牛MYOZ2基因启动子克隆及活性分析  

Cloning and Activity Analysis of Bovine MYOZ2 Gene Promoter

作　　者：王明明[1] 赵志东[1] 李安宁[1] 张亚冉[1] 段美艳 李世军[1] 吴森[1] 王晓宇[1] 昝林森[1,2] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]国家肉牛改良中心,杨凌712100

出　　处：《畜牧兽医学报》2016年第4期652-660,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家"863"计划(2013AA102505);国家现代农业产业技术体系建设专项(CARS-38);"十二五"国家科技支撑计划(2011BAD28B04-03);陕西省科技统筹创新工程计划(2014KTZB02-02);新疆建设兵团重大科技计划(2014AA001-1)

摘　　要：旨在克隆测定牛肌原调节蛋白2基因(Myozenin2,MYOZ2)启动子的全长序列,进行活性区域分析,为牛MYOZ2基因功能和表达调控机理研究提供理论依据。通过5′RACE方法确定牛MYOZ2基因转录起始位点;采用PCR技术,以牛基因组为模板克隆MYOZ2基因启动子序列。利用在线软件分析启动子区域中可能包含的转录因子结合位点。依据分析结果重新设计引物,构建7个包含不同缺失片段的双荧光素酶报告基因载体,转染C2C12细胞系,利用双荧光素酶系统检测不同片段的启动子活性。结果表明,克隆得到牛MYOZ2基因启动子序列2 065bp,确定MYOZ2基因的转录起始位点;MYOZ2基因片段-84/+125荧光素酶相对活性极显著高于空载体pGL3-Basic(P<0.01),MYOZ2基因片段-683/+125荧光素酶相对活性极显著高于基因片段-263/+125(P<0.01)。MYOZ2基因启动子核心区域位于-84/+125bp,而且MEF2,SRF,MyoD,YY1等转录因子可能参与MYOZ2基因的转录调控。The Myozenin2（MYOZ2）gene promoter of bovine was cloned and sequenced to analyze the active region,which will provide theoretical basis for function determination and expression regulation of MYOZ2 gene.Applying 5′-rapid amplification of cDNA end analysis（RACE）,we determined the transcription initiation site of MYOZ2 gene.Using genome DNA as the template,the promoter of MYOZ2 gene was obtained by PCR.Online software was used to analyze putative transcription factor binding sites.Primer were redesigned according to the results of online software analysis,seven promoter fragments in different length were obtained by unidirectional deletion and cloned into luciferase reporter gene expression vectors.Then,the vectors were transfected into C2C12 cells,their expression activity were measured using the dual reporter assay.The promoter sequence of 2 065 bp of MYOZ2 gene in bovine was obtained and the transcription initiation site of MYOZ2 gene was determined.Luciferase assays indicated that the transcriptional activity of pMYOZ2-84/＋125was significantly higher than pGL3-Basic（P〈0.01）,and the transcriptional activity of pMYOZ2-683/＋125also was significantly higher than pMYOZ2-263/＋125（P〈0.01）.The core functional promoter of bovine MYOZ2 was located in the region-84/＋125relative to the TSS,and the transcriptional factors such as MEF2,SRF,MyoD and YY1 might be involved in the transcriptional regulation of MYOZ2 gene.

关 键 词：牛 肌原调节蛋白2基因启动子 基因克隆 活性分析 5′RACE 

分 类 号：S823.8[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]