鸡快慢肌纤维分化相关IncRNA的鉴定及其调控机制的研究

The difference of chicken muscle-fiber types is one of the important factors affecting muscles growth and meat quality traits. At present, the research on chicken muscle fibers differentiation are mostly focused on the level of genome mining and screening related genes. The regulation of Non-coding RNA mediated muscle fibers differentiation is relatively few. To provide a theoretical basis for the genetic improvement of high chicken quality traits, the genetic mechanism of fast and slow muscles’ formation was investigated. In this study, the comparative analysis in muscles differentiation was analyzed on the Rose Crown Chicken and Cobb Broiler Chicken, and high-throughput sequencing techniques were used to screen differentially expressed genes and lncRNA that affect the differentiation of muscle fibers, identify new lncRNA and predict target genes for lncRNA. The regulation pathway of muscle fiber differentiation is constructed by lncRNA-mRNA integrative analysis. On this basis, screened lncRNAs were verify for functional at the cellular level, the role of lncRNA in muscle differentiation was analyzed, and the regulatory mechanism of related genes during the development and differentiation of chicken muscle fibers was also explored. Furthermore, the molecular mechanism of lncRNA on the fiber types differentiation of Rose Crown Chicken and Cobb Broiler Chicken was also initially revealed . All of these laid the foundation for further improving the molecular theory of chicken muscle development and studying the breeding of high-quality chicken meat quality traits.

鸡肌纤维类型的差异是影响肌肉生长发育及肉品质性状的重要因素之一。目前，关于鸡肌纤维分化的研究多集中在挖掘和筛选基因方面，非编码RNA调控肌肉分化的研究相对较少。探明肌肉中快慢肌形成的遗传机理，为鸡肉品质性状的遗传改良提供理论基础。本研究对玫瑰冠鸡、科宝肉鸡快慢肌分化形成的差异进行比较分析，采用高通量测序技术分别筛选影响肌纤维分化的差异表达基因和lncRNA，鉴定新的lncRNA，对lncRNA进行靶基因预测，通过lncRNA-mRNA联合分析构建肌纤维分化的调控通路；在此基础上，将筛选到lncRNA进行细胞水平的功能验证，分析lncRNA在鸡快慢肌纤维分化时的作用，探讨鸡肌纤维发育和分化过程中相关基因的调控机理，初步揭示lncRNA对玫瑰冠鸡和科宝肉鸡肌纤维类型分化的分子调控机制，进一步完善鸡肌肉发育的分子理论，为优质鸡肉质性状的育种奠定基础。

肌纤维类型组成与分化的调控机制是肌肉功能差异的分子基础，并最终决定肉品质性状。本研究针对玫瑰冠鸡、科宝鸡胚胎期快慢肌分化形成的差异，筛选影响肌纤维分化的差异表达mRNAs和lncRNAs，鉴定新的lncRNAs，通过lncRNA-mRNA联合分析构建肌纤维分化的调控通路；在此基础上，将差异lncRNA进行功能验证，分析其在鸡快肌、慢肌纤维分化时的作用。.（1）随着日龄的增加，玫瑰冠鸡、科宝鸡腿肌纤维直径和横切面积均呈极显著增长（P<0.01)趋势，肌纤维密度呈极显著下降（P<0.01)趋势，相同日龄科宝鸡的腿肌纤维直径和横切面积均极显著高于（P<0.01)玫瑰冠鸡，而腿肌纤维密度、慢肌纤维比例则极显著低于（P<0.01)玫瑰冠鸡；随着日龄的增加，玫瑰冠鸡、科宝鸡胸肌快肌纤维比例均呈增长趋势，且科宝鸡的快肌纤维比例、慢肌纤维比例均极显著高于（P<0.01)玫瑰冠鸡。.（2）筛选玫瑰冠鸡、科宝鸡胚胎期8天与14天肌肉组织中差异表达的lncRNA241个，预测916个靶基因，两种鸡胚胎期第8天差异表达lncRNA48个，预测290个靶基因，胚胎期第14天差异表达lncRNA81个，预测455个靶基因。GO和KEGG富集分析与肌肉组织形态发生、钙离子信号调节骨骼肌收缩和肌细胞分化的调控等有关，确定多个与快慢肌纤维转化相关的lncRNA和相应靶基因，分别为：LNC_002865-MEF2A、LNC_015454- MEF2C和LNC_004686- MYH1B等，并绘制lncRNA与其预测靶基因网络互作图。.（3）RACE扩增获得LNC-004686的全长序列，pcDNA3.1(+)-004686过表达重组质粒能促进成肌细胞增殖，显著抑制成肌细胞的分化(P<0.05)，快肌特异性基因FWM、MEF2A、SOX6、SIX1以及相关蛋白的表达量显著提高 (P<0.05)，慢肌特异性基因SM、MYOG、PPARD、MEF2C及相关蛋白的表达量显著降低(P<0.05)；而干扰lncRNA-004686显著抑制成肌细胞增殖 (P<0.05)，促进成肌细胞的分化，相关蛋白表达量结果恰恰相反，推测lncRNA-004686负向调控靶基因MYH1B的表达，从而影响胚胎期肌纤维类型的改变。.研究结果揭示了肌纤维生成和类型转换中发挥调控作用的重要因子，对今后对鸡肉品质的遗传改良提供新思路和策略。