拟南芥SY1蛋白抑制逆境基因表达的分子机理研究

Molecular mechanisms of gene repression by a novel gene inhibitor, SY1, will be studied in th project. In our preliminary research, a gene encoding a transcriptional repressor was identified through EMS mutagenesis, mutant screening by a salt-inducible LUC reporter gene system, and map-based cloning. The gene was designated SY1 ( abbreviated from SHINY1) because in sy1 mutant the LUC reporter gene was activated and showed higher signals than the wild type.SY1 expression is inhibited by stresses (such as salt and drought) and ABA treatment. Furthermore, in sy1 mutant, the assymetric cytosine methylation was reduced. Phenotypically, sy1 was more tolerant to ABA in seed germination than wild type and when growing at low temperature, sy1 mutant plants displayed slow growing and early senescence of leaves compared to wild type plants. SY1 protein contains five KH domains and is localized in the nucleus. Yeast two-hybrid system analysis and bi-molecular fluorescence complementation assay showed that SY1 is able to interact with FRY2/CPL1, another gene inhibitor reported previously. FRY2, a CTD phosphatase,is able to interact with DMS3, a general factor for PolV transcription in RdDM (RNA-directed DNA Methylation). Hence we suggest that SY1 and FRY2 are specific factors for RdDM pathway to regulate the expression of some stress-inductive protein-encoding genes. Under non-inductive conditions, SY1 interact with FRY2 to prompt PolV transcription thus increasing the de novo methylation of related DNA sequences and resulting in gene repression. This project aims to verify the hypothesis by further experiments. We will identify the target genes repressed by both SY1, FRY2 and PolV, analyse the correlation between target gene activation and DNA methylation, and find out the functional associations among SY1, FRY2 and PolV through real-time qPCR ,bisulfite sequencing, protein co-immuno precipitation, chromatin immunoprecipitation,etc. The aim of the project is to elucidate the role and mechanisms of SY1 and FRY2 in DNA mehtylation and gene repression. The proposed research will elucidate the role and molecular mechanisms of SY1 and FRY2 in the regulation of repression of stress-inducible genes and provide us with important information on functions of cytosine methylation in plant adaptation to environment.

本项目拟研究一种新的基因抑制因子SY1抑制逆境基因表达的分子机理。前期研究发现：SY1表达受盐等逆境抑制；sy1突变体中，逆境基因SOT12的启动子驱动的LUC报告基因的表达被激活,启动子CHH位点的甲基化水平下降；蛋白相互作用分析发现SY1能与基因抑制因子FRY2结合，FRY2能与RdDM途径中PolV的基本转录因子DMS3结合。因此，我们推测SY1通过与FRY2等因子发生作用，参与PolV在某些逆境基因位点的转录，促进DNA甲基化，从而在非诱导条件下抑制逆境基因的表达。本项目拟通过突变体表型分析、SY1与FRY2、NRPE1共同靶基因的鉴定、靶基因的表达与甲基化分析、蛋白因子SY1、FRY2、NRPE1的相互作用分析及这三种蛋白因子与靶DNA的结合分析，揭示SY1在抑制逆境基因表达中的功能。项目对阐明逆境基因的表达调控机制、理解DNA甲基化在植物逆境应答与适应中的功能具有重要意义。

基因抑制因子抑制逆境基因在非诱导条件下的表达并平衡诱导条件下的激活水平，在逆境基因的表达调控中起着重要作用。本项目以逆境基因抑制因子SY1的突变体（sy1/sy30）为材料，研究SY1等在逆境中的功能及其调控逆境基因表达的分子机制，获得了以下进展：一、SY1（含KH结构域的RNA结合蛋白）是拟南芥耐盐、碱、渗透等非生物胁迫所必需的，其功能丧失导致拟南芥对这些逆境的抗性减弱。与sy1来自同一个突变体筛选系统（Psot12-LUC报告基因，突变体中LUC激活）的FRY2、HDA6的突变体fry2/sy29也有类似sy1的表型。FRY2与SY1蛋白存在着相互作用，转录组测序分析检测到199个逆境基因在sy1、fry2突变体中均表达上升，包括RLP激酶家族蛋白、AP2/ERF、WRKY、MYB等多个家族的转录因子，它们在功能上与逆境反应中的信号转导、对逆境的耐受性有关，说明SY1、FRY2在抑制逆境基因的表达方面存在着协同作用。二、SY1参与ABA信号途径的调节。与野生型相比，sy1种子萌发对ABA敏感，而SY1互作蛋白的突变体fry2/sy29对ABA耐受。ABA信号途径的关键基因在两个突变体中均有变化，如AFP2、DEAR1、EDL1、RD29A、MYB96、NAC019、ZF2等在sy1、fry2中均表达上升，但ABA合成途径的关键基因NCED3仅在fry2中上升。另外，SY1、FRY2均有自己特异的互作蛋白。这些结果说明SY1、FRY2既共同调控某些逆境基因的表达，又在各自的特异性复合物中调控特异性靶基因的表达。三、SY1参与拟南芥对生物逆境反应的调节。sy1及其互作蛋白基因的突变体fry2/sy29对细菌性病原PstDC3000敏感，WRKY家族转录因子如WRKY18、WRKY33、WRKY40、WRKY60等在两个突变体中的表达均上升，启动子占用分析发现SY1、FRY2能够结合靶基因的启动子区域，调控靶基因的表达。与sy1、fry2相反，同系列筛选到的HDA6突变体hda6/sy1502呈现组成型病原反应，sy1502中大量病原反应基因被激活，对PstDC3000的抗性比野生型强。HDA6能够结合在病原反应基因的启动子区域，通过降低组蛋白酰基化水平抑制靶基因表达。这些进展为理解逆境基因的表达调控机制提供了重要依据，也为后续研究提出了新的科学问题。