人DEPDC7基因启动子的转录活性分析

Analysis of transcriptional activity of human DEPDC7 gene promoter regions

目的对人DEPDC7基因近端启动子进行生物信息学分析并构建该启动子的荧光素酶报告系统,初步探讨该启动子的活性。方法采用生物信息学方法对人DEPDC7基因的启动子和转录因子进行分析和预测,通过PCR法扩增DEPDC7基因近端启动子序列,PCR产物经HindⅢ和XhoⅠ双酶切后定向克隆到p GL2-Basic载体,构建一系列的启动子区域荧光素酶报告基因载体,随后经脂质体转染入293T细胞并检测各缺失片段萤光素酶活性。结果人DEPDC7基因位于11p13,翻译起始位点上游1 300 bp左右可能为启动子区域。经双荧光素酶报告基因检测后发现,所有的重组质粒均表现出荧光素酶活性,其中p GL2-DEPDC7P3活性最强。结论初步证明人DEPDC7基因启动子在-879 bp^+100 bp区域具有较强转录活性,为进一步揭示该基因生物学功能和其分子调控机制奠定基础。

Objective To analyze the bioinformatics of human DEPDC7 gene promoter and construct its firefly luciferase report gene vector and preliminary explore its transcription activity. Methods The aim of this study is to analyze and predict the promoter and transcriptional factors of human DEPDC7 gene. In addition, the DNA fragment of 5＇ flanking of DEPDC7 gene were amplified from the genomic DNA of Human embryonic kidney 293T cells （HEK 293T） by PCR, and cloned into pGL2- Basic luciferase reporter gene vector to construct pGL2-DEPDCTP recombinants. After the recombinants were transfeeted into 293T cells with TurboFect Transfection Reagent, relative luciferase activity were detected by the Dual-Luciferase Reporter Assay System. Results Human DEPDC7 gene is mapped to chromosome 11. Some important transcription factor binding sites were found in the transcription regulatory region. The expression vector pGL2-DEPDC7P was constructed successfully and the pGL2- DEPDC7P3 had strong transcriptional activity. Conclusion It confirmed that DEPDC7 promoter region （-879 bp~＋100 bp） had strong transcriptional activity. What is more important is that the direction has been pointed for further research on regulation mechanisms of transcription.