"βαβαββα"排列方式。在三维结构预测的基础上,通过原核表达技术获得了TcGSTM7重组蛋白,该重组蛋白分子量为26 kD,与预测结�【Objective】 Tetranychus cinnabarinus is an important pest mite in agriculture. Resistance of this pest mite to various pesticides has developed quickly because of long-term chemical control, thus, it is of great significance to clarify the detoxification mechanism of T. cinnabarinus in order to carry out effective resistance management. Glutathione S-transferase（GST） is one of the major detoxification enzymes in Arthropods. In previous research, a highly expressed GST gene TcGSTM7 from T. cinnabarinus was identified, and the expression of this gene is inducible by pesticide. In this case, the objective of this study is to construct the recombinant protein of TcGSTM7 with prokaryotic expression system, analyze interaction between recombinant protein and pesticides（fenpropathrin and cyflumetofen）, and test the pesticide susceptibility change of T. cinnabarinus after decreasing the expression of TcGSTM7 through RNAi.【Method】The sequence characteristics of TcGSTM7 protein were annotated by BLAST in NCBI database. The structural domain, binding site, and active site were presented in a three-dimensional structure model, which was predicted by SWISS MODEL. TcGSTM7 was expressed by prokaryotic expression in Escherichia coli. GST activity of recombinant protein was tested with specific substrate, and its interaction with fenpropathrin and cyflumetofen was analyzed through activity inhibition test. Then, specific dsRNA was synthetized according to the sequence information of TcGSTM7. T. cinnabarinus was feed on the dsRNA through leaf disc method. Gene expression change of TcGSTM7 was detected by qPCR, then, susceptibility change of T. cinnabarinus to fenpropathrin and cyflumetofen was analyzed by bioassay. 【Result】Amino acid sequence analysis of TcGSTM7 showed that the N-terminal domain contained a thioredoxin-like domain, which is a specific feature of Mu class GST. In addition, GSH binding site（G-site） was also located in the N-terminal domain. The C-terminal domain contained an alpha helical dom

关 键 词：朱砂叶螨 谷胱甘肽S-转移酶 RNAI 解毒代谢 

分 类 号：S433.7[农业科学—农业昆虫与害虫防治]