甘蓝夜蛾和八字地老虎肌动蛋白基因的克隆与序列分析
Molecular cloning and sequence analysis of two actin cDNA sequences from Mamestra brassicae and Agrotis cnigrum
朴冬花,贺玉年,樊 东*
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作者单位：东北农业大学农学院 哈尔滨 150030
中文关键词：克隆; 序列分析; 肌动蛋白;甘蓝夜蛾;八字地老虎
英文关键词：molecular cloning， sequence analysis， actin， Mamestra brassicae， Agrotis c-nigrum
中文摘要： 肌动蛋白是细胞骨架微丝的主要组成成分，在肌肉收缩、细胞骨架形成、细胞移动等方面起重要作用。以鳞翅目夜蛾科昆虫甘蓝夜蛾Mamestra brassicae L.和八字地老虎Agrotis c-nigrum 3龄幼虫整个虫体为材料提取总RNA，利用RT-PCR和cDNA末端快速扩增技术（RACE），分别扩增得到2种昆虫的肌动蛋白的cDNA序列，甘蓝夜蛾肌动蛋白的cDNA序列含有1 441个碱基，而八字地老虎肌动蛋白的cDNA序列含有1 411个碱基。2种昆虫的该基因的cDNA序列均包括1个1 131个碱基的开放阅读框，编码1个含376个氨基酸的蛋白。甘蓝夜蛾肌动蛋白分子量约为41.8 kDa；八字地老虎肌动蛋白分子量约为41.9 kDa。Prosite软件分析结果表明，甘蓝夜蛾和八字地老虎肌动蛋白氨基酸序列中存在3个肌动蛋白特征片段。GenBank数据库搜索及序列比对结果表明，甘蓝夜蛾肌动蛋白属于肌肉特异型肌动蛋白，八字地老虎肌动蛋白属于细胞质特异型肌动蛋白。2个基因的cDNA序列已经登录GenBank并获得登录号，甘蓝夜蛾肌动蛋白cDNA序列登录号为EU035314，八字地老虎肌动蛋白cDNA序列登录号为EU035315。利用RT-PCR技术在八字地老虎4龄、5龄、6龄幼虫、蛹期4个不同发育阶段和6龄期的肠道、体壁、脂肪体3种不同组织中都检测到了肌动蛋白基因在mRNA水平的表达。
英文摘要： Actin plays an important physiological role in muscle contraction, cell motility, cytoskeletal structure, cell division, intracellular transport and cell differentiation. Total RNA was isolated from the thirdinstar larval body of Mamestra brassicae L. and Agrotis cnigrum. The two actin cDNA sequences were cloned by RTPCR and rapid amplification of cDNA ends (RACE). The M. brassicae actincDNA, 1 441 base pairs in length, contained an open reading frame of 1 131 base pairs coding for a olypeptide of 376 amino acid residues with a predicted molecular weight of 41.8 kDa. The A. cnigrum actin cDNA, 1 411 base pairs in length, contained an open reading frame of 1 131 base pairs coding for a polypeptide of 376 amino acid residues with a predicted molecular weight of 41.9 kDa. The two deduced amino acid sequences of actin were predicted using the software Prosite , which showed that they have three actin signature fragments. Blast search in GenBank and sequence comparisons with actin amino acid sequences from other species suggested that the A. cnigrum actin cDNA sequence encoded a cytoplasmic actin, and the M. brassicae actin cDNA sequence encoded a muscle actin. The two actin cDNA sequences have been deposited in GenBank with the accession nos. EU035314 and EU035315, respectively. Transcript analysis of A. c-nigrum actin during arious developmental stages and in different tissues was determined by RTPCR. A specific product was obtained from the integument, midgut and fat bodies from the third to sixthinstar larval stage and the pupal stage.