一种基于搭桥pcr方法的启动子定点突变产生方法 - 第三军医大学学报.docVIP

细胞周期素D2启动子Trichostatin A效应区域的研究 郭志义1 ，郝小惠1，田 炜1，谭菲菲2，姜雪莲2 ，李潇婷2，胡倩倩2，杨 方1 (063000 河北 唐山，河北联合大学：医学实验研究中心，河北省煤矿卫生与安全重点实验室1 ，生命科学院) [摘要] 目的 研究在TSA处理条件下细胞周期素D2的调控机制。方法：细胞周期素D2的mRNA水平通过RT-PCR方法检测。细胞周期素D2的启动子片段，以A549细胞基因组DNA为模版，通过高保真PCR扩增获得，并克隆到pGL3-BASIC载体中；并以此为基础通过高保真PCR扩增出不同长度的启动子片段。启动子相对活性通过萤火虫酶报告基因方法确定。结果 在TSA处理条件下，细胞周期素D2的mRNA水平上升。共构建4个以不同长度细胞周期素D2启动子片段驱动的报告基因质粒，分别命名为pGL3-1816CD2-luc、pGL3-1358CD2-luc、pGL3-720CD2-luc和pGL3-524CD2-luc。在TSA处理条件下，启动子活性的变化在-524删切质粒时消失。结论 TSA条件下细胞周期素D2 mRNA水平上升是启动子依赖的；细胞周期素D2启动子的TSA效应区域在-720到-524之间。 [关键词] 细胞周期素D2 ；启动子；报告基因； Trichostatin A [中图法分类号] Q78 [文献标志码] A Determination of cyclin D2 promoter region in response to Trichostatin A treatment Guo Zhiyi1 ，Hao Xiaohui2 ，Tan Feifei,2 Jiang Xuelian2 , Yang Fang1（1Medical Experimental and Research center, Hebei United University, Hebei Province Key Laboratory of Occupational Health and Safety for Coal Industry， China 0630002 School of Life Science, Hebei United University, China 063000） ［Abstract］ Objective to explore the cyclin D2 expression under Trichostatin A (TSA) treatment in A549 cell line. Methods cyclin D2 mRNA level was detected by RT-PCR. Human cyclin D2 promoter fragment was amplified by High-Fidelity PCR with the genome DNA as template isolated from A549 cells, and then inserted into pGL3-BASIC vector. The truncation series of human cyclin D2 promoter fragment were amplified based on the plasmid. The relative promoter activity was evaluated by reporter gene analysis. Results cyclin D2 mRNA level increased under TSA treatment. Four truncated plasmids were constructed with name assigned: pGL3-1816CD2-luc、pGL3-1358CD2-luc、pGL3-720CD2-luc and pGL3-524CD2-luc.The change of reporter gene relative activity driven by cyclin D2 promoter fragment under TSA treatment diminished when it was truncated to -524bp. Conclusion The increased mRNA level of cyclin D2 is promoter activity dependent. The TSA response region of cyclin D2 located from the -720bp to -524bp. ［Ke