基于AMEL基因位点差异的水牛性别PCR鉴别方法

Sex identification of buffalos based on difference in AMEL gene loci by using PCR technique

【目的】建立检测不同性别水牛染色体AMEL基因位点差异的PCR鉴别方法,为水牛早期胚胎性别鉴定及水牛分离精子重分析提供简便可靠的检验手段。【方法】采用PCR扩增不同性别水牛染色体中的AMEL基因,Clone Manager 9.0软件分析序列差异,BLASTn程序分析基因同源性,MEGA 6.06软件构建系统发育进化树。【结果】以水牛血液基因组DNA为模板、amel B1为引物,引物退火温度60℃,公水牛可扩增出两条清晰条带,长度分别为376（AMEL-X基因）和304 bp（AMEL-Y基因）;母水牛仅扩增出1条清晰条带,长度为376 bp（AMEL-X基因）。与AMEL-X基因相比,水牛AMEL-Y基因缺失155-226区域的72 bp,同时存在20个基因型多态位点。临床检测结果证实,基于AMEL基因的PCR鉴别结果与实际性别的吻合率达100%,amel B1引物的灵敏度为0.5 ng模板量。BLASTn比对分析结果显示,水牛AMEL-X和AMEL-Y基因与不同哺乳动物的对应序列存在高度保守性,其相似性均接近或高于90%,与牛存在最近的亲缘关系。【结论】AMEL基因在哺乳动物中高度保守,且在不同性别水牛中呈多态性特征,以其作为分子靶标建立的水牛性别PCR鉴定方法切实可行,为水牛早期胚胎性别鉴定及水牛分离精子重分析提供了一个更便捷、高效的检验手段。

【Objective】The present experiment was conducted to establish PCR-based method for identifying differences in amelogenin（AMEL） gene loci of chromosome between female buffalo and male buffalo, in order to provide a simple and reliable detection method for sex identification of early embryos and purity reanalysis of sorted semen.【Method】The PCR technique was used to amplify AMEL gene from chromosomes of female and male buffalos respectively.Then the sequence differences and homology of AMEL gene between female buffalo and male buffalo were analyzed using Clone Manager 9.0 software and BLASTn procedure respectively. And the phylogenetic tree was constructed using MEGA6.06 software. 【 Result 】 The results showed that using amel B1 as primer, two clear gene bands were amplified from genome DNA of male buffalo blood under annealing temperature of 60 ℃, which were AMEL-X gene（376 bp in length） and AMEL-Y gene（304 bp in length）, respectively. Under the same conditions, one clear gene band was amplified from female genome DNA of female buffalo blood, that was AMEL-X gene（376 bp in length）. Compared with sequence of AMEL-X gene, there existed deletion of 72 bp（155-226 domain） and twenty polymorphic sites in the AMELY gene of buffalo. The results of clinical tests showed that, the consistency between PCR detection results and actual sex was up to 100%, the sensitivity of amel B1 was 0.5 ng DNA template. The results of BLASTn comparison indicated that, the AMELX and AMELY genes sequences of buffalo shared high similarity（more than 90%） with those of other mammals, and which had closest genetic relationship with cattle. 【Conclusion】The AMEL gene is highly conserved in the mammals and polymorphic in female and male buffalos, thus, which can be used as molecular target to identify sex of buffalo. Therefore,the established PCR-based method is feasible and efficient, which can be applied in sex identification of early embryos and purity reanalysis of sorted semen.