牛MyoG基因启动子的克隆及功能的初步分析  

Cloning and Preliminary Functional Analysis of Bovine MyoG Promoter

作　　者：王秋华[1] 曹允考[1] 李树峰[1] 佟慧丽[1] 兴孝友[1] 李光鹏[2] 严云勤[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]内蒙古大学生命科学学院,呼和浩特010021

出　　处：《畜牧兽医学报》2012年第1期37-43,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：转基因生物新品种培育科技重大专项基金(2008ZX08007-002)

摘　　要：本研究旨在比较日本和牛MyoG基因的不同长度片段启动子的活性强弱并初步探讨其中的机制。根据GenBank已公布的牛MyoG基因的启动子序列,设计特异性PCR引物扩增日本和牛MyoG基因的一系列启动子缺失序列,构建重组克隆载体pMD18-T-MyoGpro,对阳性克隆进行限制性酶切鉴定、测序及生物信息学分析,进而构建一系列启动子缺失片段的pGL3-MyoGpro双荧光素酶表达载体,转染牛肌源干细胞(MDSCs)和牛胎儿成纤维细胞,并进行双报告基因活性检测。结果表明,日本和牛MyoG基因的166～2 125bp启动子都能不同程度的启动双荧光素酶报告基因在牛肌源干细胞中的表达,且具有肌肉特异性。通过生物信息学分析得知日本和牛MyoG启动子序列中有1个TATA盒,15个E-box,并可能含有MyoD、MEF2、MEF3、MTBF、TEF1、PRDF、Sp1、多个SRF、ERE、GRE及多个Oct-1等转录因子调控结合位点,本试验通过比较不同长度启动子片段的活性并结合对上述转录因子的分析,认为这些转录因子可能对启动子活性起着重要的调控作用。对牛MyoG基因启动子的克隆与功能和序列分析为进一步研究MyoG基因的表达调控奠定了基础。This experiment was conducted to compare the activity of different fragments size of Wagyu MyoG promoter and investigate the potential mechanism. According to the sequence of Bovine myogenin published in GenBank, the primers were designed to amplify the promoter region of Wagyu MyoG by performing PCR. The PCR products were ligated to pMD18-T-MyoGpro cloning vector. Positive clones were identified by restriction endonuclease and sequencing. Then the Wagyu MyoG promoter dual-luciferase reporter gene vector（pGL3-MyoGpro） was constructed, and then was transfected into muscle derived stem cells（MDSCs） and fetal fibroblast cells of Bos traurus, and the activity of dual-luciferase reporter gene was detected. The activity and the specificity of these promoters during 166-2 125 bp were identified in MDSCs. Bioinformatics analysis showed that the promoter fragment contained one TATA box, fifteen E-box and may contained binding sites of transcription factors MyoD, MEF2, MEF3, MTBF, TEF1, PRDF, Spl, SRF, ERE, GRE and Oct-1. By comparing the activity of different promoter fragments and analyzing on these transcription factors, it initially indicate that these transcription factors may play an important role in regulation of promoter activity. Cloning and analyzing of the promoter region and function of MyoG lay a foundation for further research on the mechanism of regulation and expression of MyoG in Bos taurus.

关 键 词：MYOG基因 启动子序列 转染 双报告检测 肌肉特异性 生物信息学分析 

分 类 号：S823[农业科学—畜牧学] Q523.8[农业科学—畜牧兽医]