桑树韧皮部汁液长距离运输miRNAs结合蛋白的鉴定及功能研究

In plants, some miRNAs emerged as a non-cell autonomous information macromolecule were transported via the phloem over long distances between different tissues and organs. These miRNAs have been shown to play an important role in regulating plant growth and development as well as their adaptation to changing environmental conditions. However, the mechanisms underlying miRNAs mobility in the phloem are still poorly understood. The angiosperm phloem translocation stream contains a unique population of RNA binding proteins (RBPs), some of which function in a non-cell-autonomous manner and were implicated in the long-distance delivery of RNA molecules through the phloem. Therefore, we speculated that some RBPs in the phloem translocation stream were involved in the long-distance trafficking of miRNAs. In this study, the miRNAs that have been shown to be mobile were employed to pull-down the miRNA-binding proteins in the mulberry phloem translocation stream using the Pierce™ Magnetic RNA-Protein Pull-Down Kit. Then the proteins pulled down will be identified with LC-MS/MS and electrophoretic mobility shift assays will be carried out to verify their ability of binding miRNAs. Subsequently, the functions, post-translational modifications and proteins interacting with them will also to be explored to reveal the characteristics of the proteins acting with miRNAs and the mechanisms underlying their mediating miRNA trafficking. The information obtained in this study will provide further insights into the molecular mechanisms of miRNA mobility acting as long-distance signaling and promote the research on the molecular mechanisms for systemic gene silencing in trees.

植物韧皮部汁液中非细胞自主性miRNAs可以作为系统信号在组织或器官间传递信息，在调控生长发育和响应环境胁迫等方面具有重要作用，但韧皮部miRNAs运输的调控机制还不清楚。植物韧皮部汁液中具有非细胞自主性的RNA结合蛋白在RNA运输过程中具有重要调节作用，我们推测有些RNA结合蛋白在miRNAs长距离运输过程中也具有调控作用。本课题拟以桑树韧皮部汁液中可以长距离运输的miRNA为诱饵分子，利用RNA-Protein Pull-Down Kit分离韧皮部汁液中的miRNA结合蛋白，采用液相串联质谱技术对其鉴定，通过Super-shift EMSA实验验证其与miRNA的结合能力，结合对其生物功能、翻译后修饰和相互作用蛋白的分析，明确其与miRNA的结合特性及调节miRNA运输的机制，以期为全面揭示植物miRNA长距离传导的分子机制奠定基础，对于林木RNAi的系统性传播机制研究具有重要意义。

本项目以可长距离移动的miR172作为诱饵分子捕获韧皮部汁液蛋白，利用液相串联质谱技术鉴定到18种miR172结合蛋白，其中有4种蛋白质含有信号肽，并具有RNA结合功能域。克隆了其中2种蛋白MuL484和MuL5860的基因，EMSA试验结果表明该两种蛋白与可移动miRNAs的结合具有普遍性，拟南芥微嫁接试验证明MuL484和MuL5860蛋白在植物体内可以长距离双向运输。通过对MuL484和MuL5860基因的启动子的组织和诱导表达活性分析表明，两种基因在植物生长发育和环境胁迫应答过程中可能具有一定作用。生物学功能研究结果表明MuL484具有促进植物发育和提早开花的作用，另外，该基因可能负调控植物对逆境的抗性。而MuL5860基因对植物的生长发育可能具有负调控作用，但该基因在拟南芥中表达可以提高转基因植株盐胁迫条件下种子的萌发率及促进根的生长，提高植株对甘露醇和ABA的耐受性，增强植株对盐分和干旱胁迫的抗性。利用酵母双杂交技术筛选鉴定到13种MuL5860互作蛋白质，并利用酵母双杂交技术和双分子荧光互补实验验证了MuL5860与Mul-CAC及Mul-CSN5A蛋白的互作关系。生物信息学分析表明MuL484和MuL5860蛋白质存在多个潜在的磷酸化和乙酰化修饰位点， 通过提取转MuL484基因和转MuL5860基因拟南芥植株蛋白质，分离了MuL484和MuL5860蛋白质并进行了磷酸化位点的质谱鉴定，在MuL484蛋白质中鉴定到8个磷酸化位点，在MuL5860蛋白中鉴定到7个磷酸化位点。研究结果初步探明MuL484和MuL5860蛋白质与miRNA的结合特性及调节机制，揭示了MuL484和MuL5860蛋白介导的miRNA长距离传导机制，为全面揭示植物miRNAs信号分子长距离传导的分子机制奠定基础，对林木RNAi的系统性传播机制研究具有重要意义。