小鼠睾丸hsc70t基因的克隆与序列分析

Clone and Sequence Analysis of hsc70t Gene in Mouse Testis

为了获得小鼠睾丸hsc70t基因的开放阅读框,根据GenBank中已发表的hsc70t的基因序列设计了特异性引物(GenBank accession No:NM_013558),提取睾丸中的总DNA,利用PCR方法扩增hsc70t基因序列,连接到pMD18-T载体上,然后进行序列测定与序列分析。测序结果表明PCR扩增得到一条1926bp左右的基因片段。所测序列被GenBank收录,收录号为EU549780,与GenBank上公布的小鼠hsc70t基因序列同源性达99.9%,与大鼠(Rattus norvegicus)、猕猴(Macaca mulatta)、黑猩猩(Pan troglodytes)、蟾蜍(Xenopus)、人类(Homo)和青鳉(Oryzias latipes)的相似性分别为94.4%、85.4%、84.9%、72.7%、71.9%、67.1%;进化树构建结果表明小鼠与大鼠距离最近。证实了hsp70基因的高度保守性,并为hsc70t的进一步研究和应用奠定了基础。

To obtain the open reading frame（ORF）of hsc70t gene in mouse testis,the specific primers wered esigned according to hsc70t gene order published in GenBank（GenBank accession No：NM_013558）,Thet otal DNA was extracted from mouse testis,The hsc70t gene was amplified by PCR from total DNA,andc onnectioned to pMD18-T vector,then sequencing and analysis were accomplished.Sequencing results howed the sequence contained 1926 bp was obtained by PCR amplification.The sequence was embodied byG enBank,the Genbank accession number is EU549780,The similarity of nucleotide sequence was 99.9%a pproximately between the cloned gene and the mouse testis hsc70t gene in Genbank,Comparison of then ucleotide sequence between mouse and the Rattus norvegicus,the Macaca mulatta,the Pan troglodytes,theX enopus,the Homo,the Oryzias latipes by DNA Star,shows similarity score were 94.4%、85.4%、84.9%、7 2.7%、71.9%、67.1%,Phylogenetic analysis showed mouse had the closest genetic relationship with rattusn orvegicus.The results confirmed that the hsp70 has highly conservatism in different organisms and this trialp rovides a good foundation for further research and application of hsc70t.