二斑叶螨乙酰辅酶A羧化酶基因全长cDNA克隆及其生物信息学分析

doi:10.3864/j.issn.0578-1752.2013.08.024

摘要/Abstract

摘要： 【目的】克隆二斑叶螨（Tetranychus urticae）的乙酰辅酶A羧化酶（Acetyl-CoA carboxylase，ACCase）基因，对其进行生物信息学分析并研究其在二斑叶螨敏感和抗性品系中的表达量。【方法】利用RT-PCR技术克隆ACCase基因的cDNA 全长，采用生物信息学软件分析克隆基因的编码蛋白特性；利用实时荧光定量 PCR 方法分析ACCase基因在二斑叶螨敏感和抗螺螨酯品系中的表达差异。【结果】ACCase基因（GenBank 登录号：JX424763）的开放阅读框长度为7 068 bp, 编码2 235个氨基酸，分子量约为266.35 kD，理论等电点为6.38，包含典型的生物素羧化酶（biotin carboxylase，BC）、生物素羧基载体蛋白（biotin carboxyl carrier protein，BCCP）和羧基转移酶（carboxyltransferase，CT）3个结构域。结合其它物种ACCase基因构建系统发育树表明，该基因与体虱（Pediculus humanus corporis）ACCase基因具有较高的同源性。实时荧光定量 PCR 结果表明，ACCase基因在抗螺螨酯品系中的相对表达量较高，与明感品系间存在显著差异。【结论】利用RT-PCR技术在二斑叶螨中克隆到ACCase基因，ACCase基因的过量表达可能与二斑叶螨对螺螨酯的抗性形成有关，为二斑叶螨的抗性治理提供了依据。

关键词: 二斑叶螨 , 螺螨酯 , ACCase , cDNA克隆 , real-time PCR

Abstract: 【Objective】The objective of this study is to clone full-length cDNA sequence of the Acetyl-CoA carboxylase (ACCase) gene from Tetranychus urticae and analyze its expression in different strains. 【Method】 The full length cDNA of ACCase gene from T. urticae was cloned by using RT-PCR, and the bioinformatics software was employed to analyze the characteristics of the coded protein. Expression profiles of ACCase gene between susceptible strain (S) and spirodiclofen resistant strain (Sp-R) of T. urticae were performed by the quantitative real-time PCR. 【Result】The full-length cDNA of ACCase gene (GenBank accessin number JX424763) contained 7 068 bp encoding 2 235 amino acids. The putative protein of ACCase gene showed predicted molecular weight of 266.35 kD with a theoretical pI of 6.38. The ACCase contained three function domains, including the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyltransferase (CT). The phylogenetic tree of nine ACCase amino acid sequences from different species were constructed by MEGA5.0．The results showed a close relationship with Pediculus humanus corporis. Quantitative real-time PCR showed that the transcripts of ACCase gene were 2-fold higher in spirodiclofen (Sp-R) resistant strain compared with susceptible strain.【Conclusion】The ACCase gene was cloned from T. urticae by using the RT-PCR. The higher expression of ACCase gene was probably related with the development of resistance to spirodiclofen, which may provide a basis for the management of pesticide resistance of T. urticae.

Key words: Tetranychus urticae (Koch) , spirodeclofen , Acetyl-CoA carboxylase , cDNA cloning , real-time PCR

吕娟娟, 王进军, 高新菊, 沈慧敏. 二斑叶螨乙酰辅酶A羧化酶基因全长cDNA克隆及其生物信息学分析[J]. 中国农业科学, 2013, 46(8): 1736-1744.

LU Juan-Juan,WANG Jin-Jun, GAO Xin-Ju, SHEN Hui-Min. 二斑叶螨乙酰辅酶A羧化酶基因全长cDNA克隆及其生物信息学分析[J]. Scientia Agricultura Sinica, 2013, 46(8): 1736-1744.

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