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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 analyze the sequence of CCTη (MD-CCTη) gene of housefly chaperon, clone its c DNA sequence and detect the expression of CCTη gene in housefly. Methods MD-CCTη gene was screened from the cDNA library of housefly larvae by sequence-based tagging (EST) sequencing and sequenced. The 3-day-old larvae of housefly were extracted and the c DNA was reverse transcribed , Amplified by PCR, ligated with p MD-19T vector and transformed into E. coli DH5α. Real-time fluorescence PCR was used to analyze the expression pattern of MD-CCTη in different stages of housefly development and 3-day-old larval tissues. Results The open reading frame (ORF) of MD-CCTη gene was 1632 bp in length and encoded a protein of 543 amino acids with a theoretical molecular mass of 59.3 kDa and an isoelectric point of 6.27. The deduced amino acid sequence of MD-CCTη gene was higher than other insect homologous sequences Similarity (93%), clustering analysis showed that the phylogenetic relationship between Musca domestica and Drosophila melanogaster was the closest (100%). MD-CCTη expressed the most in egg stage, followed by 1 day old larva The expression of MD-CCTη gene was the highest in the 3rd-instar larvae of housefly, followed by the salivary gland, with the lowest expression level in the body wall. Conclusion The c-DNA sequence of MD-CCTη gene was successfully cloned. There was a significant difference in the expression patterns of MD-CCTη in different developmental stages and tissues of housefly.