摘要
目的:克隆釉原蛋白基因启动子及可能影响转录调控的序列,分析其在不同细胞中的转录模式。方法:检索并分析釉原蛋白基因的上游调控序列。利用PCR及酶切方法,从小鼠C57BL/6J基因组上扩增不同长度(包括基本启动子区域)的转录调控序列,与PGL3-Basic载体的虫荧光素酶基因连接。瞬时转染CHO、Hela、UMR-106细胞,检测荧光素酶的活性,分析不同长度的启动子片段在各种细胞中的转录活性。结果:共构建了6个不同长度的报告载体,瞬转后发现在Hela细胞中有较强的荧光素酶活性,在CHO、UMR-106细胞中活性很弱。在不同的细胞内,启动子活性随片段长度变化,其变化趋势有明显的相似性。表现为在转录起始点上游975bp与532bp的区域具有较强的转录活性,而转录起始点上游285bp区域的转录活性有所降低。结论:釉原蛋白的启动子可在Hela细胞中激活,Hela细胞可作为研究釉原蛋白启动子转录调控的细胞模型;初步判断釉原蛋白启动子上的-1693与-975之间的序列为转录抑制区域,-532与-285之间为转录增强区域。
PURPOSE： To chine different promoter region of amelogenin gene and analyze their transcriptional activity. METHODS： The upstream regulation sequences of amelogenin gene were retrieved and analyzed. Amplified by PCR from the genomie DNA of mouse C57BL/6J and digested with restriction endonucleases enzyme, different transcriptional regulation sequences of 5＇ flanking of amelogenin gene including the basal promoter were cloned and ligated with luciferase gene in PGL3-Basic vector. These report vectors were transiently transfected into CHO, Hela and UMR-106 cells and lueiferase assay was performed to analyse the transcription activation of these promoters. RESULTS： 6 promoters different in length were cloned. The activity of luciferase was very strong in Hela cells. On the contrary, the CHO and UMR-106 cells showed weak fluorescence. Luciferase activity fluctuated with the different promoter lengths in Hela cell as well as in CHO and UMR-106 cells. 975 bp and 532 bp of amelogenin 5＇ flanking DNA had a strong transcriptional activation, but 285 bp of amelogenin 5＇ flanking DNA had a weaker transcriptional activation. CONCLUSION： The sequence of amelogenin promoter can be activated in Hela cell. Hela cell can be used as a good model to study the transcriptional regulation of amelogenin promoter. According to the different activities of different lengths, it is suggested that there were some potential siliencer located between -975 and -532, and some potential activator in the region between -532 and -285. Supported by National Natural Science Foundation of China （30271418）.
出处
《上海口腔医学》
CAS
CSCD
2005年第4期402-406,共5页
Shanghai Journal of Stomatology
基金
国家自然科学基金(30271418)
关键词
釉原蛋白
启动子
转录调控
报告基因
荧光素酶
Amelogcnin
Promoter： Transcription regulation
Report gene
Lueiferase
