三种鳞翅目夜蛾科昆虫α-微管蛋白基因的克隆与mRNA表达水平
Cloning and mRNA expression levels of αtubulin cDNAs from three noctuid spelies
樊 东1，2； 秦松柏1； 朴冬花2 ；许艳丽1**
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作者单位：1. 中国科学院东北地理与农业生态研究所 哈尔滨 150040； 2. 东北农业大学农学院 哈尔滨 150030
中文关键词：夜蛾科， 昆虫， α-微管蛋白， 克隆， mRNA表
英文关键词：Noctuidae， insect， αtubulin， cloning， mRNA expression
中文摘要：根据昆虫微管蛋白的分子特征筛选对昆虫微管有效的抑制剂来控制昆虫的生长发育或不同器官的有效功能的表达来达到控制害虫的目的，在未来的害虫综合治理中具有广泛的应用前景。以预蛹期甜菜夜蛾Spodoptera exigua、小地老虎Agrotis ypsilon和八字地老虎Agrotis c-nigrum为材料提取总RNA，利用RT-PCR和cDNA末端快速扩增技术（RACE），分别扩增得到以上3种夜蛾科昆虫的α-微管蛋白基因的cDNA序列， 3种昆虫的该基因序列均包括1个1 353个碱基的开放阅读框。这3个cDNA序列均编码1个含450个氨基酸的蛋白，分子量约为50 kDa。氨基酸的142～148位存在1个微管蛋白信号片段GGGTGSG，在氨基酸序列的C-端都有1个酪氨酸残基，N-端存在1个对转录后调控非常重要的保守区MRECI序列，以上特点与其他昆虫α-微管蛋白氨基酸序列保守区序列相同。序列比对表明，克隆得到的α-微管蛋白基因的核苷酸序列是高度保守的，同源性为94.4%～97.0%，而氨基酸的序列同源性达到100%。利用RT-PCR技术在3种昆虫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α-微管蛋白基因在mRNA水平的表达。
英文摘要：Total RNA was isolated from the prepupae of Spodoptera exigua, Agrotis ypsilon and Agrotis c-nigrum, respectively. Three αtubulin cDNA sequences were cloned by RT-PCR and rapid amplification of cDNA ends (RACE). The three cDNA sequences all contain an open reading frame of 1 353 base pairs. Each cDNA sequence encodes a polypeptide of 450 amino acid residues with a predicted molecular weight of about 50 kDa. There is a tubulin signature sequence GGGTGSG at positions 142 to 148 of the deduced amino acid sequence. The C-terminal tyrosine residue is conserved. The MRECI domain, probably implicated in the posttranscriptionalautoregulatory mechanisms, was found at the N-terminus. Three αtubulin cDNA sequences and their deduced amino acid sequences share high homology with those from other insects. The identities between the three isolated nucleotides and between the deduced amino acids are 94.4%～97.1% and 100%, respectively. RT-PCR showed that α-tubulin mRNA could be detected in the 4th, 5th and 6th instars and pupae, and also in the gut, integument and fat bodies of the 6th instar larvae.