MTS_1基因β启动子E_2F_1结合位点序列突变重组质粒的构建与表达

CONSTRUCTION AND EXPRESSION OF THE RECOMBINANT PLASMID OF THE MTS_1 GENE β PROMOTER ON E_2F_ 1-BOUND LOCUS MUTATION

目的 :深入研究MTS1 基因 β 启动子的转录激活与E2F1 转录因子的相互作用关系 ,阐明该基因转录水平的调控机制。方法 :用PCR定点突变方法或酶切连接法 ,构建β 启动子0.38kbSacⅡ -SacⅠ酶切片段中E2F1 A、B、C任意2个位点或3个位点均突变的 pGL3 重组质粒。用脂质体介导的基因瞬时转染法 ,将构建的重组质粒转染MTS1 基因双等位缺失的急性T淋巴细胞白血病Jurkat细胞 ,检测pGL3 重组质粒中荧光素酶报告基因的表达。 结果 :构建的E2F1 A、B、C结合位点突变的重组质粒经SacⅠ或NaeⅠ酶切鉴定和DNA序列分析得到证实。与E2F1 位点野生型重组质粒比较 ,突变型重组质粒在Jurkat细胞中荧光素酶报告基因的表达量减少 ,以3个位点均突变的重组质粒为明显。 结论 :构建的E2F1 A、B、C2个或3个结合位点均突变重组质粒成功 ,可通过基因转染用于研究MTS1 基因的功能试验中 ;MTS1 基因β 启动子的转录活性可能与E2F1 转录因子的反式激活有关。

Purpose: To further study the effect of E 2 F 1 transcription factor on the transcriptional activation of β promoter, and explain the transcriptional regulation mechanism on MTS 1 gene. Methods:The recombinant plasmid containing 2 or 3 mutant or deleted on E 2 F 1 A, B, C_bound locus sequence on the 0.38 kb fragment cut by SacⅡand SacⅠ of the β promoter was constructed by PCR site_directed mutagenesis and enzymatic cutting and ligating. The recombinant plasmids containing 2 or 3 mutant or deleted on E 2 F 1 A, B, C_bound locus sequence were transfected into Jurkat cells, which were biallelic deletion of MTS 1 gene by transient transfection. Luciferase report gene was used to observe β promoter transcriptional activation. Results: Four new recombinant plasmids containing the mutant with two bound loci of E 2 F 1 A,B,C were obtained separately by PCR, and a recombinant plasmid containing all the three mutant on locus was constructed by enzymatic cutting and ligating, and identified by SacⅠ or NaeⅠenzymatic cutting, and sequencing. The luciferase expression of recombinant plasmid in Jurkat cells decreased, especially the mutant 3 bound locus sequence of E 2 F 1 A, B, C, as compared to the wild_type recombinant plasmid on E 2 F 1_bound locus sequence. Conclusion: These newly_constructed recombinant plasmids can be used to study the function of the transcriptional activation of MTS 1 gene by gene transfection. There is a potential relation between the transcriptional activation of MTS 1 gene β promoter and the transactivation of E 2 F 1 transcription factor.[