绵羊tnp2基因的克隆及其在体外共培养系统中圆形精子细胞的转录分析

Cloning of Sheep tnp2 Gene and Transcription Analysis of Round Spermatid Cells in the in vitro Co-culture System

过渡蛋白2基因(tnp2)是圆形精子细胞特异表达的基因。为了开展绵羊圆形精子细胞标记基因的研究,根据GenBank上已公布的牛的cDNA序列设计引物,采用RT-PCR和分子克隆方法,克隆了蒙古绵羊tnp2基因cDNA部分编码区序列。DNA序列测定结果与牛的核苷酸序列比对,同源性为95.3%。根据绵羊tnp2基因的cDNA序列设计引物,对共培养的四月龄绵羊睾丸生殖细胞进行RT-PCR鉴定。结果显示体外共培养的绵羊睾丸生殖细胞一直到第十周后仍有圆形精子细胞产生。绵羊tnp2基因的cDNA克隆和序列测定为进一步研究绵羊精子发生过程奠定了基础。

The transition protein-2 gene （tnp2） have been reported as the round spermatid-specific marker gene. However, the DNA sequence of trip2 gene in Ovis aries have not been reported. In order to study the sheep round spermatid-specific marker gene, we designed primers according to the reported Bos taurus tnp2 gene cDNA sequences in the GenBank. The partial CDS was amplified by RT-PCR. The PCR fragment was inserted into the T-A cloning vector pMD 19-T. The partial nucleotide sequences of Mongolia sheep trip2 gene were compared with the counterpart sequences of Bos taurus, and the nucleotide homology was 95.3%. Using the primers designed according to the trip2 cDNA sequence, the tnp2 gene transcription expression in the co-cultured cells derived from four-months old sheep testis was assessed by RT-PCR. The results demonstrate that the round spermatid cells are generated in the co-culture system until ten weeks in vitro. The cDNA cloning and sequencing lay down a foundation for further study on spermatogenesis of sheep.