Klotho基因启动子区域定位及活性分析

Promoter region of human klotho and its transcriptional activity in different cell lines

目的构建含不同长度klotho基因启动子片段的报告基因载体,研究其在不同细胞系中的转录活性。方法以人全血基因组DNA为模板,克隆长短不同的klotho基因启动子片段,命名为klothoⅠ、Ⅱ、Ⅲ、Ⅳ;分别克隆入荧光素酶报告基因质粒pGL3-Basic构建真核表达载体;采用Lipofectamine 2000将重组质粒分别和pRL-TK共转染HEK293和HeLa细胞,分析不同长度的klotho基因启动子片段在细胞内的转录活性。结果双酶切和测序鉴定均显示表达载体构建成功;klotho基因的核心启动子区域位于-504～-6;双荧光素酶活性检测显示klothoⅢ在2种细胞中的转录活性明显高于klothoⅠ、Ⅱ(P<0.05),而klothoⅣ的转录活性在HEK293细胞中能维持高水平,在HeLa细胞中显著下降(P<0.01)。结论 klotho基因启动子在不同细胞中的转录活性不同,-973～-504区域可能是导致HeLa细胞中klothoⅣ转录活性下降的原因。

Objective To construct vectors with report gene containing klotho promoter segments in different lengths and identify their transcriptional activities in different cell lines.Methods Genomic DNA of human red cells was used as the template,and human klotho promoter segments in different lengths were amplified by PCR.The amplified products were named klothoⅠto Ⅳ and then respectively cloned into pGL3-Basic vectors with luciferase report gene to construct eukaryotic expression vectors.The recombinant vectors were respectively co-transfected with pRL-TK vectors into HEK293 and HeLa cells with Lipofectamine 2000.The transcriptional activities were analyzed by double luciferase method.Results The different length klotho promoter segments were successfully cloned and their expression vectors were successfully constructed by double digestion and sequencing.The sequence-504 to-6 may be a core promoter region of klotho.The activities of klotho I and klotho Ⅱ in the both cells were lower,and the activity of klotho Ⅲ in the both cell lines was higher（P0.05）.The activity of klotho Ⅳ was maintained at a high level in HEK293 cells,but significantly the activity was decreased in HeLa cell（P0.01）.Conclusion Its expression level of klotho is cell-specific and the sequence-973 to-504 may be responsible for the activity decrease of klotho Ⅳ in HeLa cells.