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目的:探讨软骨分化的负性转录因子Gas6基因启动子调控的分子机制。方法:采用5′RLM-RACE技术克隆Gas6的转录起始位点,利用生物信息学的方法对Gas6潜在启动子区域在小鼠、人与大鼠之间进行同源性比较,对Gas6上游序列进行系列缺失突变,构建Luciferase基因报告载体,转染细胞后检测Luciferase的表达水平。结果:Gas6的转录起始位点为碱基C,在小鼠、人与大鼠之间存在两个高度保守序列A-box与B-box,缺失A-box,B-box的后1/2区域以及NF-Y结合位点后,潜在启动子的转录活性明显下降。结论:A-box,B-box及NF-Y结合位点与Gas6的转录活性有关。
Objective: To investigate the molecular mechanism of promoter regulation of Gas6 gene, a negative transcription factor of cartilage differentiation. METHODS: The transcriptional start site of Gas6 was cloned by 5’RLM-RACE technique. The homology of Gas6 potential promoter region was compared between the mouse, human and rat using bioinformatics method. The sequence of Gas6 upstream sequence A series of deletion mutants were constructed and Luciferase gene reporter vector was constructed. The expression of Luciferase was detected after transfection. Results: The site of transcription of Gas6 was base C, there were two highly conserved sequences A-box and B-box in mice, human and rat, the last 1 / 2 region and NF-Y binding sites, the potential promoter transcriptional activity decreased significantly. Conclusion: The A-box, B-box and NF-Y binding sites are related to the transcriptional activity of Gas6.