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目的对NFBD1启动子区域顺式作用元件CHR进行定点突变分析,以分析CHR在NFBD1转录调控中的作用。方法以原有NFBD1核心启动子荧光素酶报告基因重组体NFBD1-PS1-325为模板,采用PCR介导的基因定点突变技术对NFBD1启动子区域中的CHR位点进行定点突变,构建相应的CHR定点突变重组体;采用荧光素酶双报告基因分析技术检测各重组体的启动子活性,分析CHR定点突变对NFBD1启动子活性的影响;结合阿霉素处理,分析CHR在DNA损伤后NFBD1转录下调中的潜在作用。结果CHR定点突变后能显著降低NFBD1的启动子活性;CHR定点突变后显著减弱了DNA损伤后NFBD1的转录下调比例。结论CHR参与NFBD1的基础转录调控及DNA损伤后的转录下调。
Objective To analyze the role of CHR in NFBD1 transcriptional regulation by site-directed mutagenesis of the cis-acting element CHR in NFBD1 promoter region. Methods The original NFBD1 core promoter luciferase reporter gene recombinant NFBD1-PS1-325 was used as a template for site-directed mutagenesis of the NFBD1 promoter region by site-directed mutagenesis using PCR-mediated gene knockdown to construct the corresponding CHR The recombinant plasmid was sequenced. The luciferase reporter assay was used to detect the promoter activity of each recombinant. The effect of CHR site-directed mutagenesis on NFBD1 promoter activity was analyzed. Combined with doxorubicin treatment, the transcription of NFBD1 In the potential role. Results After site-directed mutagenesis of CHR, the promoter activity of NFBD1 was significantly reduced. After site-directed mutagenesis of CHR, the transcriptional ratio of NFBD1 was significantly decreased after DNA damage. Conclusion CHR participates in basic transcriptional regulation of NFBD1 and down-regulation of transcription after DNA damage.