中国农业科学 ›› 2014, Vol. 47 ›› Issue (5): 984-994.doi: 10.3864/j.issn.0578-1752.2014.05.015

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

山羊MyoG启动子的克隆和活性检测

 李硕, 郝斐, 吴海青, 毕兆伟, 张志鹏, 刘东军, 仓明   

  1. 内蒙古大学生命科学学院/哺乳动物生殖生物学与生物技术教育部重点实验室,呼和浩特 010070
  • 收稿日期:2013-05-13 出版日期:2014-03-01 发布日期:2013-09-17
  • 通讯作者: 仓明,E-mail:ndcangming@sina.com
  • 作者简介:李硕,E-mail:sure4598@126.com
  • 基金资助:

    国家转基因生物新品种培育重大专项(2014zx08008-003)

Cloning and Activity Analysis of Hircine MyoG Promoter

 LI  Shuo, HAO  Fei, WU  Hai-Qing, BI  Zhao-Wei, ZHANG  Zhi-Peng, LIU  Dong-Jun, CANG  Ming   

  1. College of Life Science, Inner Mongolia University/Key Laboratory of Mammal Reproductive Biology and Biotechnology, Ministry of Education, Huhhot 010070
  • Received:2013-05-13 Online:2014-03-01 Published:2013-09-17

摘要: 【目的】肌细胞生成素(myogenin,MyoG) 是生肌调节因子(MRFs)基因家族中唯一能在骨骼肌细胞发育与生长过程中均可表达的调控因子, 在肌肉细胞分化过程中起着中心调控的作用,它正调控着骨骼肌卫星细胞向成熟肌细胞分化的过程,是唯一不可代替的生肌调节因子。MyoG基因在复制、扩增、基因激活、转录、翻译等多级水平上对肌肉发育进行调控。基因转录的起始阶段是机体生长发育因子进行调控的开端,而此阶段调控的实质是通过启动子和上游调控序列的相互作用,调控目的基因的表达。因此克隆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,为治疗人类相关疾病以及改良家畜肉质研究提供实验依据。本研究克隆山羊Myogenin(MyoG)基因的启动子区域,检测其在哺乳动物骨骼肌细胞内的启动活性。【方法】克隆山羊MyoG基因的启动子序列,连入pDsRed2基本骨架构建了以红荧光蛋白基因为标记基因的真核表达载体pDsRed-GoatMyoG(5.3 kb)。表达载体pDsRed-GoatMyoG经酶切和测序鉴定后,分别转染体外培养的绵羊肌卫星细胞、肌管细胞和成纤维细胞,观察红色荧光蛋白表达情况,然后利用实时荧光定量PCR、Western blot和冰冻切片、免疫组化等技术检测细胞转染后标记基因mRNA 和蛋白在体外培养细胞中的的表达活性。pDsRed-GoatMyoG表达载体对小鼠进行肌肉注射,检测GoatMyoG启动子在体内组织中的启动特异性和效率。肌肉注射5 d后,分别取小鼠的注射腿肌肉组织、非注射腿肌肉组织、睾丸组织、肠组织和肝脏组织,通过实时荧光定量 PCR 检测标记基因DsRed在不同组织中的表达情况。【结果】克隆得到的MyoG启动子序列测序正确,载体pDsRed-GoatMyoG经酶切和测序鉴定,证实载体构建成功;转染质粒 pDsRed-GoatMyoG后,肌卫星细胞和肌管细胞在显微镜下均可观察到细胞发红色荧光,成纤维细胞没有红色荧光。通过实时荧光定量PCR检测得出,转基因肌管细胞内GoatMyoG 启动DsRed 基因表达mRNA 的相对量为14.07;通过Western blot技术检测出转基因肌管细胞含有GoatMyoG启动的DsRed蛋白质,在转基因成纤维细胞内没有检测到 DsRed 蛋白质,说明GoatMyoG启动子可在肌肉组织特异性启动外源基因表达。小鼠肌肉注射pDsRed-GoatMyoG质粒后,注射腿肌肉组织内GoatMyoG 启动DsRed 基因转录mRNA 的相对量为212.32,非注射腿肌肉组织内mRNA的相对量为39.76,注射腿肌肉组织和非注射腿肌肉组织内mRNA 的量均是其它组织的1.99倍以上。通过免疫组化技术在注射腿肌肉组织和非注射腿肌肉组织内均可检测到红色荧光蛋白,在睾丸、肠和肝脏内均未检测到DsRed 蛋白。【结论】山羊MyoG启动子可以特异性的在骨骼肌组织驱动外源基因的表达,是一种有效的肌肉特异性启动子。

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