| 摘要: |
| 为研究胞内氯离子通道5基因(Chloride intracellular channel 5,CLIC5)广泛参与调节细胞内的各项生理活动与生化反应,并探讨该基因自身的表达调控机制,以小鼠基因组序列为模板,利用PCR技术扩增小鼠CLIC5基因5'上游调控序列,将其插入荧光素酶报告基因表达载体(pGL3-Basic)中,同时采用5'侧翼区缺失的方法构建了7个缺失不同DNA片段的荧光素酶表达载体。重组质粒与海肾荧光素酶载体(phRL-TK)共同瞬时转染HEK-293细胞,经双荧光素酶报告基因活性分析后,确定CLIC5基因的核心启动子区。利用生物信息学方法预测其中转录因子结合位点及启动子区甲基化状况。结果表明,CLIC5基因启动子缺乏TATA盒,但含有典型的GC盒及其他潜在转录因子结合位点;双荧光素酶报告基因活性分析表明,CLIC5基因-329~+1、-624~+1、-917~+1和-2 230~+1区域的启动子活性较高,其中-624~+1区域的启动子活性最强。进一步分析表明,启动子区-624~-329存在负性调控元件,预测存在转录因子结合位点RXR heterodimer binding sites与GC-Box factors Sp1/GC,-420~-283范围内存在CpG岛位点。 |
| 关键词: CLIC5 表达调控 启动子 转录因子 CpG岛 |
| DOI：10.11841/j.issn.1007-4333.2012.05.022 |
| 投稿时间：2012-02-21 |
| 基金项目:国家自然科学基金资助项目(30972113) |
|
| Promoter identification and transcriptional regulation of mouse CLIC5 |
|
JU Ting-ting, LV Wen-tao, YU Bo-yang, LIU Yang, JIANG Mei-hua, XU Lin, ZHAO Ze-ping, YIN Jin-dong
|
| (College of Animal Science and Technology, China Agricultural University, Beijing 100193, China) |
| Abstract: |
| The CLIC5 gene (chloride intracellular channel 5) has been proved to be one of the major genes regulating cell biology,however,the molecular mechanism underlying the expression regulation is not clear.PCR was used to amplify the promoter region from mouse DNA and cloned into pGL3-basic vector to construct CLIC5 promoter reporter.Serial deletions of CLIC5 promoter reporters were constructed by DNA blunting method.HEK-293 cell line was used for transfection,and the dual-luciferase reporter assay system was used to identify the core promoter.Bioinformatic analysis and luciferase reporter assay indicated that,CLIC5 promoter contained classical GC box as well as other putative transcriptional factor binding sites though lacking TATA box,its-329~+1,-624~+1,-917~+1,-2 230~+1 truncated fragment exhibited the higher transcriptional activity,and its-624 to+1 region showed the maximal transcriptional activity.Its-624 to-329 region existed potential inhibitory motifs that had varying degrees of inhibition of transcriptional activity.RXR heterodimer binding sites and GC-Box factors Sp1/GC might be involved in the transcriptional regulation of CLIC5 gene.In addition,there is a CpG island located in the region of-420~-283. |
| Key words: CLIC5 expression regulation promoter transcriptional factor CpG islands |
