牛肌细胞生成素基因(MyoG)启动子的活性及组织特异性分析  

Activity and Tissue-specific Analysis of Bovine(Bos) Myogenin Gene(MyoG) Promoter

作　　者：李硕[1] 郝斐[1] 梁浩[1] 王鹤霏[1] 吴海青[1] 诺明途[1] 刘东军[1] 仓明[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010070

出　　处：《农业生物技术学报》2014年第1期87-93,共7页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2014ZX08008-003)

摘　　要：肌细胞生成素(myogenin,MyoG)在肌肉细胞分化过程中起着中心调控的作用。为了研究牛(Bos)MyoG基因启动子的启动效率和组织特异性。本研究首先克隆了牛MyoG基因启动子区域,以pDsRed2载体为外源载体,红色荧光蛋白为目的基因,构建了pDsRed-BosMyoG真核表达载体。利用脂质体介导转染体外培养的绵羊(Ovis aries)骨骼肌卫星细胞和成纤维细胞,最后通过实时定量PCR和Western blot检测红色荧光蛋白的表达,从而确定牛MyoG基因启动子的启动效率及组织特异性。结果显示,pDsRed-BosMyoG转染绵羊骨骼肌卫星细胞后在显微镜下可观察到红色荧光蛋白的表达,但在转染后的成纤维细胞中并没有观察到红色荧光蛋白的表达。在mRNA水平上,转基因骨骼肌卫星细胞中红色荧光蛋白mRNA表达量明显高于转基因成纤维细胞(P<0.01)。Western blot结果显示,转基因骨骼肌卫星中高表达红色荧光蛋白,而在转基因成纤维细胞中并未检测到此蛋白。结果表明,本研究克隆得到的牛MyoG基因启动子可以特异性的在绵羊骨骼肌卫星细胞中驱动外源基因的高表达,是一种有效的肌肉细胞特异性启动子。克隆MyoG基因的启动子,探讨启动子区域的启动活性,有助于从理论上揭示MyoG基因表达的关键调控位点,同时也有利于揭示肌肉发育调控的相关机理,为改良家畜肉质研究提供实验依据。Myogenin （MyoG） plays a central control role in the process of muscle differentiation. The aim of this study is to research the activity efficiency and tissue specificity of bovine（Bos） myogenin （MyoG） gene promoter. In order to construct a eukaryotic expression vector pDsRed-BosMyoG, promoter fragment of bovine MyoG was obtained by PCR, and pDsRed2 was used as an exogenous vector in which red fluorescence was as a target gene. Then pDsRed-BosMyoG was transfected into a sheep（Ovis aries） skeletal muscle satellite cells and fibroblasts using lipofectamine. Red fluorescence was detected by RT-PCR and Western blot to study activity efficiency and tissue specificity of bovine MyoG-promoter. The results showed that red fluorescence could be observed in transgenic skeletal muscle satellite cells under microscope but not in transgenic fibroblasts. In mRNA level, red fluorescence mRNA expression value in transgenic muscle cells was significant higher than that in the transgenic fibroblasts（P〈0.01）. Similarly, Western blot result showed that red fluorescence protein of transgenic skeletal muscle satellite cells was high, but there was not red fluorescence protein of transgenic fibroblasts. The conclusion was that bovine MyoG-promoter can specifically promote the expression of exogenous genes in skeletal muscle cells, and was an effective muscle cells-specific promoter. Cloning bovine MyoG-promoter and exploring the activity of promoter region, was good to reveal a key expression sites of MyoG-promoter and the underlying regulation mechanism of muscle development, and to provide experimental evidences of improvement of meat quality in livestock.

关 键 词：肌细胞生成素 MyoG基因启动子 肌肉卫星细胞 肌管 

分 类 号：S828.2[农业科学—畜牧学]