畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (8): 1567-1575.doi: 10.11843/j.issn.0366-6964.2019.08.005

• 遗传育种 • 上一篇    下一篇

MSTN启动子与MEF2C转录因子相互作用的研究

杨涛1,2,3, 许厚强1,2,3,4*, 陈伟1,2,4, 周迪1,2,4, 王圆圆1,2,3, 朱晓锋1,2,3   

  1. 1. 贵州大学高原山地动物遗传育种与繁殖教育部重点实验室, 贵阳 550025;
    2. 贵州大学贵州省动物遗传育种与繁殖重点实验室, 贵阳 550025;
    3. 贵州大学动物科学学院, 贵阳 550025;
    4. 贵州大学生命科学学院, 贵阳 550025
  • 收稿日期:2019-01-15 出版日期:2019-08-23 发布日期:2019-08-19
  • 通讯作者: 许厚强,主要从事细胞分子生物学研究,E-mail:gzdxxhq@163.com
  • 作者简介:杨涛(1993-),男,贵州铜仁人,硕士生,主要从事动物遗传育种与种质资源创新研究,E-mail:1037901148@qq.com
  • 基金资助:
    国家科技支撑计划(2015BAD03B02-3);教育部促美大项目(【2015】2062号)

Study on Interaction between MSTN Promoter and MEF2C Transcription Factor in Cattle

YANG Tao1,2,3, XU Houqiang1,2,3,4*, CHEN Wei1,2,4, ZHOU Di1,2,4, WANG Yuanyuan1,2,3, ZHU Xiaofeng1,2,3   

  1. 1. Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region of Ministry of Education, Guizhou University, Guiyang 550025, China;
    2. Key Laboratory of Animal Genetics, Breeding and Reproduction in Guizhou, Guizhou University, Guiyang 550025, China;
    3. College of Animal Science, Guizhou University, Guiyang 550025, China;
    4. College of Life Science, Guizhou University, Guiyang 550025, China
  • Received:2019-01-15 Online:2019-08-23 Published:2019-08-19

摘要: 旨在解析牛MSTN启动子与MEF2C之间的相互调控作用,进一步探讨MSTN基因在肉牛肌肉生长发育方面的调控机制。首先,通过PCR扩增关岭牛MSTN启动子序列2 267 bp和MEF2C基因CDS区1 425 bp。其次,构建pGL3-Basic-MSTN和pcDNA3.1(+)-MEF2C双荧光素酶报告载体,采用脂质体法共转染至小鼠C2C12成肌细胞,24 h后检测其双荧光素酶活性。最后,通过PCR扩增MSTN启动子核心片段MSTN-P1,重新构建以MSTN-P1片段替代(CMV)区的重组表达载体pEGFP-N3-MSTN-P1-MEF2C,将其分别转染至小鼠C2C12成肌细胞和大鼠下颌腺上皮细胞,24 h后观察绿色荧光蛋白发光情况,48 h后提取细胞总RNA,利用qRT-PCR检测MEF2C基因在不同细胞中的表达情况。结果显示,在小鼠C2C12成肌细胞中,共转染pcDNA3.1(+)-MEF2C试验组较pGL3-Basic-MSTN试验组能显著增强MSTN启动子的活性(P<0.05),较pGL3-Basic对照组能极显著增强MSTN启动子的活性(P<0.01);MSTN启动子核心片段MSTN-P1能驱动MEF2C基因在小鼠C2C12成肌细胞中的表达量极显著上调(P<0.01),且能驱动MEF2C基因在大鼠下颌腺上皮细胞中的表达量显著上调(P<0.05)。结果表明,在转录水平,MSTN启动子与MEF2C可能同时参与了牛肌肉生长和分化的调控。

关键词: 牛, MSTN启动子, MEF2C转录因子, 互作

Abstract: The aim of this study was to explore the regulation mechanism of MSTN on muscle growth and development by analysis of the interaction regulation between the MSTN promoter and MEF2C in cattle. Firstly, the 2 267 bp upstream promoter region of MSTN and 1 425 bp coding region of MEF2C were amplified by PCR. Secondly, pGL3-Basic-MSTN and pcDNA3.1(+)-MEF2C dual luciferase reporter vectors were constructed and co-transfected into C2C12 myoblasts by liposome transfection method. The dual luciferase activity were measured after 24 hours. Finally, the core fragment with high activity of MSTN promoter was amplified by PCR. The recombinant expression vector pEGFP-N3-MSTN-P1-MEF2C which replaced (CMV) area with MSTN-P1 fragment was reconstructed, and it was transiently transfected into C2C12 myoblasts and rat mandibular gland epithelial cells, respectively, the expression of green fluorescent protein in cells was observed after 24 hours. Total RNA was extracted from cells after 48 hours. The expression levels of MEF2C in different cells were detected by qRT-PCR.The results showed that the activity of MSTN promoter was enhanced significantly by co-transfected of pcDNA3.1(+)-MEF2C(P<0.05), and it was enhanced extremely significantly compared with pGL3-Basic in C2C12 myoblasts(P<0.01); MSTN-P1 promoter core fragment could drive the expression of MEF2C, and the mRNA expression levels were extremely significantly up-regulated(P<0.01) in C2C12 myoblasts, and significantly up-regulated(P<0.05) in rat mandibular gland epithelial cells. The results indicated that both MSTN promoter and MEF2C could be involved in the regulation of growth and differentiation of muscle at the transcriptional level.

Key words: cattle, MSTN promoter, MEF2C transcription factor, interaction

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