人EFTUD2启动子的鉴定及初步分析

Structural and functional analysis of the human EFTUD2 promoter

目的:鉴定并分析EFTUD2(elongation factor Tu GTP-binding domain-containing 2)基因的启动子区域。方法:应用生物信息学技术对EFTUD2基因结构及潜在启动子区域进行分析,预测4个EFTUD2启动子序列。使用全基因合成技术以及高保真PCR扩增得到目的启动子序列,通过BglⅡ和NheⅠ双酶切,将目的条带克隆到双荧光素酶报告基因载体psiCHECK-2中,得到4个长度不同并覆盖EFTUD2基因转录起始位点附近约2 kb的EFTUD2基因启动子双荧光素酶报告基因重组体。荧光素酶分析检测启动子活性。结果:经酶切、测序鉴定,成功构建了人EFTUD2启动子荧光素酶报告基因重组质粒。通过荧光素酶活性检测,重组体EFTUD2-0.5的相对荧光素酶活性增加(P <0.05),提示EFTUD2基因核心启动子序列位于转录起始位点附近约500 bp的区域内。结论:EFTUD2基因转录起始位点上游500 bp具有启动子活性,初步判定其核心启动子位于该区域。

Objective:To identify and analyze the elongation factor Tu GTP-binding domain-containing 2(EFTUD2)promoter region for the further study of the gene in transcriptional control and expression. Methods:The EFTUD2 gene structure and potential promoter regions were analyzed by bioinformatics methods,and based on which four EFTUD2 promoter sequences were predicted.Whole-genome synthesis technology and high-fidelity PCR amplification were used to obtain the target promoter sequence. The target band was cloned into the dual luciferase reporter vector psiCHECK-2 by Bgl Ⅱ and Nhe Ⅰ double digestion,and four different EFTUD2 gene promoter luciferase reporter recombinants covering about 2 kb upstream of the EFTUD2 gene transcription initiation site were obtained. Luciferase assay was used to detect promoter activity. Results:Luciferase reporter assays demonstrated that,compared with the control group,the relative luciferase activity of the EFTUD2-0.5 recombinant increased(P < 0.05),suggesting that the EFTUD2 core promoter may be located in a region of 500 bp near the transcription initiation site. Conclusion:It was identified that the500 bp upstream of the EFTUD2 gene transcription initiation site has promoter activity,and it is speculated that its core promoter is located in this region.