大豆蚜羧酸酯酶基因AgCarE的克隆、表达及活性分析

doi:10.3864/j.issn.0578-1752.2012.18.008

摘要/Abstract

摘要： 【目的】克隆大豆蚜羧酸酯酶基因，了解该酶的分子特性，为研究大豆蚜抗药性机理奠定基础。【方法】利用RT-PCR和RACE技术，克隆了1个大豆蚜羧酸酯酶基因的全长cDNA序列，命名为AgCarE，利用生物信息学软件及网上工具进行序列分析，以pET-21b为载体构建原核表达系统进行表达，并以α-醋酸萘酯溶液为底物进行活性测定。【结果】测序分析表明，羧酸酯酶基因AgCarE（GenBank登录号：JF970181）全长1 946 bp，编码526个氨基酸。羧酸酯酶AgCarE的等电点（pI）为6.08，蛋白活性中心包括3个氨基酸残基（催化三联体）：Ser186、Glu313、His434，5个N-联糖基化位点，具备羧酸酯酶的结构特征，属羧酸酯酶家族（EC: 3.1.1.-）。将该基因编码序列与pET-21b载体重组，经IPTG诱导表达HIS标签融合蛋白、SDS-PAGE分析和Western-blot检测，在大肠杆菌BL21中成功表达约59 kD的AgCarE蛋白，以α-醋酸萘酯为底物，检测表达的羧酸酯酶活性为0.036 mmol/100 μL酶液。【结论】成功克隆、表达了大豆蚜羧酸酯酶蛋白AgCarE，以α-醋酸萘酯为底物，检测表达的羧酸酯酶活性为0.036 mmol/100 μL酶液，有助于进一步了解羧酸酯酶的结构特征及水解作用，为设计研发新型杀虫剂提供可能。

关键词: 大豆蚜, 羧酸酯酶, 基因克隆, 活性分析

Abstract: 【Objective】 The objective of this study is to clone carboxylesterase gene and provide a molecular basis for insecticide resistance of Aphis glycines. 【Method】 A full-length cDNA was cloned by using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, and the carboxylesterase gene was named AgCarE, and inserted into Escherichia coli. The carboxylesterase activity of AgCarE was characterized with the α-NA as a substrate and its enzyme activity was analyzed. 【Result】 The AgCarE is 1 946 bp in length, and the open reading frame encodes 526 amino acids (GenBank accession number is JF970181). The predicted isoelectric point of AgCarE is 6.08. The AgCarE possesses a catalytic triad, consisting of a Ser, a His and a Glu residue (Ser186, Glu313 and His434), which is characterized by carboxylic esters (EC: 3.1.1.-). The AgCarE was recombined into pET21b vector. Then the HIS fusion protein was expressed by induction with IPTG. The result of SDS-PAGE and Western-blot analysis showed that the AgCarE protein from A. glycines was expressed in E. coli BL21 which induced by IPTG, and its MW was found to be about 59 kD, similar as the predicted result. The activity for AgCarE is 0.036 mmol/100 μL enzyme buffer. 【Conclusion】 A cDNA encoding carboxylesterase from A. glycines was cloned and expressed. The results will further explore the three-dimensional structure, hydrolysis of carboxylesterase and provide a possibility for the design of new types of pesticides.

Key words: Aphis glycines, carboxylesterase, gene clone, activity analysis

杨帅, 王玲, 赵奎军, 韩岚岚. 大豆蚜羧酸酯酶基因AgCarE的克隆、表达及活性分析[J]. 中国农业科学, 2012, 45(18): 3755-3763.

YANG Shuai, WANG Ling, ZHAO Kui-Jun, HAN Lan-Lan. Cloning, Expression and Activity Analysis of Carboxylesterase Gene from Aphis glycines (Hemiptera: Aphididae)[J]. Scientia Agricultura Sinica, 2012, 45(18): 3755-3763.

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