牦牛Agouti基因的克隆及编码区多态性研究

Cloning of Agouti Gene in Yak and Polymorphisms Detection

以Agouti基因作为牦牛毛色调控的候选基因,探索Agouti基因编码区序列多态性,以期为阐明Agouti与毛色形成的相关性及毛色形成机理奠定基础。根据GenBank已发表序列(NM_206843)设计1对引物,以天祝白牦牛和黑色被毛甘南牦牛皮肤总RNA为模板,采用RT-PCR技术扩增,成功获得了牦牛Agouti基因编码区序列。采用生物信息学方法预测分析Agouti基因及其编码蛋白的基本理化性质、信号肽、疏水性、二级结构等;采用DNA混合池测序法和直接测序法,检测牦牛Agouti基因编码区的序列变异。结果表明,扩增得到的Agouti基因的编码区是一条长为593 bp的DNA序列。黑色甘南牦牛和天祝白牦牛皮肤的Agouti基因序列相同,并与黄牛基本一致。将牦牛的Agouti基因序列命名为YAK ASIP,并且在NCBI数据库中注册该基因的登录号为KJ630463。该基因含有一个长度为402 bp的开放性阅读框,编码133个氨基酸。Agouti基因编码蛋白属于亲水性蛋白,有一个明显的信号肽,含有多个磷酸化位点。其二级结构主要以α螺旋和无规则卷曲为主。Agouti基因编码产物氨基酸邻接系统树表明,牦牛Agouti与黄牛、绵羊等物种的Agouti氨基酸具有高度相似性,并且牦牛Agouti基因编码区中无多态性位点。

In yak,Agouti gene is a potential candidate gene in yak skin＇s color formation and regulation,investigate in Agouti gene sequence polymophism lay the foundation for revealing yak skin and coat color genetic mechanisms. The presented study using DNA cloning techniques to obtain yak Agouti gene encoding sequences. Using bioinformatics tools to forecast the protein＇s basic physical,chemical properties,hydrophobicity,signal peptide and secondary structure. The polymorphisms of Agouti gene CDS were identificated by DNA pools and direct sequencing in Yak. The results showed that the obtained Agouti was a 593 bp DNA sequence. Gannan black yak Agouti gene was identical with that of Tianzhu white yak. Also,yak Agouti gene was similar with Bos. The yak Agouti gene was named YAK ASIP and registered number in NCBI was KJ630463. This gene encode a hydrophilic protein which containing 133 amino acids. It contains a clear signal peptide and several phosphorylation sites. The secondary structure of the protein was α helix secondary structure and random coil. The Agouti and related proteins of Bos grunniens,Bos taurus,Ovis aries are close in the phylogenetic tree,which showed highly homology. The gene sequence detection showed that the yak Agouti gene was without any polymorphisms.