辽宁新品系绒山羊ZFX、ZFY基因片段的进化研究与性别鉴定

Cloning and Evolution Analysis of ZFX,ZFY Partial Gene in New-breeding Cashmere Goat and Identification of Its Sex

根据摩弗伦羊(Mouflon)和人(Human)的ZFX、ZFY基因序列以及相关文献设计引物,以雌雄辽宁新品系绒山羊的基因组DNA为模板,用PCR方法扩增并克隆其ZFX、ZFY基因第11号外显子的部分片段,进行序列测序。结果用NCBI的BLAST,生物软件ClustalX.exe、Mega、BioEdit.exe、DNAClub等进行分析。对辽宁新品系绒山羊(New-breeding cashmere goat)、藏系绵羊(Tibetan sheep)、摩弗伦羊(Mouflon)、马(Horse)、山狗(Coyote)、猪(Pig)、鲸(Whale)、人(Human)和牛(Cow)的ZFX、ZFY基因的外显子同源性进行序列对比分析。用N-J方法构建分子系统树。分析结果表明:ZFX、ZFY基因两者核苷酸同源性达92%,推导的氨基酸同源性为95%。经分析可知,辽宁新品系绒山羊与摩弗伦羊亲缘关系最近,与其他物种的亲缘关系较远。利用限制性内切酶AvaII对辽宁新品系绒山羊ZFX、ZFY基因的PCR产物进行酶切,确定AvaII为ZFX基因上的特异酶切位点,以此对辽宁新品系绒山羊性别进行鉴定。

According to the Mouflon and the human sex differentiation related ZFX and ZFY genes,a pair of primers were designed. The fragments were amplified from the genomic DNA of male or female New-breeding cashmere goat. Subsequently the portion amplified fragments of exon 11 were cloned and sequenced. The comparison of these sequences with those of other animals （Tibetan sheep,Mouflon,Horse,Coyote,Pig,Whale,Human and Cow） was performed using the bio-software such as, ClustalX. exe, Mega through blastn of NCBI. Using many ways to build molecule system trees, for example, the N-J method can show that the new-breeding cashmere goat is the closest to Mouflon,while it is the furthest to other species. The results of comparison indicated that the homologies of nucleotide sequence is 92 % ,the homologies of transmitted amino acid sequence is 95%. The result of PCR＇s product which is digested by restricted enzyme （Avail）show that; restriction site of Avail was found to be specific to ZFX gene. So the combination of PCR with Avail digestion is a simple and sensitive way to identify the new-breeding cashmere goat sex.