人NaDC1基因启动子序列转录调控元件的初步分析  

Positional identification of transcription regulation elements in human NaDC1 promoter

作　　者：杨聚荣[1] 何娅妮[1] 张剑凯[2] 李新伦[1] 梁海君[1] 林静[1] 吴小玮[1] 王惠明[1] 李开龙[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所肾内科,重庆400042 [2]广东医学院基础学院人体解剖学教研室,湛江524023

出　　处：《第三军医大学学报》2007年第18期1787-1789,共3页Journal of Third Military Medical University

摘　　要：目的对hNaDC1基因启动子序列转录调控元件进行初步分析。方法构建hNaDC1基因5′侧翼序列系列缺失质粒,以双荧光素酶报告基因检测系统检测hNaDC1基因5′侧翼区(-2232/+136)的转录调控元件与转录调控蛋白之间的相互作用。结果pGL3-hNaDC1A质粒的转录活性最高,为(2.98±0.45);pGL3-hNaDC1D的转录活性最低,为(0.45±0.14)。以pGL3-hNaDC1A质粒的转录活性为基准(100%),其他4种质粒相应的转录活性分别为:pGL3-hNaDC1B86.7%,pGL3-hNaDC1C89.4%,pGL3-hNaDC1D15.8%,pGL3-hNaDC1E61.2%。MatInspector5.0软件预测结果显示hNaDC1基因5′侧翼启动子序列共含有22个14种转录因子结合位点。结论hNaDC1基因5′侧翼区-1084/-254和-12/+136区域可能存在有正性作用转录因子的结合位点。Objective To determine the positions of transcription regulation elements in 5＇ flanking region of human Na ^＋ dependent dicarboxylate transporter 1 （ hNaDC 1 ） gene. Methods The DNA fragments hNaDC 1 A-E（ -2 232/＋ 136）of its 5＇ flanking were amplified from the genomic DNAs of HK-2 cells by using PCR. The PCR products were directionally subcloned into pGL3-basic vector. The recombined clones were identified by agarose gel electrophoresis after restriction endonuclease digesting and DNA sequencing. The recombined vectors as well as pGL3-basic vector were respectively transfected into HK-2 cells with Lipofectamine 2000. To normalize transfection efficiencies, 0.4 μg of a Renilla luciferase positive control vector （ pRL-CMV plasmid） was used for co-transfection. The interaction between transcription regulation elements and transcription regula tion proteins in 5＇ flanking region of hNaDC1 gene（ -2 232/＋ 136）was measured by analyzing relative light unit （RLU） with the help of Dual Luciferase Report Gene Assay System （DLR）. Results The RLU was significantly lowered in HK-2 cells transfected with pGl3-basic when compared with the cells harboring pGL3- hNaDC1 A-E （P 〈0. 05 or P 〈0. 001 ）. Compared with pGL3-hNaDC1 A group, RLU was significantly lowered in pGL3-hNaDC1 D group （P 〈0. 01 ） and pGL3-hNaDC1 E group （P 〈0. 05）. MatInspector 5.0 system indicated that there were 22 binding sites of 14 trans-acting factors（ Sore 〉 90 ） in the regions - 1 084/-254 and - 12/＋ 136. Conclusion Transcription enhancer elements, which have interacted with transcription acti- vation proteins, exist in both region -1 084/-254 and -12/＋ 136. These results may offer an approach for further study on the enhancers of hNaDC1 gene.

关 键 词：hNaDC1基因 双荧光素酶报告系统 转录调控 

分 类 号：R394-33[医药卫生—医学遗传学] R394.2[医药卫生—基础医学]