真鲷肌肉生长抑素（MSTN）基因的克隆及表达分析

采用同源克隆的策略,设计了3对引物,首次分离、克隆了2820bp的真鲷肌肉生长抑素（MSTN）基因组序列,该序列在GenBank上注册（注册登录号为AY965686）.经过剪接、比较,分析了真鲷MSTN基因的序列和结构特征.真鲷的MSTN基因具有3个外显子、2个内含子,整个开放阅读框编码着384个氨基酸,第1个外显子上有一个连续11个Q氨基酸的残基,C-末端具有9个保守的半胱氨基酸及一个RVRR的蛋白酶解加工位点.在第1、第2内含子和3＇非翻译区上分别具有一个微卫星重复序列.RT-PCR分析表明,MSTN基因在真鲷不同组织中都有表达,但表达量具有明显的差异;MSTN在肌肉中表达量最高,其次是脑、小肠、头肾、鳃和性腺,心脏、眼睛、肝脏中只有极少量表达,脾脏中没有检测到MSTN的表达.

猪MSTN基因双筛选标记打靶载体的构建及其功能鉴定

【目的】利用正负筛选策略,构建猪肌肉生长抑素(Myostatin,MSTN)基因的双筛选标记打靶载体。【方法】以猪胎儿成纤维细胞DNA为模板,PCR扩增MSTN基因同源长、短臂;采用PCR和Overlap PCR扩增打靶载体正筛选标记嘌呤霉素和绿色荧光蛋白基因及负筛选标记单纯疱疹病毒胸苷激酶基因。以pUC57载体为骨架载体,在其多克隆位点连接一段包括Frt序列在内的酶切位点的多克隆位点序列,在2个Frt序列之间连接正筛选标记嘌呤霉素基因和绿色荧光蛋白基因,在Frt序列两侧分别连接5.7和1.9kb的MSTN基因同源长、短臂;在同源短臂后连接负筛选标记单纯疱疹病毒胸苷激酶基因,将目的片段与载体定向连接克隆。用脂质体法将打靶载体转染猪肾细胞(PK15细胞),用嘌呤霉素和丙氧鸟苷进行正负筛选,验证正负筛选标记基因的功能。【结果】成功克隆了猪MSTN基因同源长、短臂及正筛选嘌呤霉素和绿色荧光蛋白基因及负筛选单纯疱疹病毒胸苷激酶基因,构建了猪MSTN基因双筛选标记打靶载体,打靶载体长14kb。在打靶载体转染的PK15细胞中,正负筛选标记基因均有生物学活性。【结论】成功构建了猪MSTN基因双筛选标记打靶载体。

Sequencing Analysis of Myostatin Gene （MSTN） for Meat Cattle in Mongolia

In developed countries, study on special or candidate genes, which are useful for identifying species, breed and productivity of livestock, was conducted at high level and the results have already been used in practice. Such advanced technology and innovation that we are facing is necessary to adopt in Mongolia. In this study, the myostatin gene （MSTN） was investigated as a candidate gene for meat animal in Mongolian breeds of cattle. The conventional phenol-chloroform method and FavorPrepTM tissue DNA extraction kit were used for DNA isolation, and the polymerase chain reaction （PCR） and sequencing analysis were used for further study. The nucleotide sequences of MSTN gene from Selenge, Kazakh white head breeds and Mongolian cattle were sequenced and reported on the DDBJ/EMBL/GenBank database （LC142726, LC146648, LC146649）, and Selenge breed showed the result of single nucleotide mutation in MSTN gene.