MTS1基因ARF启动子基础转录调控区转录活性研究

目的　研究MTS1基因ARF启动子基础转录调控区的转录激活及其与E2 F1转录因子之间的关系。方法　以含ARF启动子基础转录调控区E2 F1结合位点野生型序列的W6重组质粒为模板 ,设计该片段中E2 F1A、B、C结合位点序列突变或缺失的引物 ,用聚合酶链反应构建该区域中E2 F1A、B、C结合位点序列突变或缺失的ZE、ZF、ZG重组质粒。再将W6、ZE、ZF、ZG重组质粒转染进Jurkat细胞 ,检测其荧光素酶报告基因的表达。结果　构建的ZE、ZF、ZG重组质粒经SacⅠ或NaeⅠ酶切鉴定和DNA序列分析得到证实。与E2 F1位点野生型重组质粒W6比较 ,突变型重组质粒ZE、ZF、ZG在Jurkat细胞中荧光素酶报告基因的表达量减少 ,以E2 F1A位点突变的重组质粒减少明显。结论　构建ARF启动子E2 F1A、B、C结合位点序列突变的重组质粒成功 ;MTS1基因ARF启动子基础转录调控区的转录激活可能与E2 F1转录因子的作用有关。

Study on transcriptional activity of basic transcriptional regulatory region in ARF promoter of MTS1 gene

Objective To study the effect of E2F1 transcription factor on the transcriptional activity of basic transcriptional regulatory region in the ARF promoter of MTS1 gene.Methods The primers containing mutant or deleted sequence of the E2F1 A,B,C- bound loci in the 0.38kb fragment from ARF promoter were designed. W6 recombinant plasmid of wild-type sequence of E2F1-bound loci in the fragment from the regulatory region of ARF promoter was used as template.ZE,ZF and ZG recombinant plasmids containing mutant sequence in loci bound to E2F1A,B,C were constructed by PCR.ZE,ZF and ZG recombinant plasmids were transfected into Jurkat cells by transient transfection.Luciferase report gene was determined to observe ARF promoter transcriptional activity.Results The new recombinant plasmids containing mutant or deleted sequence in loci bound to E2F1 A,B,C were identified by SacI or NaeI enzymatic cutting and sequencing.The Luciferase expression of recombinant plasmids containing mutant sequence in loci bound to E2F1 A,B,C in Jurkat cell was decreased,especially the mutant sequence in the locus bound to E2F1A,as compared with the wild-type sequence in the locus bound to E2F1 in recombinant plasmid.Conclusions The recombinant plasmids were successfully constructed.There was a potential corelation between the transcriptional activity of the basic transcriptional regulatory region of RF promoter of MTS1 gene and the effect of E2F1 transcription factor.