CoCoSF调控的可变剪接在植物冷胁迫下的分子作用机制研究

The studies on molecular mechanisms of plants in response to low temperature are focused on gene transcription and regulation, and however, little is known about the roles of RNA splicing in the process. In our previous investigation, transcriptome-scale analysis showed that more than 67% of expressed splicing factors in Arabidopsis thaliana and Populus trichocarpa are up-regulated under chilling treatment for 24 hours, novel splicing events emerge in large numbers and especially, the number of differentially AS events is rapidly increased to more than 2100, altogether demonstrating that AS events are largely activated by low temperature in both A. thaliana and P. trichocarpa. Further studies identified two splicing factors of CoCoSF(Conserved and Cold-induced highly expressed Splicing Factors), which are conserved in A. thaliana and P. trichocarpa and quickly responded and continuously induced at a high expression level under chilling treatment. Accordingly, we hypothesize that CoCoSF are involved in the adaptation of plants to low temperature by regulating AS events of cold-resistant transcripts. In the proposal, by high-throughput sequencing and bioinformatics analyses in A. thaliana and P. trichocarpa, we will investigate CoCoSF-regulated AS events and CoCoSF-binding motifs to reveal the molecular mechanism that CoCoSF regulates AS events of transcripts in response to cold stress. Finally, by integrating phenotypes of wildtypes, CoCoSF knockout mutants and reversible mutants under chilling treatment, we will try to decipher the molecular roles of CoCoSF-regulated AS events in response to low temperature, which probably contribute to selecting genotypes with superior performance under low temperature conditions and engineering cold-tolerant crops and trees.

植物响应低温胁迫的分子机制研究主要集中于基因转录调控，较少涉及RNA可变剪接。我们发现植物在低温胁迫下，可变剪接响应异常活跃：毛果杨和拟南芥在冷处理24小时后，大多数剪接因子(>67%)上调表达，同时涌现出大量新的剪接事件；并且，超过2100个剪接事件发生了显著性差异变化。进一步分析，在毛果杨和拟南芥中筛选到两个保守的、快速响应冷胁迫且持续高表达的剪接因子CoCoSF。申请人推断毛果杨和拟南芥在冷胁迫诱导下具有保守的分子作用机制：剪接因子CoCoSF调控下游基因的可变剪接，产生与冷适应性相关的关键剪接异构体。据此，本项目拟寻找CoCoSF靶RNA及其调控的剪接事件，揭示CoCoSF调控的分子剪接机理，并结合野生型、CoCoSF缺失型突变体和恢复型植株的冷逆境表型差异，阐述CoCoSF在植物低温胁迫下的分子作用机制过程，为培育抗低温作物和林木提供分子理论基础。

经过长期自然选择，植物进化出了多种基因表达逆境分子适应机制。基因表达的调控具有多个层次，如基因水平、转录水平、转录后水平、翻译水平和翻译后水平等。本项目围绕在逆境胁迫下（特别是低温），被子植物转录因子—剪接因子的分子作用，开展植物的抗逆性分子机理及其进化研究，主要揭示了被子植物在低温胁迫下的基因转录和转录后调控的作用及其分子进化。目前，获得了以下结果：①揭示低温相关的AP2/ERF基因家族在全球大降温时期趋同扩增。AP2/ERF家族是冷响应最强烈的转录因子家族，含有冷响应通路的关键转录因子CBFs，分子系统发育证实在全球大降温时期（如K-Pg期间和晚新生代冰期），该家族在被子植物中发生了趋同进化；②揭示CBF冷响应转录因子起源及其调控网络在被子植物中的进化。研究发现CBF产生于祖先被子植物，经历两次革新，在全球大降温时期又发生了显著性扩增，低温基因表达调控网络随之被重塑和升级；③揭示低温胁迫下生物节律核心因子介导的层次式基因调控网络。我们发现生物节律核心因子CCA1/LHY, RVE7, RVE4和RVE6，其转录和转录后的可变剪接在低温胁迫下都发生了差异化调控；并建立植物在低温胁迫下的基因转录—转录后的层次式调控网络；④揭示剪接因子SCL30在低温胁迫下剪接调控和分子作用机制：我们发现SCL30基因过表达可导致植株冷敏感，其调控了生物节律核心因子LHY和其他冷相关基因ICE2, COR15A和CP29的可变剪接，进而介导冷耐受过程；⑤被子植物转录因子—剪接因子及其调控网络在全球大降温时期的共进化。在全球大降温时期，和低温应答相关的转录因子（如CCA1/LHY、RVE4/8、CBFs）和剪接因子（如GRP7/8）发生了共进化，这些转录因子、剪接因子的共进化重塑和升级了冷响应基因转录和转录后的调控网络。综上，这些研究系统性拓展了我们对被子植物在低温胁迫下的分子响应及其适应进化的认识，并为农林作物抗逆性品种的培育，提供了一批抗寒基因。