LncRNA gga-lnc-0181对胚胎期肉鸡骨骼肌发育的影响及机制研究

Long non-coding RNAs (lncRNAs) are important regulators of muscle development. A large number of lncRNAs were found in chicken muscle by high-throughput technology, while selecting a key lncRNA from a large database and studying its regulatory mechanism is an urgent task currently. We selected a lncRNA gga-lnc-0181 highly expressed in chicken muscle from previous study. However, the role of gga-lnc-0181 in embryonic muscle of chicken has not been reported, its mechanism remains to be investigated. China is a major producer of broiler, however, the production efficiency, especially for native breeds, is relatively low. So, to improve the yield of chicken is not only necessary for the national strategy, but also a important scientific problem. Therefore, in this project, the Lushi chicken (a native breed of Henan province) are used as a research modle, Rose chicken as control, to observe the expression of gga-lnc-0181 in different embryonic stages, and to explore whether or not there is a difference in the expression of gga-lnc-0181 between breeds. Further, gga-lnc-0181 overexpression and RNA interference lentiviral vectors are designed and constructed, and are transfected into chicken embryo primary muscle satellite cells, to study the function of gga-lnc-0181. Several technologies, including metabolomics, morphology, epigenetics, will be applied to clarify the mechanism of gga-lnc-0181 regulating chicken muscle development, therefore, to provide a theoretical basis for improving poultry production.

LncRNAs（long non-coding RNAs）是近年发现的调控动物肌肉发育的重要因子。目前，应用高通量技术在鸡肌肉中发现有大量lncRNAs表达，而从庞大的数据库中筛选关键lncRNA并深入研究其调控机理亟需进行。申请人前期研究发现lncRNA gga-lnc-0181在鸡胚肌肉中具有高水平表达，且呈现品种差异和发育阶段性，提示其很可能在鸡胚肌肉发育中发挥调节作用。但其对鸡胚肌肉发育的具体作用及其机制尚不清楚。因此，本项目拟以河南地方品种卢氏鸡为研究对象，从在体和体外两个方向，从确认gga-lnc-0181对胚胎期肉鸡肌肉发育的影响、筛选可能介导gga-lnc-0181调节功能的关键基因、应用RNA免疫沉淀、染色质免疫共沉淀等技术对该lncRNA调节关键基因表达的作用途径3个层次深入研究，揭示其调控鸡胚肌肉发育的机制，为提高鸡肉产量提供理论依据。

LncRNAs是调控肌肉发育的重要因子。本项目通过品种比较、应用LPS构建急性炎症反应、饮水添加甜菜碱等探讨长链非编码RNA gga-lnc-0181在肌肉发育以及炎症反应中的作用。具体内容如下：.1.两个品种肉鸡胚胎期gga-lnc-0181及肌肉生长发育相关基因表达规律.分别在10胚龄、18胚龄以及出雏当天，采集卢氏鸡和罗斯肉鸡腿肌，发现长链非编码RNA ggal-lnc-0181的表达在三个时间节点呈现高-低-高的规律，且1日龄时表达量远高于两个胚胎期，另在1日龄时，罗斯肉鸡腿肌中gga-lnc-0181的表达显著低于卢氏鸡。调控肌肉生长发育的基因MyoG、MyoD、IGF1、mTOR、MSTN以及DNA甲基化酶DNMT3A的表达与gga-lnc-0181的表达模式一致。提示，gga-lnc-0181可能是通过对染色质甲基化的调节来调控肌肉生长发育相关基因的表达的。.2. LPS诱导的肉鸡急性炎症中腿肌lncRNA的表达变化.通过腹腔注射LPS诱导肉鸡急性系统性炎症反应。发现LPS处理2 h，lnc0035、 lnc0119、lnc0181的表达出现显著差异，且均与细胞因子IL-6的基因表达呈显著相关性。进一步研究发现，饮水中添加甜菜碱能够缓解LPS引起的肌肉IL-6过度表达，并且能抑制LPS对lnc0119以及TLR4基因表达的效应。MeDIP实验结果显示，LPS能够显著降低IL-6启动子CpG岛的甲基化水平，提高TLR4启动子CpG岛的甲基化水平，而甜菜碱的添加能够缓解LPS对TLR4启动子甲基化的影响。提示，lnc0035、lnc0119以及lnc0181与肉鸡急性期系统性炎症反应有关，其中lnc0119可能参与IL-6及TLR4的基因表达调控。.3.饮水中添加甜菜碱对肉鸡肌肉发育的影响.新生罗斯肉鸡饮用添加0.1%甜菜碱的饮水3周，其腓肠肌横肌纤维明显肥大。抑制肌纤维分化发育的基因MSTN表达显著降低，而促进肌纤维分化发育的Myf5以及MyoG的mRNA水平显著升高；但是长链非编码RNA lnc0181、lnc0035以及lnc0119的表达无显著差异。腿肌RNA总m6A修饰水平显著升高，与该修饰相关的酶FTO，METTL3，YTHDF2等的蛋白表达出现显著差异。这些结果提示m6A修饰在甜菜碱对肉鸡肌肉发育的影响中发挥了调控作用。