关键词: 肌细胞生成素 , MyoG启动子 , 肌肉卫星细胞 , 肌管

Abstract: 【Objective】Myogenin (MyoG), as a number of myogenic regulatory factor (MRFs) gene family, is the only factor that can express in all skeletal muscle cells, plays a central regulation role in muscle cell differentiation, and positively regulate the process which of skeletal muscle satellite cell differentiation into mature muscle cells, and is the only irreplaceable myogenic regulatory factor. MyoG gene regulates muscle cell development in multiple levels of gene replication, amplification, activation, transcription and translation. The initial stage of gene transcription is the beginning of the regulation of growth factor, and the essence of this regulation is the interaction between promoter and upstream sequence to regulate the expression of target gene. Therefore MyoG gene promoter cloning and promoter region activity investigation not only could contribute to theoretically understand the critical control points of MyoG gene expression, but also reveal the mechanism of regulation of muscle development, so as to provide a experimental basis for the treatment of human muscle lesions and improvement of domestic animal meat quality. To study the basic mechanism of muscle development and growth, MyoG gene promoter was cloned and its promoter activities in mammal skeletal muscle cell were tested in the study. 【Method】 The promoter sequences of hircine MyoG gene were cloned respectively, MyoG promoter was connected to pDsRed2 framework, respectively, to construct eukaryotic expression vectors of pDsRed-GoatMyoG(5.3 kb). After the vector was identified by enzyme digestion, the expression vector was transfected into cultured ovine muscle satellite cells, myotube cells and fibroblasts, respectively, then the expression of red fluorescent protein in those cells were observed, and the expression efficiency of marker gene mRNA and protein was detected by real-time PCR, Western blot and immunohistochemistry methods. The vector with GoatMyoG was injected into mice muscle, and the promoting specificity and efficiency in in vivo tissues were detected. After 5 days of muscle injection, the injected leg muscle tissue, non-injected leg muscle tissue, testicular tissue, intestines and liver tissue were collected, and the expression of DsRed in different tissues were detected by real-time PCR.【Result】The sequence of the cloned MyoG promoter proved to be correct by DNA sequencing. Vector pDsRed-GoatMyoG by restriction analysis and sequencing confirmed that the vector was successfully constructed. Red fluorescence could be observed in muscle satellite cells and myotube cells under microscope after pDsRed-GoatMyoG, but not in fibroblasts. The results of real-time PCR detection indicated that the relative expression value of mRNA promoted by GoatMyoG was 14.07. The results of Western blot indicated that the quantity of DsRed protein promoted by GoatMyoG was deteced in myotube cells, and DsRed protein was not deteced in fibroblasts. The results indicated that the promoting efficiency of GoatMyoG was significantly higher than that of myotube cells, the promotor could promote foreign gene expression in muscle tissues specifically. To investigate the promoter activity in vivo, the relative expression value of DsRed mRNA promoted by MyoG was 212.32 in injected leg muscle tissue, and was 39.76 in non-injected leg muscle tissue. The value of DsRed mRNA in injected leg muscle tissue and non-injected leg muscle tissue were more than 1.99 times than that of in other tissues. The expression of red fluorescent protein could be observed by immunohistochemical method in injected leg muscle tissue and non-injected leg muscle tissue, but not in testicular tissue, intestines and liver tissue. 【Conclusion】 MyoG promoter is an effective muscle-specific promoter to express the desired transgenes in mammal skeletal muscle cell.

Key words: Myogenin , MyoG promoter , muscle satellite cell , myotube