绵羊Nanog基因部分编码序列的克隆及分析

Cloning and Analysis of Partial Coding Sequences of Nanog Gene in Sheep

利用NCBI中GenBank里查询到已登录的人、猪、牛和山羊的NanogmRNA序列,通过多重同源比较,获得高度保守区域序列,设计了同源引物,并首次对绵羊Nanog部分编码序列进行了分子克隆。经过PCR扩增,获得绵羊Nanog基因第二外显子168 bp(GenBank Accession:EF436277)、第四外显子124 bp(GenBank Acces-sion:EF596905)和第二外显子至第四外显子498 bp(GenBank Accession:EU016097)三个序列片段。经测序,三个序列已登录GenBank。绵羊Nanog第二显子168 bp与山羊相应序列相似性最高达97%,与人相应序列相似性最低达84%;绵羊Nanog第四显子124 bp与山羊相应序列相似性最高达100%,与人相应氨基酸序列相似性最低达73%;经判断,绵羊Nanog第二外显子至第四外显子498 bp序列片段应该是一段假基因序列,与山羊mRNA相应序列相似性最高达98%,与人mRNA相应序列相似性最低达83%。本研究为进一步研究绵羊Nanog基因表达谱提供了序列信息。

The present study is using the accessed sequences of.human, pig, cattle and goat Nanog mRNA that checked from GenBank of NCBI, through multiple alignment, high conservative sequences were found, and homologized primers were designed based on them. This is the first time to clone Nanog gene from sheep genome. By PCR amplifica tion, exon2 168 bp （GenBank Accession： EF436277）, exon4 124 bp （GenBank Accession： EF596905）, and exon2- exon4 498 bp （GenBank Accession： EU016097） partial coding sequences （CDS） of sheep Nanog gene were obtained. Through sequencing, these three sequences had been submitted to GenBank. The similarity of 168 bp sequence at exon2 in sheep Nanog gene is the highest to 97% and lowest to 84% compared with corresponding sequences in goat and human, respectively. The highest similarity of 100% for 124 bp sequence at exon4 is found between sheep and goats, and the lowest similarity of 73% for corresponding amino acid sequence exists between sheep and human. But the researchers of the study think that the sequence of exon2--exon4 should be a pseudogene sequence. Compared with the corresponding sequences, the similarity of 498 bp sequence is highest to 98% between sheep and goats and lowest to 83% between sheep and human. Our present sdudy will provide information of sheep Nanog CDS for further studying of Nanog gene expression in sheep.