| 摘要: |
| 【目的】鉴定牛MYOZ1基因转录起始位点，确定牛MYOZ1基因核心启动子区域，为进一步研究牛MYOZ1基因的转录调控机制奠定基础。【方法】以秦川牛肌肉5′ RACE准备cDNA为模板，设计5′ RACE扩增试验，确定牛MYOZ1基因转录起始位点。以秦川牛外周血基因组DNA为模板，通过PCR克隆获得牛MYOZ1基因转录调控区-1 628/+61目的片段。通过生物信息学分析软件预测可能包含的转录因子结合位点，设计逐段缺失引物，获得7个亚克隆，将其分别与pGL3-Basic载体连接，得到牛MYOZ1基因启动子双荧光素酶报告基因重组质粒，通过脂质体法转染C2C12细胞系，检测7个重组质粒的荧光素酶活性，分析启动子活性。【结果】确定了牛MYOZ1基因的转录起始位点，成功克隆获得7个系列缺失的牛MYOZ1基因启动子双荧光素酶报告基因重组质粒：pMYOZ1-1 628/+61、pMYOZ1-1 430/+61、pMYOZ1-1 179/+61、pMYOZ1-932/+61、pMYOZ1-676/+61、pMYOZ1-437/+61和pMYOZ1-116/+61，其中重组质粒pMYOZ1-116/+61启动子活性极显著高于pGL3-Basic，推测牛MYOZ1基因-116/+61区域可能包含核心启动子；重组质粒pMYOZ1-1 628/+61启动子活性极显著高于pMYOZ1-1 430/+61片段活性 (P＜0.01) ，表明牛MYOZ1基因启动子区域-1 628/-1 430片段可能包含启动子活性增强元件。生物信息学分析发现，牛MYOZ1基因启动子-116/+61片段可能包含SP1、GC Box、CAAT等多个重要转录因子结合位点；-1 628/-1 430片段可能包含SP1、MyoD等多个重要转录因子结合位点。【结论】成功构建了7个系列缺失的牛MYOZ1基因启动子双荧光素酶报告基因重组质粒，且初步确定了牛MYOZ1基因的核心启动子区域位于-116/+61。 |
| 关键词: 牛 MYOZ1 5′ RACE 启动子 双荧光素酶报告载体 |
| DOI： |
| 分类号: |
| 基金项目:“十二五”国家“863”计划项目（2013AA102505，2011AA100307-02）；国家现代农业产业技术体系建设专项（CARS-38）；“十二五”国家科技支撑计划项目（2011BAD28B04-03） |
|
| Activity of bovine MYOZ1 gene promoter |
|
WANG Mingming,LI Anning,ZHAO Zhidong,et al
|
| Abstract: |
| 【Objective】The research determined the transcription initiation site and core promoter region of bovine MYOZ1 gene by detecting the activities of MYOZ1 promoter constructs in C2C12 cell lines to lay foundation for further research on transcriptional regulation of bovine MYOZ1 gene.【Method】The 5′ Rapid amplification of cDNA ends (5′ RACE) was used to determine the transcription initiation site of MYOZ1 gene,and the 5′ flanking region (-1 628/+61) of bovine MYOZ1 gene was obtained by PCR.By bioinformatics prediction of transcription factor binding sites of bovine MYOZ1 gene promoter,7 sub-clones were amplified by PCR before being inserted into pGL3-Basic vectors.The recombinant plasmids were transfected into the C2C12 cells,and the relative transcriptional activities were measured using Dual-Luciferase Reporter Assay System.【Result】The translational start site was determined,and seven recombinant plasmids were constructed successfully by restriction enzyme digestion and sequence analysis including pMYOZ1-1 628/+61,pMYOZ1-1 430/+61,pMYOZ1-1 179/+61,pMYOZ1-932/+61,pMYOZ1-676/+61,pMYOZ1-437/+61,and pMYOZ1-116/+61.Luciferase activity assay indicated that luciferase reporter plasmid pMYOZ1-116/+61 had significantly higher luciferase activity than negative control pGL3-Basic,indicating that it may contain core promoter region of bovine MYOZ1 gene.Luciferase activity of pMYOZ1-1 628/+61 was significantly higher than its downstream plasmid pMYOZ1-1 430/+61 (P＜0.01),indicating that the -1 628/-1 430 region upstream of MYOZ1 gene promoter might contain transcriptional enhancer factors. Bioinformatics analysis found that pMYOZ1-116/+61 might contain transcriptional combining sites such as SP1,GC Box and CAAT,and pMYOZ1-1 628/-1 430 might contain SP1 and MyoD.【Conclusion】The luciferase report gene vectors of bovine MYOZ1 gene promoter were successfully constructed and the core promoter of bovine MYOZ1 gene was determined within -116/+61 region. |
| Key words: bovine MYOZ1 gene 5′ RACE promoter dual-luciferase reporter |
