摘要
目的:研究Smad3对Amelotin启动子转录活性的影响。方法:用基因克隆技术获得含有不同长度Amelotin启动子片段的荧光素酶报告基因载体,然后将其与Smad3瞬时转染小鼠成釉细胞,并用双荧光素酶报告基因检测其活性;生物学信息软件对人和小鼠的Amelotin基因启动子序列的同源性进行BLAST分析;凝胶迁移实验(EMSA)观察Smad3和Amelotin启动子特异性结合位点之间的相互作用;基因定点突变构建Smad3结合位点突变型Amelotin启动子,并用双荧光素酶报告基因检测系统分析Smad3对野生型和突变型Amelotin启动子转录活性的影响。结果:Smad3转染后,Amelotin启动子(-160^+196)转录活性明显增强(P<0.05);将人与小鼠的Amelotin启动子序列(-160^+196)进行Blast序列比对,发现两序列在(-160^-1)具有很大的同源性;EMSA结果显示,Smad3与Amelotin启动子(-160^+196)的GTCTG有相互作用;将Amelotin启动子上Smad3结合位点特征性序列"GTCTG"突变为"CCCTG"后,Smad3对突变型Amelotin基因启动子的转录活性无显著影响(P>0.05)。结论:转录因子Smad3可通过与Amelotin启动子特定序列结合而调控Amelotin的基因表达,从而在釉质发育过程中发挥重要作用。
AIM：To study the regulatory effects of Smad3 on the promoter activity of Amelotin gene in amelo-blasts. METHODS：Luciferase reporter gene vectors containing different lengths of Amelotin promoters were constructed by gene cloning and then the constructs were co-transfected into ameloblasts with Smad3 respectively. Dual luciferase a-nalysis was used to observe the effects of Smad3 on the transcriptional activity of Amelotin gene promoter in ameloblasts. On the basis of above results, the new functional promoter region was found. Electrophoretic Mobility Shift Assay（ EM-SA） was carried out to identify the interaction between Smad3 and Smad3 binding site on the Amelotin promoter. The Smad3 binding site on Amelotin promoter were mutated by site-specific mutagenesis and the effect of Smad3 on the tran-scriptional activity of the wild or mutant type Amelotin promoter was observed by Dual luciferase analysis. RESULTS：Transfection of Smad3 increased the Amelotin gene promotor （-160~ ＋196） transcriptional activity（P0. 05）. CONCLUSION： Smad3 enhances the transcriptional activity of Amelotin promoter by interacting with Smad3 binding site on Amelotin promoter.
出处
《牙体牙髓牙周病学杂志》
CAS
2015年第6期329-334,共6页
Chinese Journal of Conservative Dentistry
基金
国家自然科学基金(81170927)
关键词
SMAD3
Amelotin
EMSA
双荧光素酶报告基因检测
Smad3
amelotin
EMSA
dual luciferase reporter assay [Chinese Journal of Conservative Dentistry,2015,25（6）：329
