牦牛CAPN1基因的克隆与序列分析

CAPN1是影响肌肉嫩度的数量性状位点 (QTL)的候选基因。根据GenBank发表的普通牛CAPN1基因序列设计特异性引物，以天祝白牦牛cDNA为模板，分段进行PCR扩增，克隆，测序。应用生物软件BioEdit对各测序结果进行序列拼接共获得牦牛CAPN1 cDNA 片段2267bp，其中包含一个2151bp的完整的开放阅读框(ORF),以及3’和5’末端非编码区的部分序列(77bp和166bp) 。分析表明：牦牛CAPN1基因编码区全长2151bp,共编码716个氨基酸。与已报道的牛，猪，人小鼠的序列进行比较，核苷酸同源性分别为99.3%，93.9%，90.0% ，85.5% 。预测氨基酸的同源性分别为99.4%，96.1%，94.6%，89.0%，并且对牦牛CAPN1四个结构域分别进行NCBI BLAST发现四个结构域在以上四个物种中都显示出很好的保守性，最为保守的在结构域Ⅳ(>96%)。牦牛与牛产生的 14个核苷酸突变中，有3个产生了氨基酸突变，均发生在结构域Ⅲ。构建分子系统进化树表明：聚类结果与传统分类学相符。

Molecular Cloning and Sequence Analysis of CAPN1 Gene in Yak

CAPN1 was investigated as a potential candidategene for a quantitative trait locus(QTL) affecting meat tenderness.According to the CAPN1 gene sequence of Cattle in GenBank,the specific primers were designed.Using the Tianzhu White yak cDNA as template,PCR amplifying,cloning and sequencing for every fragment.Using the BioEdit to splice sequences and a 2267bp yak cDNA of CAPN1 gene was obtained.Including a complete open reading frame and partical sequence of 3' end and 5' end UTR.Analysis of the gene revealed that the yak CAPN1 gene is encoded by 2151bp and the 716 AA potein predicted from this sequence.The nucleotide sequence and amino acid sequence of yak calpain were compared with those of cattle,pig,human,and mouse calpain.Identities for nucleotide and amino acid sequences were 99.3% and 99.4% for cattle,93.9% and 96.1% for pig,90.0% and 94.6% for human,and 85.5% and 89.0% for mouse calpain.Search using NCBI BLAST of yak 4 domains respectively found the 4 domains in the above 4 species have shown good conservative,the most conservative in domain IV(> 96%).There are 14 nucleotide mutations between yak and cattle and 3 of that were predicted to alter the protein sequence,both of which occurred in the domain III.Construction of molecular phylogenetic tree shows:The cluster results coincided with the traditional taxonomy.