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家蝇伴侣蛋白CCTη基因序列分析与表达模式

DOI: 10.11847/zgggws2015-31-08-18, PP. 1043-1046

Keywords: 家蝇,伴侣蛋白(CCT),克隆,实时荧光,表达模式

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Abstract:

?目的对家蝇伴侣蛋白CCTη(MD-CCTη)基因进行序列分析,克隆其cDNA序列并检测家蝇CCTη基因的表达情况。方法采用表达序列标签(EST)测序技术从已构建的家蝇幼虫cDNA文库中筛选MD-CCTη基因,对其进行序列测定和分析;提取家蝇3日龄幼虫RNA,逆转录cDNA为模板,PCR扩增,并与pMD-19T载体连接,转化大肠杆菌DH5α中;采用实时荧光PCR技术分析MD-CCTη基因在家蝇不同生长发育阶段及3日龄幼虫组织中表达模式。结果MD-CCTη基因开放阅读框(ORF)全长1632bp,编码543个氨基酸,理论分子量59.3kDa,等电点6.27;该基因推导的氨基酸序列与其他昆虫同源序列比较有较高的相似性(93%),聚类分析结果显示,家蝇与地中海实蝇和果蝇的亲缘关系最近(100%);MD-CCTη在发育阶段以卵期表达量最高,其次为1日龄幼虫,雄性成虫表达量最低;MD-CCTη基因在家蝇3日龄幼虫组织中气管表达量最高,其次为唾液腺,在体壁中表达量最低。结论成功克隆出MD-CCTη基因的cDNA序列,MD-CCTη在家蝇不同发育阶段及组织中表达模式存在明显差异。

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