徐淮山羊Oct4启动子功能的初步分析  

Functional analysis of Oct4 promoter in Xuhuai goat

作　　者：韦光辉[1] 李东[1] 左其生[1] 张亚妮[1] 朱睿[1] 张蕾[1] 刘志永[1] 邱峰龙[1] 李碧春[1] 

机构地区：[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

出　　处：《遗传》2014年第8期793-799,共7页Hereditas（Beijing)

基　　金：国家转基因重大专项(编号:2013ZX08008-003)资助

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1μmol/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。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 ac- tivity. 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 130 bpfragment 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 do- mains 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.

关 键 词：OCT4 启动子活性 TSA VPA 诱导 

分 类 号：S827[农业科学—畜牧学]