TBX1基因启动子活性的初步研究

Preliminary study on TBX1 gene promoter activity

目的研究心脏发育关键转录因子T-box家族成员TBX1基因启动子的活性并确定关键区域。方法利用聚合酶链反应(PCR)技术扩增TBX1基因转录起始位点上游启动子不同长度片段,酶切连接至pGL3-Basic报告基因载体,构建含不同长度TBX1基因启动子调控荧光素酶的报告基因。利用FuGene HD转染试剂将不同长度的TBX1基因启动子调控荧光素酶的报告基因载体瞬时转染293T、COS7细胞,42 h后经荧光素酶检测,比较不同长度TBX1基因启动子的活性。结果不同长度TBX1基因启动子活性不同,其中5 kb和1.2 kb的TBX1基因启动子活性较高;TBX1基因启动子在不同细胞中表现的活性不同,COS7细胞中5 kb的基因启动子活性最高,297T细胞中1.2 kb的基因启动子活性最高。结论 TBX1启动子在转录水平上可以驱动报告基因表达,不同长度启动子的活性不同,推测转录起始位点上游1.2 kb区域是TBX1基因启动子关键区域。

Objective To determine the activity of TBX1 gene promoter, and identify the critical region of TBX1 gene promoter. Methods TBX1 gene promoters with different length were isolated and amplified by PCR, digested and inserted into pGL3-Basic reporter gene vector. 293T and COS7 cells were transiently transfected with reporter plasmids containing TBX1 gene promoters with different length using FuGene HD. Forty-two hours later, the activity of TBX1 gene promoters with different length was compared with luciferase detection. Results There were differences in activity among TBX1 gene promoters with different length, and the activity of TBX1 gene promoters with 5 kb and 1.2 kb in length was higher. The activity of TBX1 gene promoters varied in different ceils, with the highest activity of TBX1 gene promoter with 5 kb in length in COS7 cells and that with 1.2 kb in length in 297T cells. Conclusion In transcriptional level, TBX1 promoter could activate the expression of reporter gene. The activity varies among promoters with different length. It is suspected that 1.2 kb upstream region of transcript start site is critical to TBX1 gene promoter.