遗传 ›› 2014, Vol. 36 ›› Issue (8): 793-799.doi: 10.3724/SP.J.1005.2014.0793

• 研究报告 • 上一篇    下一篇

徐淮山羊Oct4启动子功能的初步分析

韦光辉, 李东, 左其生, 张亚妮, 朱睿, 张蕾, 刘志永, 邱峰龙, 李碧春   

  1. 扬州大学动物科学与技术学院, 江苏省动物遗传繁育与分子设计重点实验室, 扬州 225009
  • 收稿日期:2014-01-22 出版日期:2014-08-20 发布日期:2013-07-19
  • 通讯作者: 李碧春,教授,博士生导师,研究方向:动物胚胎工程与生物技术。E-mail:yubcli@yzu.edu.cn
  • 作者简介:韦光辉,在读博士,研究方向:分子克隆和基因表达调控机制。E-mail: weiguanghui2006@gmail.com
  • 基金资助:
    国家转基因重大专项(编号:2013ZX08008-003)资助

Functional analysis of Oct4 promoter in Xuhuai goat

Guanghui Wei, Dong Li, Qisheng Zuo, Yani Zhang, Rui Zhu, Lei Zhang, Zhiyong Liu, Fenglong Qiu, Bichun Li   

  1. Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
  • Received:2014-01-22 Online:2014-08-20 Published:2013-07-19

摘要: 为确定徐淮山羊Oct4(Octamer-binding transcription factor 4)基因启动子活性区域, 并初步探讨TSA (Trichostatin A)和VPA(Valproic acid)对Oct4基因启动子活性的调控作用, 文章采用Primer5.0设计包含Oct4基因启动子不同长度片段的特异性PCR引物, 扩增并定向克隆至PGL3-Bacic荧光素酶报告载体, 分别转染gEF、P19和COS7细胞, 通过TSA和VPA诱导, 进行双荧光报告基因活性检测。同时用Oct4启动子-1516~+30 bp片段替换pEGFP-N1中的CMV启动子, 通过GFP荧光表达检测Oct4启动子的活性。结果表明:在gEF、P19和COS7细胞中Oct4启动子各片段均表现出不同程度的活性, 且最强活性区域为上游-1516~+30 bp, 基本活性区域为-238~+30 bp; 在-1516~-946 bp、-615~-96 bp区域存在正调控元件, 在-1936~-1516 bp、-946~-615 bp区域存在负调控元件; 终浓度为1 μmol/L的TSA和4 mmol/L的VPA为诱导的最佳浓度, 均能显著增强Oct4基因启动子的活性; -1516~+30 bp片段能够启动GFP的表达。研究结果为揭示Oct4基因的转录调控机制奠定了基础。

关键词: Oct4, 启动子活性, TSA, VPA, 诱导

Abstract: The aim of this study was to determine the activity region of Oct4 (octamer-binding transcription factor 4) promoterin Xuhuai goat, and to investigate the effect of TSA (trichostatin A) and VPA(valproicacid) on Oct4 promoter activity. Specific PCR primers of Oct4 promoter including different lengths of fragments were designed by Primer 5.0, then were amplified and cloned into PGL3-Bacic luciferase reporter vector. All the reconstruction vectors were transfected into gEF, P19 and COS7 cells, respectively. After TSA and VPA treatment, the activity of dual-luciferase reporter gene in these three transfected cells was detected. In addition, the CMV promoter of pEGFP-N1 was replaced by the -1516—+30 bp fragment of Oct4 promoter, GFP fluorescence was used to detect the activity of Oct4 promoter. The results indicated that different fragments of Oct4 promoter showed different degrees of activity in gEF, P19 and COS7 cells, and the maximal activity region of Oct4 promoter was -1516—+30 bp, the basal activity region was -238—+30 bp. Positive regulatory domains existed in the region of -1516—-946 bp and -615—-96 bp, while negative regulatory domains existed in the region of -1936—-1516 bp and -946—-615 bp. The optimum induction concentration to enhance the activity of Oct4 promoter was 1 μmol/L of TSA and 4 mmol/L of VPA. The GFP expression can be started by the fragment of -1516—+30 bp. This study provides an experimental basis for revealing the mechanism of expression and regulation of Oct4 in goat.

Key words: Oct4, promoter activity, TSA, VPA, inducing