连环蛋白基因功能区截短突变体的构建及其功能

Construction and functional study of truncated β catenin genes

目的 :观察连环蛋白 (βcatenin)基因功能截短体的亚细胞定位及转录活性的变化。方法 :采用反转录PCR克隆全长野生型βcatenin基因。在此基础上 ,分别构建 βcatenin基因N端、C端及Armadillo区缺失突变体的真核基因表达载体和融合绿色荧光蛋白 (EGFP)的表达载体。应用双荧光素酶报告系统 ,在2 93细胞中检测不同功能截短体的转录活性。在倒置荧光显微镜下观察不同功能截短体在COS7细胞中的亚细胞定位。结果 :成功地构建了 βcatenin基因不同功能截短体的真核表达载体及与绿色荧光蛋白基因融合的表达载体。在 2 93细胞中检测到Armadillo区缺失的突变体蛋白丧失了转录活性 ,N端及C端缺失突变体蛋白的转录活性较野生型分别下降了80 %和 90 %。在COS7细胞中观察到C端缺失突变体蛋白主要定位于胞浆中 ,N端缺失突变体蛋白以粗颗粒形式定位于胞核中 ,Armadillo区缺失突变体的定位情况类似野生型 βcatenin ,以细颗粒形式定位于胞核中。结论 :βcatenin基因不同功能区亚细胞定位的不同 ,提示其在转录激活过程中发挥着不同的作用。

AIM: To observe the subcellular location and the change of the transcriptional activity of truncated β catenins in mammalian cells. METHODS: The full length wild type β catenin gene was cloned by RT-PCR. The truncated β catenins were constructed by deleting the N terminal, the C terminal or the Armadillo domain sequences, and were inserted into the EGFP expression vector. After they were transfected into 293 cells, the transcriptional activity of truncated β catenins in 293 cells was detected by dual luciferase reporter system. The subcellular location of truncated β catenin in transfected COS7 cells was observed under fluorescence microscope. RESULTS: The eukaryotic expression vector containing truncated β catenin gene and expression vector of truncated β catenin gene fused with EGFP gene were constructed successfully. C terminus-deleted β catenin localized mainly in the cytoplasm of transfected COS7 cells, and the transcriptional activity of the N terminus- and the C terminus- deleted β catenins was lower than that of the wild type β catenin in transfected 293 cells. CONCLUSION: The different locations of truncated β catenins in transfected COS7 cells suggest that the truncated ones play different roles in the transcriptional activity.