家蝇伴侣蛋白CCTη基因序列分析与表达模式

Molecular cloning,sequencing and expression pattern of chaperonin gene in housefly-Musca domesitca

目的对家蝇伴侣蛋白CCTη(MD-CCTη)基因进行序列分析,克隆其c DNA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c DNA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c DNA为模板,PCR扩增,并与p MD-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1 632 bp,编码543个氨基酸,理论分子量59.3 k Da,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c DNA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。

Objective To clone and analyze a novel cDNA,named as Musca domesitca chaperonin containing tall- less complex polypeptide ,η （MD-CCTη）and to detect relative expression levels of MD-CCT-η by real time quantitative reverse transcriptase PCR（QRT-PCR）. Methods The total RNA was extracted from Musca domesitca larvae of 3rd day ;MD-CCTη gene was cloned with PCR and then ligated into vector pMD-19T and transformed into Escherichia coli DH5a competent cells. The relative expression levels of MD-CCT-η were detected in different tissues of the larvae of 3rd day and during different developmental stages by QRT-PCR. Results Sequence analysis indicated that the open reading frame was 1 632 bp, encoding a putative protein consisting of 543 amino acids, with a predicted molecular weight of 59. 3 kDa and an isoelectric point（pI）of 6 27. The analysis using National Center for Biotechnology Information-Basic Local Alignment Search Tool（ NCBI-BLASTi confirmed that the deduced amino acid sequence shared high identity with the reported homologous sequences from other insects （93%）. Cluster analysis results showed the genetic relation of Musca domesitca was more close to that of C. capitata and Drosophila（ 100% ）. QRT-PCR showed that the expression of MD-CCTη was the highest in the egg, followed by that of in the larvae of 1 st day, and lowest in male adult during devel- opment;whereas the the expression of MD-CCTη was the highest in the trachea, medium in the salivary glands, and the lowest in the body wall of 3rd day larvae. Conclusion MD-CCTη was cloned and mRNA expression levels of MD-CCTη were different during different development period and in the different tissues of Musca domesitca.