GHR基因缺失突变导致鸡肌纤维数量减少和直径变小的机制研究

Fewer muscle fiber number and smaller muscle fiber diameter are generally existed in sex-linked dwarf chickens which caused by GHR gene mutation. However, GHR gene is not involved in muscle fiber number regulation in mammalian study, even though it can also enlarge muscle fiber diameter. Therefore, the mutation of GHR gene not only inhibits the GHR function, but also changes its effects. Our previous studies have found that many genes, non-coding RNAs, pathways and molecular interaction networks may involve in the cause of inhibition of skeletal muscle development in dwarf chicken. However, there is still a lack of systematic understanding of the GHR mutation mediated regulatory networks that are related to muscle fiber number and diameter. In order to elucidate how GHR gene mutation leads to the reduction of muscle fiber number and muscle fiber diameter, and to explore and verify genes and pathways involved in the regulation of chicken muscle fiber number and diameter, the normal chicken and the GHR gene deletion mutation dwarf chicken will be used as the research objects in this project. The differences of types and functions of the GHR transcript between the skeletal muscles of these two chicken breeds will be analyzed. The impact of GHR gene deletion mutation in chicken myoblast development process, and the regulation of GHR gene deletion mutation in the GHR downstream signaling pathway and molecular interaction network will also be detected in this project. The purpose of this project is to elaborate the molecular mechanism of how the GHR gene deletion mutation influence the GHR gene functions and chicken muscle fiber development.

由GHR基因突变所导致的性连锁矮脚鸡普遍表现出肌纤维数量减少和直径变小的现象，但在哺乳动物研究中GHR基因可促进肌纤维直径变粗，而并不参与调控肌纤维数量。因此GHR基因的突变一方面抑制了GHR功能，另一方面可能改变了它的作用。本课题组前期研究发现许多基因、非编码RNA、通路和分子互作网络可能参与导致矮脚鸡骨骼肌发育受阻，但对于GHR基因突变所介导的肌纤维数量和直径的调控网络仍缺乏系统认识。为阐明GHR基因突变如何导致鸡肌纤维数量减少和直径变小，挖掘和验证与鸡肌纤维数量和直径相关的基因和通路，本项目拟以正常型鸡和GHR基因缺失突变型矮脚鸡为研究对象，分析两种鸡骨骼肌中存在的GHR转录本类型和功能的差异，检测GHR基因缺失突变对鸡成肌细胞发育进程的影响，以及对GHR下游信号通路和分子互作网络的调控，以此综合阐述缺失突变影响GHR基因功能和鸡肌纤维发育的分子机制。

相比于正常鸡，GHR基因缺失型矮脚鸡具有更少的肌纤维数量和更细的肌纤维直径。为揭示其中的分子调控机制，本项目围绕GHR基因缺失突变调控鸡肌纤维形成和肥大的方式、GHR基因突变所影响的下游基因和调控通路、正常鸡和GHR缺失突变型鸡骨骼肌中存在的GHR基因转录本类型、GHR基因各转录本对鸡肌纤维形成和肥大的影响等四个方面进行研究，获得了以下主要结果：发现GHR缺失突变通过抑制JAK-STAT信号通路、FOXO3信号通路和MAPK信号通路活性，影响了肌肉细胞增殖、分化、蛋白质合成，最终导致GHR缺失突变型鸡出现肌纤维数量减少和肌纤维直径变粗。发现c-Myc和TP63基因是GHR缺失突变所影响的主要下游基因，GHR缺失突变通过影响c-Myc和TP63表达，进而抑制了成肌细胞增殖和肌纤维肥大。同时，GHR缺失突变可通过影响IGF2BP3、SOCS2和CISH基因表达，刺激JAK-STAT信号通路和细胞周期通路，促进脂肪沉积。利用三代全长转录组测序，发现正常鸡和GHR缺失型矮脚鸡骨骼肌中各存在7种GHR基因转录本，矮脚鸡中的GHR转录本普遍缺失了1773 nt序列。对这些转录本进行功能研究，发现两条截短型GHR转录本、长链非编码RNA lnc-GHR和GHR天然反义转录本GHR-NAT均可以调控鸡成肌细胞增殖和分化，影响肌纤维的形成。最后，我们还发现GHR基因缺失突变区的两个SNPs与鸡的生长、屠体和肉质性状显著关联，可作为肉鸡育种的分子标记。项目已发表了4篇SCI收录论文和2篇中文核心论文，培育博士1名，硕士2名。研究结果对于肉鸡育种和dw基因的应用具有重要参考意义。