红色蚋细胞色素氧化酶COI基因克隆及鉴定

Cloning,identification and sequence analysis of Simulium rufibasis mitochondrial cytochrome oxidase subunit I(COI) gene

目的对红色蚋细胞色素氧化酶亚基I(COI)基因进行克隆、鉴定与序列分析。方法以红色蚋成虫基因组DNA为模板,设计特异性引物进行PCR扩增、纯化基因片段后连接、转化大肠杆菌DH5a,筛选阳性克隆、测序并与NCBI数据库中的核酸序列进行同源性比对分析,掌握其基因序列及其蛋白质结构特征。结果克隆序列的结果表明COI基因全长1542 bp,编码513个氨基酸,编码蛋白等电点为5.84,相对分子量为56 KDa,具有一个细胞色素氧化酶亚基I的核心保守结构;其基因碱基组成A、T、C、G分别为28.60%,36.90%,17.76%,16.74%,G+C含量为34.5%,A+T含量为65.5%。结论成功克隆出红色蚋CO I基因序列,可为进一步研究奠定基础。

Objective To clone and identify the cytochrome C oxidase subunit I（CO I） gene of Simulium rufibasis（S.rufibasis） in order to analyze the gene sequence.Methods A pair of primers were designed and the template of adult S.ru-fibasis total DNA was used to amplify the gene with polymerase chain reaction and the amplification products were purified and cloned into the E.coli DH5α.The inserts of the positive clones were identified first with double digestion,then with col-ony PCR and further sequenced.The data were analyzed with nucleotide BLAST software of NCBI,and the features of COI gene sequence were determined.Results Sequence analysis reveales that the open-reading frame of COI gene in S.rufibasis containes 1 542 bp,encoding 513 amino acids and the molecular weight is about 56 kDa and the isoelectric point is 5.84.Structural predication shows that the protein possesses a COI conservative domain and its composition（A,C,G and T） is 28.60%,36.90%,17.76%,and 16.74%,respectively.Conclusion The COI sequence of S.rufibasis was cloned and identified.It will provide a base for further research.