牦牛高原低氧适应关键基因CREB1的功能鉴定及分子机制研究

牦牛是我国青藏高原的重要畜种，其骨骼肌中高比例的氧化型(I型)肌纤维是适应高寒低氧环境的重要特征。申请人前期通过选择信号和基因渗入分析发现转录因子CREB1是牦牛高原低氧适应的关键候选基因，其调控的miR-381会降低I型肌纤维比例和线粒体拷贝数，通过RIP-seq实验发现Wnt信号通路中的关键转录因子LEF1是miR-381的靶基因。在前期工作的基础上，本项目拟以牦牛骨骼肌卫星细胞为模型，利用功能获得和缺失试验验证CREB1在牦牛骨骼肌纤维类型转化过程中的功能；通过Chip-qPCR和EMSA等技术研究CREB1对miRNA-381表达的调控方式，明确CREB1和Wnt信号通路与肌纤维类型之间的关系，并阐释CREB1通过miR-381/Wnt信号轴调控牦牛骨骼肌低氧适应特征形成的分子机制，以期揭示牦牛低氧适应的分子机制，为牦牛分子育种及功能基因组学研究奠定理论依据。

Yak is the key animal species in the Qinghai-Tibetan Plateau. The higher content of type I muscle fiber is one of the important adaptive characteristics of yak in response to extreme conditions (cold and low oxygen). According to our previous research, cyclic AMP response element binding protein1 (CREB1) was selected as candidate gene for the hypoxic adaptation of yak by assessing signatures of selection and gene introgression. MiR-381 is one of the downstream genes of CREB1, which could reduce the content of type I muscle fiber and the copy number of mitochondrial. Furthermore, by using RNA immunoprecipitation combined with high-throughput sequencing (RIP-seq), we identified 1ymphoid enhancer factor 1 (LEF1), a key transcription factor of Wnt/β-catenin pathway, as a target of miR-381. In the present study, we use satellite cells of yak as experimental material. We will study the effects on conversion of muscle fiber types by increasing or reducing the expression of CREB1. Next, chromatin immunoprecipitation (ChIP) followed by qPCR (ChIP-qPCR) assays and electrophoretic mobility shift assay (EMSA) technology were employed to study the regulation mode of miR-381 expression and clarify the relationship among CREB1, Wnt signaling pathways and muscle fiber types. Our results would elucidate the roles of CREB1 in regulation of hypoxia adaptation through miR-381/Wnt axis, and provide a theoretical basis for molecular breeding and functional genomics studies of yak.