miRNA靶向调控刺参响应高温低氧逆境胁迫的分子机制研究

miRNA介导的调控机制已成为动物逆境响应分子机制研究的焦点，而关于miRNA介导的刺参非生物胁迫响应调控机制的研究还鲜有报道。本项目拟从刺参不同组织（肠道，呼吸树和体腔液细胞）在高温，缺氧以及高温缺氧协同作用下的miRNA动态表达谱的构建入手，基于PacBio Sequel三代测序平台获得的全长转录组预测差异表达miRNA的靶标基因；结合靶标基因在刺参不同组织不同胁迫条件下构建的mRNA和蛋白水平的动态表达谱，从整体上分析miRNA介导的抗逆应答模式，构建差异miRNA和靶标基因之间的调控网络；基于此，利用双荧光报告系统验证差异miRNA和靶标基因之间的互作位点；利用miRNA过表达和抑制表达功能实验从细胞和活体层面验证miRNA和靶标之间的调控关系，查明差异miRNA参与调控的信号通路，发掘胁迫响应关键miRNAs，初步揭示miRNA靶向调控刺参响应高温低氧逆境胁迫的分子机制。

Exploration of the molecular mechanisms of microRNAs (miRNA) in mediating stress responses is a current hot topic in animal stress adaptation research. Current knowledge of miRNA roles in the sea cucumber abiotic stress response and their regulatory mechanisms are very limited. The proposed research begins with the construction of microRNA expression profiles of different tissues (intestine, respiratory tree, coelomocyte) of the sea cucumber (Aposticus japonicus) in response to environmental stresses (high temperature, hypoxia, and the interaction between high temperature and hypoxia). The miRNA-targeted genes will then be predicted based on a full-length transcriptome constructed using PacBio Sequel. By mapping the collective targets of stress-responsive miRNA with their known protein interactions, an intuitive representation of the miRNA-regulated functional network will be constructed applying the spatial analysis of functional enrichment (SAFE) tool within the Cytoscape software. Interactive locations between miRNA and targeted genes will be analyzed using a double fluorescent report system. To further validate functional roles of stress-responsive miRNAs, gain-of-function and loss-of function experiments will be conducted in vivo and in vitro at organismal and cellular levels. Results will be used to develop a specific perspective for marine invertebrate stress physiology research with particular applications to commercial aquaculture.