肌肉LIM蛋白增强生肌素对AChRγ启动子的反式激活作用

Muscle LIM Protein Enhances trans-Activation of Myogenin to the AChR γ-Subunit Gene Promoter

采用RT PCR技术克隆了大鼠肌肉LIM蛋白 (MLP) 6 40bp的全长cDNA序列 .以此cDNA为探针进行的Northern印迹表明 ,MLP于C2C12细胞在分化的第 3d至第 5d表达 .将MLPcDNA亚克隆至pcDNA3,构建真核表达质粒pcDNA3 MLP ,同时构建AChRγ启动子序列 (96 0bp)调控的荧光素酶报告基因真核表达质粒pGL3 γ .C2C12细胞转染及荧光素酶活性分析表明 ,复合转染pcD NA3 MLP和pGL3 γ的分化肌细胞表达的荧光素酶活性约为对照的 4倍 ;而在 3T3或未分化肌细胞复合转染pcDNA3 MLP和pGL3 γ均未检出报告基因表达 ,说明MLP可促进生肌素对AChRγ亚基基因启动子的反式激活作用 .

A full length sequence of rat cDNA (640 bp) coding muscle LIM protein (MLP) was amplified with RT PCR. Northern blot analysis indicated that MLP gene was transcribed from the third day when C2C12 cells were cultured in differentiation medium. The eukaryotic expression vector pcDNA3 MLP was constructed by subcloning MLP cDNA from pUC19 MLP into pcDNA3. The 960 bp upstream sequence of the acetylcholine receptor (AChR) γ\|subunit gene was subcloned into pGL3 Basic to generate the eukaryotic expression plasmid pGL3 γ, in which the luciferase reporter gene was driven by the AChR γ subunit gene promoter. C2C12 cells and NIH 3T3 were cotransfected with pcDNA3 MLP and pGL3 γ, and luciferase activities in the cell extracts were tested. The luciferase activity in myotubes was as about four times higher than the control. However, the luciferase activities in both myoblasts and NIH 3T3 cells were only in background levels. These results suggest that MLP enhances myogenin trans activation of the AChR γ subunit gene promoter during C2C12 differentiation.