剪接因子ROA1调控植物低温胁迫应答的机制分析

Plants are immobile and bound to adapt themselves to stress conditions. Low temperature, one of the major environmental stresses for crop plants, significantly constraints the agricultural productivity by adversely affecting the growth and development of crop plants. Increasing evidences proposed that post-transcriptional alternative splicing (AS) plays a vital role in plant response to various abiotic stresses. AS generates multiple transcripts from the same genetic locus and hence produces potentially different protein isoforms for expanding proteomic diversity and functional complexity. Several cold-stress responsive genes were reported to be involved in cold signaling by AS and transcriptional regulation of splicing factors, which compose the main functional component of the spliceosome. Recently, few splicing-related RNA-binding proteins including splicing factors were reported to be involved in plant stress responses. However, the mechanisms how splicing factors control RNA processing of their target genes and the functional analysis on how AS variants regulate stress signaling are far from understanding. We have reported that splicing factor ROA1 regulates abscisic acid signaling by control AS of protein phosphatase 2C HAB1 gene, then we found quickly responsive chilling hypersensitivity due to defect of ROA1 gene, which expression can be induced by cold stress. We hypothesized that ROA1 is responsible for splicing of some vital cold-responsive genes, and this is what we try to consider in this proposal. This study of the mechanism how ROA1 involve in plant cold stress response by control AS of its targets will be important and necessary for selecting genotypes with superior performance under low temperature conditions and for the engineering of cold-tolerant crop plants.

由低温导致的逆境胁迫是农作物最主要的自然灾害之一，对农业和社会造成极大损失。位置不能移动迫使植物进化形成了多种复杂灵活的响应机制，其中选择性剪接就是植物应对低温等环境胁迫的一种重要手段。通过选择性剪接，数目有限的遗传位点可以产生无限可能性的功能性产物。参与RNA前体选择性剪接的剪接（辅助）因子，其基因自身及其靶基因也会产生复杂的选择性剪接。许多冷胁迫应答相关基因都有选择性剪接现象，并且冷胁迫也可以诱导多个胁迫应答基因发生选择性剪接，但关于选择性剪接产物的功能研究较少。本申请基于此前研究剪接因子ROA1调控植物脱落酸信号转导途径之工作基础上的新发现：ROA1基因表达受低温处理诱导，其功能缺失突变体对低温异常敏感。因此，研究ROA1参与调控冷胁迫应答可以进一步揭示选择性剪接在植物应对逆境胁迫中的重要作用，从而为培育抗低温农作物提供理论基础。

项目背景：植通过长期进化和自然选择形成了多种适应环境变化的响应机制，除了基因的表达调控植物，其中RNA的选择性剪接（又称可变剪接，alternative splicing，AS）可以既可以影响功能基因的表达水平，还可以对转录翻译产生调控，使一个遗传位点可能产生翻译成不同蛋白的多种转录本。ROA1基因突变以后导致突变体对低温异常敏感，可能影响了多个靶基因的表达调控。.主要研究内容、重要结果和关键数据：通过转录组测序和表达验证分析，解析了正常情况与低温等逆境胁迫下野生型和roa1突变体内ROA1及其靶基因的剪接和表达特征。其中，ABA3和AAO3基因是ABA生物合成的关键基因，在roa1突变体中AS增强，说明ROA1负责这两个基因的正常剪接。ABA3被低温胁迫抑制表达，但roa1突变体中其正常转录本相比于野生型水平升高。HOS15基因编码的WD-40蛋白可以参与组成PWR-HOS15-HD2C复合物通过组蛋白乙酰化修饰负调控冷胁迫诱导（COR）基因的表达，该基因表达受低温胁迫轻微抑制，但roa1突变体中发生剪接紊乱，而且低温处理条件下其正常转录本表达低于野生型。CPK1基因编码一个钙依赖的蛋白激酶，正调控植物对非生物胁迫的抗性，该基因被低温胁迫诱导上调，ROA1基因突变后导致其AS增强，但低温处理后在roa1-1突变体中的内含子保留转录本有所下降。roa1突变体与野生型在各类非生物胁迫处理前后的对比显示更多胁迫应答相关基因的差异表达，例如ABF1、F3HL、MYB33、LC6D、TGL等基因在多种具有显著抗逆性差异的植物中表现出参与调控冷胁迫应答的功能。.科学意义：鉴定这些逆境胁迫应答相关基因的功能，揭示植物生长发育和逆境胁迫响应的机理，可以为实现作物抗性育种提供一定的材料和理论基础。