摘要
目的 克隆同源盒基因IRX1的核心启动子序列,并探讨其转录调控机制.方法 通过生物信息学预测IRX1基因启动子范围：利用PCR方法扩增并构建含有IRX1基因启动子不同片段的真核报告基因质粒,瞬时转染GES-1胃黏膜细胞,通过检测不同片段的荧光素酶报告质粒活性,判断不同片段的启动子活性.结果 生物信息学预测IRX1基因启动子位于转录起始点上游700 bp范围内.通过PCR技术扩增获得IRX1基因5＇侧翼区8个截短片段报告基因质粒,其活性由大到小依次为p-416＞p-584＞p-715＞p-350＞p-687＞p-320＞p-188＞p-92 除p-92与p-188片段外,其余各片段与PGL3-basic空载体的差异均有统计学意义（均P＜0.05）,其中以p-416片段活性最强,片段p-320与片段p-188之间出现较大活性落差.结论 IRX1基因上游序列启动子以p-416转录活性最高,核心启动子位于-320～-188区间,该区间具有Sp1、TFⅡD等转录因子结合序列,是下一步转录调控机制研究的重要位点.
Objective To clone core promoter regions of iroquois homeobox gene 1 （IRX1） gene and evaluate the regulatory mechanism of IRX1 transcription.Methods Upstream sequence from transcriptional start site was predicted using bioinformatics methods.Serial deleted fragments from IRXI promoter sequences were amplified by PCR and luciferase reporter plasmids were constructed.The luciferase intensity was analyzed after transferring reporters into GES-1 gastric mucosa cell line.Results The promoter of IRX1 was predicted to be within 700 bp from the 5＇-flanking region of IRX1 gene.Eight serial deleted luciferase reporter plasmids were constructed.The transcriptional activity of different fragments was expressed as following：p-416〉p-584〉p-715 〉p-350〉p-687 〉p-320〉p-188 〉p-92.Except p-320 and p-188,the transcriptional activity of other 6 fragments was higher than that of PGL3-basic plasmid.The transcriptional activity was the highest in p-416 and decreased sharply from p-320 to p-188.Conclusions The fragment p-416 shows the strongest promoter activity.The sequence from -320bp to -188 bp is identifie as core promoter region,which is focusd as key sequence for further regulatory analysis,since some binding sites for important transcriptional factors such as Sp1 and TF ⅡD are predicted.
出处
《中华胃肠外科杂志》
CAS
北大核心
2010年第11期846-850,共5页
Chinese Journal of Gastrointestinal Surgery
基金
基金项目：国家自然科学基金（30770961,30973486）
国家863重大项目（2006AA02A402,2006AA02A301）
上海市科委重点基础研究项目（09JCl409600）
上海市浦江人才计划项目（PJ200700367）
上海市慈善基金会癌一症研究专项基金项目
关键词
基因
IRX1
启动子
报告基因
胃黏膜细胞
Iroquois homeobox gene 1 Promoters Reporter genes Gastric mucosa cell line
