ABRACL基因启动子不同片段重组质粒构建及转录活性研究  

Construction and transcriptional activity of different fragments of the ABRACL gene promoter

作　　者：朱俊闻 杜祎萌 霍楠 薛春源 亢小峰 房廖鑫 徐小洁 张清媛[1] ZHU Jun-wen;DU Yi-meng;HUO Nan;XUE Chun-yuan;KANG Xiao-feng;FANG Liao-xin;XU Xiao-jie;ZHANG Qing-yuan(Harbin Medical University Cancer Hospital,Harbin,150081,China;Department of Genetic Engineering,Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]哈尔滨医科大学附属肿瘤医院,哈尔滨150081 [2]军事科学院军事医学研究院生物工程研究所基因工程研究室,北京100850

出　　处：《军事医学》2023年第5期341-345,共5页Military Medical Sciences

基　　金：国家自然科学基金项目(81972446)。

摘　　要：目的构建肌动蛋白结合Rho激活C末端样(ABRACL)基因启动子不同截短片段重组质粒,并检测其转录活性。方法以含ABRACL启动子全长质粒为模板,利用PCR方法分别扩增ABRACL启动子不同截短片段;将扩增片段分别插入pGL3.0-basic载体,构建ABRACL启动子不同截短片段重组质粒;经双酶切及序列鉴定正确后,将重组质粒转染ZR75-1和A549细胞,采用双荧光素酶报告基因检测系统测定ABRACL启动子不同截短片段的双荧光素酶活性;检测转录因子MYB原癌基因样2(B-MYB)对ABRACL启动子不同截短片段转录活性的影响。结果成功构建含ABRACL基因启动子不同截短片段重组质粒,双荧光素酶活性测定发现ABRACL基因启动子-400 bp片段活性较高,转录因子B-MYB可以增强ABRACL启动子-600 bp和-1000 bp片段的活性。结论发现ABRACL基因启动子转录活性较高区域,为进一步研究ABRACL基因上游调控机制奠定基础。Objective To construct recombinant plasmids with different truncated fragments of the actin-binding Rho activating C-terminal like(ABRACL)gene promoter,and to detect their transcriptional activity.Methods A full-length plasmid containing the ABRACL gene promoter was used as the template,and different truncated fragments of the ABRACL promoter were amplified by PCR before being inserted into the pGL3.0-basic vector respectively to construct recombinant plasmids with different truncated fragments of the ABRACL promoter.After being confirmed by double digestion and sequencing results,the recombinant plasmids were transfected into ZR75-1 and A549 cells.The luciferase activity of different truncated fragments of the ABRACL gene promoter was determined by dual luciferase reporter assay.The effect of transcription factor MYB proto-oncogene like 2(B-MYB)on the transcriptional activity of different truncated fragments of the ABRACL promoter was detected.Results Recombinant plasmids containing the ABRACL gene promoter with different truncated fragments were constructed before the luciferase activity was measured by dual-luciferase assay.The results showed that the ABRACL gene promoter-400 bp fragment exhibited a higher activity,and that the transcription factor B-MYB could enhance the activity of the ABRACL gene promoter of-600 bp and-1000 bp fragments.Conclusion The discovery of regions with high transcriptional activities of the ABRACL gene promoter can contribute to studies on the upstream regulatory mechanism of the ABRACL gene.

关 键 词：ABRACL基因 启动子 转录因子 重组质粒 荧光素酶报告基因 

分 类 号：Q78[生物学—分子生物学]