人SMYD3基因启动子荧光素酶报告载体的构建与分析  

Construction and analysis of the luciferase reporter vectors of human SMYD3 gene promoter

作　　者：谢景航[1] 邹佳宁[1] 唐聪[1] 杨家森[1] 罗学刚[1] 奚涛[1] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009

出　　处：《中国药科大学学报》2009年第2期173-177,共5页Journal of China Pharmaceutical University

基　　金：国家重点基础研究发展计划("九七三"计划)资助项目(No.G199805119)~~

摘　　要：目的:克隆5′长度不同的SMYD3基因启动子序列并进行活性鉴定。方法:采用PCR方法克隆出SMYD3基因转录起始位点上游不同长度的基因片段,构建T载体并利用酶切与测序进行鉴定;将该基因亚克隆至pGL3-Basic荧光报告基因载体上;瞬时转染Hep3B细胞后用双荧光报告检测系统检测不同片段的荧光活性。结果:T载体酶切与测序结果正确,成功构建了pGL3-Basic+370～1400bp荧光报告载体并利用双荧光素酶报告基因检测系统分析了重组质粒的荧光活性。结论:pGL3-Basic+370～1400bp重组质粒转染组与阳性对照组相比并未表现出显著的荧光活性,本研究认为SMYD3基因的启动子核心区域位于-1334bp的上游。Aim： To clone and analyze the 5＇ region of human SMYD3 gene promoter. Methods： Different fragments of the SMYD3 promoter were cloned and inserted into PTG19-T vectors and then analyzed by enzyme di- gestion and sequence detecting. The correct sequences were later inserted into pGL3-Basic luciferase reporter vec- tors. Transient transfection of the plasmids and β-galaetosidase was carried out simultaneously with Hep3B cell line. Dual-light reporter assay was used to detect the luciferase activities. Results： The enzyme digesting and se- quence detecting of the recombinant PTG19-T vectors were proved to be correct. The pGL3-Basic ＋ 370-1 400 bp lueiferase reporter vectors were successfully constructed by the dual-light system assay. Conclusion： The pGL3- Basic ＋ 370-1 400 bp transfected groups have no significant luciferase activities. It is speculated that the potential core region of the SMYD3 promoter should locate on the upstream of- 1 334 bp.

关 键 词：SMYD3 荧光素酶 报告载体 启动子 

分 类 号：Q78[生物学—分子生物学]