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小鼠DAZL基因启动子核心区域分析 及甲基化对其启动活性的影响
王颖洁,张文慧,朱睿,左其生,李东,王飞,路镇宇,纪艳芹,王曼,张亚妮,李碧春
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(扬州大学动物科学与技术学院,扬州 225009; 江苏省动物遗传繁育与分子设计重点实验室,扬州 225009)
摘要:
旨在确定小鼠DAZL启动子基本活性区,并分析启动子区域甲基化对其启动子活性的影响,探究小鼠DAZL的表达调控机制。采用PCR法扩增不同长度的小鼠DAZL启动子,插入到pEGFP-N1和pGL3-Basic载体,构建重组载体。将重组载体转染GC-1细胞系,并通过添加适量DNA甲基化转移酶抑制剂(5-azacytidine,5-aza-C)逆转各试验组启动子片段的甲基化水平,然后利用双荧光素酶报告基因检测系统检测DAZL活性变化。双荧光素酶活性检测结果表明,小鼠Dazl启动子基本活性区域为5’侧翼区 -370 ~ -36 bp,在-370 ~ -166 bp区域存在不可或缺的重要调控元件;经5-aza-C处理后,各缺失片段载体转染的GC-1细胞DAZL的启动子活性与对照组相比均有不同程度的提升,但差异不显著。本试验确定了小鼠DAZL启动子基本活性区域,证明降低小鼠睾丸DAZL启动子区域甲基化水平,有助于DAZL在生殖细胞中特异性表达,为进一步探究DAZL生物学功能和调控机制提供参考。
关键词:  DAZL  启动子甲基化  5-aza-C  基因活性
DOI:10.13610/j.cnki.1672-352x.20160512.024
基金项目:国家自然科学基金(31301959, 31472087), 高等学校博士学科点专项科研基金(20123250120009)和江苏高校优势学科建设工程项目(苏政办发[2011]137号)共同资助。
Study?on?the core?region?of?DAZL?gene?promoter?and?the effect of methylation?on?promoter?activity?in?mice
WANG Yingjie,ZHANG Wenhui,ZHU Rui,ZUO Qisheng,LI Dong,WANG Fei,LU Zhenyu,JI Yanqin,WANG Man,ZHANG Yani,LI Bichun
(College of Animal Science & Technology, Yangzhou University, Yangzhou 225009; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, Yangzhou 225009)
Abstract:
This study was aimed to determine the basic activity region of the mice DAZL gene promoter, analyze the effect of promoter methylation on gene activity, and explore the regulation mechanism of the DAZL gene expression. Different fragments of the mice DAZL gene promoter were cloned by PCR, and then recombined into pEGFP-N1 or/and pGL3-basic plasmids. The recombined plasmids pGL3-DAZL was transferred into GC-1 cells treated with 5-aza-C. The Dual-Luciferase? Reporter Assay System was performed to identify the activity of the DAZL gene. The result showed that the mice DAZL core promoter region was -370--36 bp, which indicated that there might be important regulatory elements within -370--166 bp; with the 5-aza-C treatment, the activity of the mice DAZL gene promoter fragment with different lengths were increased, but no significant difference was observed. In general, the effect of methylation of the mice DAZL gene promoter on the gene activity was studied and the results would lay a foundation for further exploring the regulation and biological function of the mice DAZL gene.
Key words:  Dazl  promoter methylation  5-aza-C  gene activity

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