一个新的人NFBD1启动子的鉴定与分析

Identification and analysis of a novel human NFBD1 promoter

目的:鉴定分析NFBD1启动子,为进一步研究其转录调控奠定基础.方法:综合应用生物信息学分析和异构体特异性的巢式RT-PCR技术鉴定NFBD1转录起始位点和转录变异体;高保真PCR方法扩增NFBD1基因启动子区域并分别定向克隆入pGL3-basic和pGL3-enhancer载体,构建NFBD1启动子荧光素酶报告基因重组体;荧光素酶分析检测启动子活性;生物信息学方法预测转录因子结合位点.结果:鉴定了一个新的NFBD1转录起始位点和转录变异体,构建了2个含有长度分别为2.5和1.2kb的NFBD1启动子序列的荧光素酶报告基因重组体以及2个相应的含有外源增强子的NFBD1启动子荧光素酶报告基因重组体.启动子活性分析表明,该启动子区域具有启动子活性,且能被外源增强子有效驱动,是一个新的人NFBD1启动子.转录因子结合位点预测分析表明,该启动子区域缺乏典型的CCAAT盒和GC盒,但含有TA-TA盒以及STAT1,MZF1和MAZ等潜在的转录因子结合位点.结论:鉴定了一个新的NFBD1启动子.

AIM： To identify and analyze NFBD1 promoter region. METHODS： NFBD1 transcriptional start site was identified by bioinformatic analysis and isoform-specific RT-PCR. NFBD1 promoter reporters were constructed by high-fidelity PCR and directional cloning into PGL3-basic and PGL3-enhancer vector. Promoter activity was measured by luciferase assay. Transcription factor binding sites were analyzed by bioinformatic analysis. RESULTS： A novel NFBD1 transcriptional variant with a new alternative transcriptional start site was successfully identified. Two fragments of 2.5 and 1.2 kb in length, upstream of the transcription start site, were amplified and cloned into both pGL3- basic and pGL3-enhancer vectors to make NFBD1 promoter reporters with and without exogenous enhancer, respectively. Luciferase reporter assay indicated that this region contained a novel NFBD1 promoter which indeed had promoter activity and could be enhanced by exogenous enhancer. Transcriptional factor binding analysis indicated that, this novel NFBD1 promoter lacked of CCAAT box and GC box, but contained TATA box as well as other putative binding sites for transcription factors such as STATI, MZFI and MAZ. CONCLUSION： A novel NFBD1 promoter has been identified and analyzed.