拟南芥热激因子AtHsfA1a对细胞程序性死亡的调控

Arabidopsis thaliana heat shock transcription factor AtHsfA1a is an important factor on regulation of stress tolerance. Programmed cell death (PCD) is a common phenomenon under stresses. Regulation of AtHsfA1a on PCD was found. However, detailed regulation mechanisms of Arabidopsis thaliana AtHsfA1a on PCD are not clear. In this study, Arabidopsis transgenic plants overexpressing AtHsfA1a and different knockout mutant of Arabidopsis AtHsfA1 class members will be used as experimental materials. Influence of AtHsfA1a on Arabidopsis PCD will be analyzed by comparing the morphological and biochemical features at the physiological level, which will reveal independent or collaborative regulatory modes of AtHsfA1a on PCD. To study direct or indirect regulatory pathways of AtHsfA1a on PCD, PCD-related genes expression of different Arabidopsis genotypes will be selected by using gene chip. The binding of AtHsfA1a to the promoter regions of PCD-related genes will be investigated by using chromatin immunoprecipitation and electrophoretic mobility shift assay. Further, relationship of AtHsfA1a and PCD-related genes will be studied through constructing expression vectors of the promoter mutation/reporter gene to show that new target genes of AtHsfA1a involve in PCD regulatory. These studies are important for understanding the mechanism of plant stress tolerance.

拟南芥热激因子AtHsfA1a是与耐逆境有关的主要转录调控因子，细胞程序性死亡(PCD)是逆境中普遍存在的现象,前期研究发现AtHsfA1a对 PCD有调控,但是AtHsfA1a对PCD调控机制还不完全清楚。本项目拟采用过量表达AtHsfA1a及与AtHsfA1a相关的基因敲除突变型的拟南芥为材料，从细胞形态和生理生化水平来研究AtHsfA1a对逆境中PCD的影响，揭示AtHsfA1a对PCD独立或协同的调控模式；为了研究AtHsfA1a对PCD相关基因直接或间接的调控途径，拟利用基因芯片技术筛选PCD相关基因，采用染色质免疫沉淀及电泳迁移率变动技术分析AtHsfA1a与PCD相关基因启动子区的结合情况，通过构建启动子突变的报告基因表达载体鉴定AtHsfA1a与PCD相关基因表达的关系，揭示AtHsfA1a 新靶基因及其参与逆境中PCD调控的新途径，该研究对认识植物耐逆境机理有重要意义。

为了研究拟南芥热激因子AtHsfA1a对PCD的调控机制，本项目采用过量表达AtHsfA1a及与AtHsfA1a相关的基因敲除突变型的拟南芥幼苗为材料，利用DAPI法从细胞形态水平研究AtHsfA1a对逆境中PCD的影响，研究发现AtHsfA1a对热胁迫中PCD形态有影响；采用分光光度法从生理水平研究AtHsfA1a对高温、低温、过氧化氢及PEG渗透胁迫下Caspase-3酶活性和过氧化氢含量的影响，结果表明逆境胁迫下拟南芥AtHsfA1a能够抑制Caspase-3蛋白酶活性和过氧化氢含量增高，且AtHsfA1b和AtHsfA1d共同对AtHsfA1a的抑制作用有影响；采用凝胶电泳法从分子水平研究AtHsfA1a对高温、低温、过氧化氢及PEG渗透胁迫下DNA的影响，结果发现逆境处理后DNA片段化程度与AtHsfA1a呈负相关，说明AtHsfA1a能抑制DNA片段化，AtHsfA1a的抑制作用与AtHsfA1b和AtHsfA1d共同作用相关，于是初步推测AtHsfA1a与AtHsfA1b和AtHsfA1d协同作用，通过抑制Caspase-3蛋白酶的活性、过氧化氢含量增高和DNA片段化程度而对PCD起抑制作用。为了研究AtHsfA1a对PCD相关基因直接或间接的调控途径，利用基因芯片技术筛选AtHsfA1a与PCD相关基因的靶基因，针对基因芯片结果用倍数法进行差异基因的筛选、用GO-Analysis进行差异基因显著靶向性功能分析、用Pathway-Analysis进行差异基因参与的信号转导通路分析、用Signal-Net进行基因间相互作用网络构建，与PCD信号转导途径相结合，筛选出热激因子AtHsfA1a与PCD相关的靶基因。采用染色质免疫沉淀及电泳迁移率变动技术体内外分析AtHsfA1a与PCD相关基因启动子区的结合情况，结果表明AtHsfA1a能够与CSD1，CSD3，GPX，MDHAR，CAT3，AtBI-2，STZ/ZTA 10， EREBP-4 ，Caspase-3, TNFR1, Fas 等基因启动子区结合，调控这些基因的表达，从而抑制PCD。通过构建启动子突变报告基因表达载体鉴定AtHsfA1a与PCD相关基因CSD1表达的关系，但此结果还有待于进一步研究。该研究揭示了AtHsfA1a靶基因及其参与逆境中PCD调控的途径，对认识植物耐逆境机理有重要意义。