广西巴马小型猪肌肉生长抑制素(Myostatin)cDNA克隆和序列分析

cDNA Cloning and Sequence Analysis of Myostatin Gene from Guangxi Bama Minipig

目的通过RT-PCR扩增广西巴马小型猪Myostatin cDNA序列,经过连接、转化、克隆测序后,与NCBI中发表的猪Myostatin序列进行同源性比较,分析广西巴马小型猪Myostatin的cDNA序列结构特点及与其他物种间的聚类关系,为日后构建Myostatin高效表达载体及进一步研究Myostatin对广西巴马小型猪肌肉生长的影响奠定基础。方法以广西巴马小型猪的肌肉组织总RNA为模板,应用逆转录-聚合酶链式反应(RT-PCR),利用合成的特异性引物,扩增得到巴马小型猪肌肉生长抑制素(Myostatin)cDNA的全序列。该扩增片段经纯化后,连接到pMD18-T载体上扩增,经一系列鉴定后,测序。结果本实验克隆到巴马小型猪Myostatin基因cDNA序列长度为1128 bp,与GenBank报道的猪Myostatin基因cDNA序列同源性高达99.7%,与人、奶牛、家鼠等物种的cDNA序列间同源性可达到92.3%以上,证明Myostatin基因具有较高的保守性。同时发现广西巴马小型Myostatin基因cDNA序列有三处存在碱基突变,两个为同义突变,一个为错义突变。

The total RNA was purified from Guangxi Bama minipig muscle tissue. The complementary DNA （cDNA） encoding Myostatin protein was amplified by the reverse transcription polymerase chain reaction （RT-PCR） method . The amplified cDNA fragment was ligated into pMD18-T vector after purification. The recombinant was verified by the method of PCR, restrictive cleavage and sequencing, respectively. The result showed that the Bama minipig Myostatin gene cDNA was successfully cloned in this study. The homologous analysis indicated that the homologous rate was 99.7% compared with the porcine Myostatin gene reported in GenBank and this gene has high conservativeness among mammalian animals（ 〉 92.3%）. Two synonymous mutations and one missense mutation were detected in Myostatin gene cDNA sequence of Bama minipig.