朱砂叶螨TcGSTM7的体外表达及其功能

doi:10.3864/j.issn.0578-1752.2018.05.009

摘要/Abstract

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，与预测结果一致，并且具有GST蛋白的催化活性。TcGSTM7重组蛋白酶活性为673.26 nmol·min^-1·mg^-1，其动力学常数K_m为0.71 mmol·L^-1，V_max为109.54 nmol·min^-1·mg^-1。药剂抑制试验结果表明甲氰菊酯和丁氟螨酯均可抑制TcGSTM7重组蛋白的酶活性，IC₅₀分别为0.038和0.2 mmol·L^-1。进一步利用叶碟饲喂法让朱砂叶螨取食dsRNA，对TcGSTM7的表达进行了干扰，qPCR检测表明取食48 h后，dsRNA对TcGSTM7的干扰效率达到52.88%。药剂敏感性测定结果显示，沉默TcGSTM7的表达后，朱砂叶螨对LC₃₀和LC₅₀的甲氰菊酯敏感性分别上升了9.0%和12.3%，对LC₃₀和LC₅₀的丁氟螨酯敏感性分别上升了12.9%和11.0%，且均达到显著水平（P＜0.05）。【结论】TcGSTM7的表达可以影响朱砂叶螨对甲氰菊酯和丁氟螨酯的敏感性，且其重组蛋白和这两种药剂存在互作效应，表明TcGSTM7可能在朱砂叶螨对甲氰菊酯和丁氟螨酯的代谢过程中具有重要作用。

关键词: 朱砂叶螨, 谷胱甘肽S, RNAi, 解毒代谢 -转移酶

Abstract: 【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 TcGSTM7proteinwere 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 ofTcGSTM7 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 domain, and the substrate binding pocket (H-site) was included. This protein consisted of nine α-helices and four β-strands and contained the typical βαβαββα motif of the thioredoxin fold. Based on the three-dimensional structure prediction, recombinant protein of TcGSTM7 was constructed through prokaryotic expression. Molecular weight of this protein was 26 kD, which was the same as prediction. Its GST specific activity was 673.26 nmol·min^-1·mg^-1, and kinetic parameters were calculated as 0.71 mmol·L^-1 for K_m and 109.54 nmol·min^-1·mg^-1 for V_max. Analysis of interaction between pesticides and the recombined protein showed that both fenpropathrin and cyflumetofen could inhibit the activity of TcGSTM7. IC₅₀ was 0.038 mmol·L^-1 for fenpropathrin, and 0.2 mmol·L^-1 for cyflumetofen. Then, dsRNA was feed to T. cinnabarinus by leaf disc feeding method. qPCR data showed the expression of TcGSTM7 was significantly decreased by 52.88%, and bioassay was carried out to test susceptibility of the mites to these two pesticides. Results showed that susceptibility of T. cinnabarinus to fenpropathrin significantly increased by 9.0% and 12.3% at LC₃₀ and LC₅₀, and to cyflumetofen, it also significantly increased by 12.9% and 11.0% at LC₃₀ and LC₅₀(P＜0.05).【Conclusion】The expression of TcGSTM7 could affect the susceptibility of T. cinnabarinus to fenpropathrin and cyflumetofen, and the interaction between recombinant protein of TcGSTM7 and these two pesticides was identified. These data indicate that TcGSTM7 is important for T. cinnabarinus during its metabolism process of fenpropathrin and cyflumetofen.

Key words: Tetranychus cinnabarinus, glutathione S-transferase (GST), RNAi, detoxification

申光茂，李恒，梁金辉，何林?. 朱砂叶螨TcGSTM7的体外表达及其功能[J]. 中国农业科学, 2018, 51(5): 905-913.

SHEN GuangMao, LI Heng, LIANG JinHui, HE Lin. In Vitro Expression of TcGSTM7 of Tetranychus cinnabarinus and Its Function[J]. Scientia Agricultura Sinica, 2018, 51(5): 905-913.

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