棉铃虫蛋白酶体β5基因Habeta5的克隆与序列分析

[目的]运用分子生物学技术克隆棉铃虫蛋白酶体B5亚基新基因,为进一步研究其功能奠定基础。[方法]以棉铃虫中肠总RNA为模扳,利用快速扩增cDNA末端（RACE）技术克隆棉铃虫蛋白酶体B5亚基新基因的cDNA序列并对所获序列进行分析。[结果]成功克隆了棉铃虫蛋白酶体瞄亚基全长947bp的cDNA序列,命名为Habeta5,包含1个843bp的完整开放读码框（ORF）序列,编码蛋白质为280个氨基酸残基,预测分子量为30．87kD,等电点为6．53。Habeta5蛋白在74～261氨基酸残基位置为蛋白酶体B5亚基的保守区域。Clustal W进行多序列比对发现,Habeta5蛋白质与果蝇等昆虫蛋白酶体B5具有62％以上的同源性,蛋白酶体B5的保守区域高度一致。邻近法NJ分子进化分析也显示,Habeta5蛋白质与其他生物蛋白酶体茚进化上同源。[结论]序列分析比对的结果表明所克隆的基因为棉铃虫蛋白酶体B5亚基基因（GenBank登陆号：FJ358434）。     

棉铃虫生物钟蛋白质TIME-EA4同源基因克隆及分子进化分析

时间间隔测定酶(TIME-EA4)是近年在家蚕中首先发现的一种生物测时蛋白质,具有正常的SOD酶活性与酯酶ATPase瞬时活性,参与家蚕滞育卵的活化调控.利用RACE技术克隆了棉铃虫TIME-EA4的同源基因Haea4(GenBank登录号:GU143551),全长cDNA 799 bp,编码199个氨基酸,蛋白质等电点为5.45,相对分子质量18 768.88,含有1个Cu/Zn SOD核心结构域.RT-PCR分析发现,Haea4基因在棉铃虫的头、中肠、脂肪体、生殖腺、体壁、丝腺中均有高量表达.氨基酸遗传距离和同源性分析结果,棉铃虫的TIME-EA4与家蚕和野蚕间的遗传距离分别为0.536、0.548,同源性均为44%;分子进化分析表明,棉铃虫和家蚕、野桑蚕的TIME-EA4同源蛋白质都与Cu-Zn/SOD蛋白的进化距离较近,蛋白质二级结构分析也显示,棉铃虫与家蚕TIME-EA4基本特性有很大相似性.     

野桑蚕性信息素结合蛋白(PBP)亚家族基因的克隆与进化分析

蛾类的性信息素信号传递是研究昆虫化学通讯的模型,性信息素结合蛋白(pheromone-binding protein,PBP)亚家族是研究热点之一。克隆了野桑蚕PBP亚家族的3个基因,命名为pbp1、pbp2、pbp3(GenBank登录号:GQ246497、GQ468569、GQ468570)。分析野桑蚕和家蚕pbp基因的编码区,发现碱基突变主要以转换为主(14/18),氨基酸变异主要为同义突变(16/18),成熟蛋白质的理化性质变化小,二级结构无变化,推测野桑蚕向家蚕的进化过程中PBP亚家族的基因功能没有变化。对具有PBP亚家族3个已知基因的6个昆虫物种进行氨基酸遗传距离和同源性分析,结果表明野桑蚕和家蚕间PBP1、PBP2、PBP3的遗传距离分别为0、0.02、0,同源性分别高达100%、98.6%和100%,物种间基因的进化速率很慢,进化分析显示PBP亚家族在这几个物种分化前已经产生。     

棉铃虫蛋白酶体β5基因Habeta5的克隆与序列分析(英文)

[Objective] Using molecular biotechnology to clone the proteasome β5 gene from cotton bollworm (Helicoverpa armigera), this research aimed to provide basis for further research on the function of proteasome β5 gene in cotton bollworm. [Method] Total RNA was extracted from midgut of cotton bollworm. The full length cDNA of Habeta5 gene was cloned by using rapid amplification of cDNA ends (RACE) technology, then sequence analysis was carried out. [Result] The full length cDNA sequence was successfully cloned and isolated, named as Habeta5. It was 947 bp in length, contained an ORF (843 bp) and encoded 280 amino acid residues, with the predicted mass of 30.87 kD and isoelectric point(pI) of 9.60. In the deduced amino acid sequence, a proteasome β5 subunit domain lies between 74th to 261st amino acid residues. It has more than 62% identity to other insects such as Drosophila melanogaster. The proteasome β5 subunit conservative regions were very similar with each other. Molecular evolution by Neighbor Joining method indicated that Habeta5 was homologous with other proteasome β5 subunit of species. [Conclusion] Sequence alignment shows that the cloned fragment is a proteasome β5 subunit gene (GenBank accession number: FJ358434).