牛MYOZ2基因启动子克隆及活性分析

doi:10.11843/j.issn.0366-6964.2016.04.003

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (4): 652-660.doi: 10.11843/j.issn.0366-6964.2016.04.003

牛MYOZ2基因启动子克隆及活性分析

王明明^1# ，赵志东^1# ，李安宁¹，张亚冉¹，段美艳¹，李世军¹，吴森¹，王晓宇¹，昝林森^1，2*

（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）

收稿日期:2015-05-13 出版日期:2016-04-23 发布日期:2016-04-23
通讯作者: 昝林森，教授，博士生导师，主要从事肉牛奶牛遗传改良与健康养殖方面的教学、科研及技术推广工作，E-mail：zanlinsen@163.com
作者简介:王明明（1989-），男，山东鱼台人，硕士生，主要从事肉牛基因转录调控研究，E-mail：785336874@qq.com；赵志东（1985-），男，甘肃榆中人，博士生，主要从事生物技术与动物育种研究，E-mail:zzd622@126.com。王明明和赵志东为共同第一作者
基金资助:
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)

Cloning and Activity Analysis of Bovine MYOZ2 Gene Promoter

WANG Ming-ming^1#，ZHAO Zhi-dong^1#，LI An-ning¹，ZHANG Ya-ran¹，DUAN Mei-yan¹，LI Shi-jun¹，WU Sen¹，WANG Xiao-yu¹，ZAN Lin-sen^{1，2 *}

(1.College of Animal Science and Technology，Northwest A & F University，Yangling 712100，China；2.National Beef Cattle Improvement Center in China，Yangling 712100，China)

Received:2015-05-13 Online:2016-04-23 Published:2016-04-23
Supported by:

摘要/Abstract

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2 065 bp，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/+125 bp，而且MEF2，SRF，MyoD，YY1 等转录因子可能参与MYOZ2基因的转录调控。

Abstract:

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/+125 was significantly higher than pGL3-Basic (P＜0.01)，and the transcriptional activity of pMYOZ2-683/+125 also was significantly higher than pMYOZ2-263/+125 (P＜0.01).The core functional promoter of bovine MYOZ2 was located in the region -84/+125 relative to the TSS，and the transcriptional factors such as MEF2，SRF，MyoD and YY1 might be involved in the transcriptional regulation of MYOZ2 gene.

中图分类号:

王明明，赵志东，李安宁，张亚冉，段美艳，李世军，吴森，王晓宇，昝林森. 牛MYOZ2基因启动子克隆及活性分析[J]. 畜牧兽医学报, 2016, 47(4): 652-660.

WANG Ming-ming，ZHAO Zhi-dong，LI An-ning，ZHANG Ya-ran，DUAN Mei-yan，LI Shi-jun，WU Sen，WANG Xiao-yu，ZAN Lin-sen. Cloning and Activity Analysis of Bovine MYOZ2 Gene Promoter[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(4): 652-660.

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牛MYOZ2基因启动子克隆及活性分析

Cloning and Activity Analysis of Bovine MYOZ2 Gene Promoter

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