心脏和骨骼肌特异表达基因KLHL31在心脏左右不对称形态发育中的功能研究

项目名称： 心脏和骨骼肌特异表达基因KLHL31在心脏左右不对称形态发育中的功能研究

项目编号： No.30871417

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 建筑科学

项目作者： 李永青

作者单位： 湖南师范大学

项目金额： 36万元

中文摘要： 心脏病严重威胁着人类健康。鉴定新的心脏发育相关基因具有重要意义。本项目对人类新基因KLHL31在心脏发育中的功能进行较系统地研究。我们研究了KLHL31蛋白的表达谱及其在COS-7和原代培养的乳鼠心肌细胞中的亚细胞定位。证明KLHL31具有转录抑制效应，可剂量依赖性抑制c-Jun表达，这种抑制效应主要由BTB 结构域决定。利用酵母双杂交、GST pull-down、Co-IP、共转染等实验证实了KLHL31通过BTB结构域与NOMO在体外和真核细胞中相互作用。在斑马鱼体内进行敲减、过表达KLHL31以及rescue实验，通过RT-PCR、Western blot和胚胎原位杂交等方法，证明KLHL31主要作用于心脏左右不对称发育；与NOMO彼此拮抗，参与调节心脏Lefty-Nodal-Pitx2左右不对称信号通路。并推断出了其在该通路中的定位。还发现KLHL31基因可能通过调节斑马鱼胚胎KV的面积和其中纤毛的数量来影响胚胎心脏左右不对称发育。总之，本课题组在世界上首次系统研究了KLHL31基因在心脏发育中的功能，基本阐明了该基因调控心脏左右不对称发育的分子机制。

中文关键词： KLHL31基因；NOMO；心脏左右不对称发育；Lefty-Nodal-Pitx2通路；斑马鱼

英文摘要： Heart diseases are seriously affecting human health. In order to understand the molecular mechanism underlying heart development and diseases, it is necessary to identify the novel genes assosiated with heart development and diseases. A novel human gene KLHL31, is systemly been studied in this project. We have described its expression patterning and analyzed the subcellular localization in COS-7 and primary heart cells from a neonatal mouse. We found that overexpression of KLHL31 inhibits the transcriptional activity of the report gene through BTB domain and significantly reduces the level of c-Jun protein in a dose-dependent manner. By using yeast two-hybrid, GST pull-down, Co-IP and co-transfection, we have demonstrated that KLHL31 interacts with NOMO via BTB-domain in vitro and in eukaryotic cells. The experiments of knock-down, overexpression, and rescue for KLHL31 in vivo were performed in zebra fish embryos. All of the results of RT-PCR, Western blot, and embryo in situ hybridization, corresponding to each other, showed that KLHL31 has important roles in heart left-right asymmetrical development and that it antagonizes to NOMO to regulate the heart left-right asymmetrical pathway of Lefty-Nodal-Pitx2. The location of KLHL31 in the pathway was determined. Moreover, KLHL31 was shown that it might affect heart left-right asymmetrical development by controlling the left-right diameter of Kupffer's Vesicle（KV）and the numbers of cilia in the KV. In summary, the fuctions of KLHL31 in heart development were systemly studied firstly in the world, and the molecular mechanism of the gene regulating heart left-right asymmetrical development was revealed in this project.

英文关键词： KLHL31 gene; NOMO; left-right asymmetrical development of heart; Lefty-Nodal-Pitx2 pathway；zebra fish