拟南芥RCD1基因在RNA介导的DNA甲基化途径及胁迫应答中的分子机理研究

RNA-directed DNA methylation is one of the most important epigenetic pathways of gene expression in plant, which plays different roles in diverse biological processes, such as growth, development and stress responses. However, little is known about the detailed mechanism of RdDM so far. It has been acknowledged that RCD1 serves as a hub for essential cellular functions by interacting with many other proteins. Strikingly, loss-of-function mutant of RCD1 is quite similar to small RNA pathway mutants in terms of developmental defects and sensitivities to various stresses, suggesting that RCD1 may participate in small RNA pathways. Our preliminary results show that both siRNA accumulation and DNA methylation level are affected in rcd1 mutant, compared to those in wild type plant. As such, in this research, investigating the function of RCD1 in RdDM pathway will be the focus. To reach this objective, the rcd1 mutant will be crossed to other RdDM pathway mutants, and the resulting molecular differences will be compared between wild type and the mutants, including the accumulation of 24nt siRNA, the levels of DNA methylation and gene expression at RdDM loci, and the distributions of H3K9me2 and PolIV. Further, the genome-wide distribution of RCD1 and the effects of various stresses on these changes will also be examined. The results of this project will not only improve our understanding on the mechanism of non-coding RNA biogenesis and activity, but also contribute to dissect the underlying connection between RdDM pathway and stress responses. More importantly, per the conservation of RdDM pathway in planta, the outcomes will provide new foundation for the genetic improvement of crops against stresses.

由RNA介导的DNA甲基化是表观遗传调控的主要途径，在诸多生物学过程起重要作用，但此途径中24nt siRNA的合成降解及RdDM的作用机制尚不清楚。RCD1是细胞内众多蛋白的结合中心，其突变体表型和对胁迫的应答均与小RNA通路中一些重要基因的突变体相似，说明RCD1可能参与小RNA通路。本课题组前期研究发现：该突变体中24nt siRNA的丰度和DNA甲基化水平均较野生型发生明显变化。本项目拟研究RCD1基因调控RdDM途径的分子机制，通过创建该基因与RdDM途径基因的多重突变体，比较不同突变体中24nt siRNA的丰度、DNA甲基化水平、PolIV、H3K9me2和RCD1的分布情况，及不同突变体在相应胁迫条件下的应答模式。研究结果将进一步揭示非编码RNA的合成与作用机制，并能建立RdDM途径与胁迫应答的内在联系。鉴于RdDM通路的保守性，本研究也将为逆境作物遗传改良提供理论基础。

由RNA介导的DNA甲基化（RdDM)是表观遗传调控的主要途径，siRNA是该途径中的关键因子，其合成与作用方式依赖于植物中特有的两个RNA 聚合酶PolIV和PolV，因此该途径是植物特有的DNA甲基化方式，也是表观遗传学的重要组成部分。RdDM途径参与多种胁迫应答过程DNA甲基化对着丝粒区异染色质转座子的转录抑制作用可能是植物胁迫应答的一个重要机制。然而，此途径中24 nt siRNA的合成降解及RdDM的作用机制尚不清楚。RCD1是植物特有的蛋白，是细胞内众多蛋白的结合中心，其突变体表型和对胁迫的应答均与小RNA通路中一些重要基因的突变体相似，说明RCD1可能参与小RNA通路。RCD1是植物胁迫应答、激素通路和生长发育等过程的重要集成点，但RCD1在这些调控途径中的分子作用机制尚不清楚，且RCD1在不同途径中的桥梁联接作用未见报道。本项目拟研究RCD1基因调控RdDM途径的分子机制，探究RdDM途径与胁迫应答的内在联系。本项目的研究目标为：鉴定RCD1蛋白在RdDM途径中的新功能及作用机制；揭示24nt siRNA 代谢的调控新机制；探索RCD1参与RdDM途径与胁迫应答过程的内在联系。在本项目的资助下，利用模式植物拟南芥为实验材料，创建了RCD1与RdDM途径基因的突变体遗传材料，通过小RNA文库，DNA甲基化文库，转录组等高通量数据分析了RCD1在染色体不同区域影响24 nt siRNA和DNA甲基化的水平；利用双分子荧光互作技术鉴定了RCD1与NAC17转录因子互作并抑制其酶活；通过盐胁迫处理发现NAC17的核质定位受盐胁迫调控，揭示了NAC17参与盐胁迫响应途径的新功能。本项目研究结果不仅完善了24 nt siRNA和DNA甲基化的调控机制，并发现了一条新的植物响应盐胁迫调控途径，揭示了转录因子NAC17的新功能。鉴于RdDM通路的保守性，本研究也将为逆境作物遗传改良提供理论基础。