梨小食心虫谷胱甘肽S-转移酶GmolGST6的基因克隆、原核表达和酶学特征

doi:10.16380/j.kcxb.2018.04.002

摘要/Abstract

摘要： 【目的】克隆一种梨小食心虫Grapholita molesta触角中谷胱甘肽S-转移酶（glutathione S-transferase, GST）基因并分析其结构特征，明确其表达特点及该基因编码蛋白的酶学特征，以期为该基因的功能研究提供理论依据。【方法】根据梨小食心虫雌虫触角转录组数据，利用RT-PCR克隆梨小食心虫GST基因；通过实时荧光定量PCR测定该基因在成虫触角、头（去触角）、胸、腹、足和翅中的转录水平；利用原核表达系统和Ni离子亲和层析技术表达和纯化梨小食心虫GST重组蛋白，并对重组蛋白的稳定性和酶促动力学参数进行分析。【结果】获得了梨小食心虫谷胱甘肽S-转移酶基因GmolGST6（GenBank登录号: MF503496）的完整编码序列，开放阅读框为645 bp，编码214个氨基酸，分子量为24.21 kD，理论等电点为5.10；进化树分析和三维结构模拟表明GmolGST6属于Delta亚家族。qPCR测定结果表明，GmolGST6基因在雌雄成虫触角中高丰度表达，且在雄虫触角中的表达量显著高于雌虫。重组蛋白GmolGST6具有催化通用底物CDNB的活性，并且在pH 7.5， 40℃时具有最高的酶活力。重组蛋白GmolGST6的米氏常数K_m为0.21±0.06 mmol/L，最大反应速度V_max为14.02±1.40 μmol/min·mg。【结论】根据GmolGST6的表达特点及其重组酶对模式底物CDNB的催化活性，推测在触角中高丰度表达的GmolGST6具有参与降解外源物质、保护嗅觉系统免受外源性物质毒害的功能。

关键词: 梨小食心虫, 谷胱甘肽S转移酶, 嗅觉, 表达分析, 酶活力

Abstract: 【Aim】This study aims to clone the glutathione S-transferase (GST) gene from the oriental fruit moth, Grapholita molesta, to analyze its structure properties, and to clarify the expression profiles and enzymatic characteristics of protein coded by this gene, so as to provide a theoretical basis for fundamental function research of this gene. 【Methods】 Based on the transcriptome data of female antennae from G. molesta, the complete coding sequence of GST gene was cloned using RT-PCR. The expression levels of this gene in antennae, head (without antennae), thorax, abdomen, leg and wing were measured by real-time fluorescence quantitative PCR (qPCR). The recombinant protein of GST was prokaryotically expressed, and then purified by Ni²⁺ affinity chromatography. In addition, the stability and kinetic parameters of the recombinant GST were analyzed. 【Results】 The complete coding region sequence of GmolGST6 (GenBank accession no.: MF503496) was obtained, and its ORF is 645 bp in length, encoding 214 amino acids with the predicted molecular mass of 24.21 kD and the theoretical isoelectric point of 5.10. The phylogenetic tree and the three-dimensional structure analysis indicated that GmolGST6 belonges to Delta subfamily of GSTs. qPCR results showed that GmolGST6 was primarily expressed in the antenna of male and female adults, and its expression level in the male antenna was significantly higher than that in the female antenna. The recombinant GmolGST6 showed catalytic activity towards the general substrate CDNB, and had the highest activity at pH 7.5 and 40℃. The Michaelis constant (K_m) of the recombinant GmolGST6 was 0.21±0.06 mmol/L, and the maximum reaction rate (V_max) was 14.02±1.40 μmol/min·mg. 【Conclusion】 Based on the expression profiles of GmolGST6 and the catalytic activities of the recombinant GmolGST6 towards CDNB, we speculate that GmolGST6 may be involved in the catabolism of exogenous substances and protecting the olfactory system from toxification of exogenous substances.

Key words: Grapholita molesta, glutathione S-transferase, olfactory, expression analysis, enzymatic activity

李帅, 苏丽, 李伯辽, 李怡萍, 李广伟, 仵均祥. 梨小食心虫谷胱甘肽S-转移酶GmolGST6的基因克隆、原核表达和酶学特征[J]. , 2018, 61(4): 398-409.

LI Shuai, SU Li, LI Bo-Liao, LI Yi-Ping, LI Guang-Wei, WU Jun-Xiang. cDNA cloning, prokaryotic expression and enzymatic characteristics of the glutathione S-transferase GmolGST6 in Grapholita molesta (Lepidoptera: Tortricidae)[J]. , 2018, 61(4): 398-409.

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链接本文: http://www.insect.org.cn/CN/10.16380/j.kcxb.2018.04.002

http://www.insect.org.cn/CN/Y2018/V61/I4/398

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梨小食心虫谷胱甘肽S-转移酶GmolGST6的基因克隆、原核表达和酶学特征

cDNA cloning, prokaryotic expression and enzymatic characteristics of the glutathione S-transferase GmolGST6 in Grapholita molesta (Lepidoptera: Tortricidae)

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