大豆热激转录因子GmHsf-25在响应疫霉菌侵染过程的功能和调控机制研究

In this project, we will identify the possible function and regulatory mechanism of heat shock transcription factor GmHsf-25 involved in soybean–Phytophthora sojae interaction. Based on the digital gene expression profiling database of soybean transcriptome constructed in our group, we previously found that GmHsf-25 was transcriptionally induced by P. sojae infection, suggesting that it could play a role in regulating soybean-P. sojae interaction. To dissect the exact function and regulatory mechanism of this gene, soybean-P. sojae and tobacco-P. nicotianae interaction systems will be employed, and transgenic taobacco and soybean plants expressing sense and antisense GmHsf-25 will be generated in this work. The biological functions of GmHsf-25 during plant-Phytophthora interaction will be investigated at gene, protein, cell and tissue levels. The molecular mechanism of GmHsf-25 contributing to plant-Phytophthora interaction will also be examined by identifying their DNA binding elements, target genes and interaction proteins by ChIP-seq, CO-IP and further confirmed by RT-PCR, yeast hybrid, EMSA and Pull-down assays. The completion of this project will not only reveal the biological function of GmHsf-25 and the molecular mechanism of plant-Phytophthora interaction, but also provide research bases for genetic engineering of soybean with improved resistance to P. sojae.

本项目拟开展大豆中转录因子GmHsf-25在大豆和大豆疫霉互作过程中的功能和调控机制研究。申请者前期筛选分析发现GmHsf-25受大豆疫霉侵染诱导表达，推测其可能参与大豆响应疫霉菌侵染过程的调控。我们拟利用大豆-大豆疫霉、烟草-烟草疫霉互作系统，分别在大豆中和烟草中过表达和抑制表达GmHsf-25来改变其转录调控网络，并从基因、蛋白、细胞、组织等方面阐明其在互作过程中的功能。然后通过ChIP-seq、酵母杂交、CO-IP等蛋白-DNA、蛋白-蛋白互作试验寻找GmHsf-25结合的DNA元件、靶标基因及其互作蛋白，并通过验证GmHsf-25对抗大豆疫霉相关基因的转录调控，综合分析揭示GmHsf-25在响应大豆疫霉侵染过程的转录调控网络。研究结果不但可以阐明大豆中GmHsf基因在与疫霉菌互作过程中的生物学功能及其分子机制，还将为设计改良大豆对大豆疫霉抗性的分子育种策略提供理论依据。

基于大豆与大豆疫霉互作不同时期的数字基因表达谱数据挖掘大豆中积极响应疫霉菌侵染的转录因子，明确其生物学功能和调控响应疫霉菌侵染的分子机制是本项目研究的重点。项目组首先基于大豆-大豆疫霉互作数字基因表达普数据库挖机大豆中响应疫霉菌侵染的转录因子，发现多个Hsf转录因子积极响应大豆疫霉菌的侵染，其中GmHsf-25受大豆疫霉侵染强诱导表达。本项目通过不同处理条件下GmHsf-25的转录分析，发现其不仅响应疫霉菌侵染，对多种激素处理都有积极响应；通过荧光蛋白标记明确其亚细胞定位于细胞核；进一步利用大豆-大豆疫霉、拟南芥-辣椒疫霉、烟草-烟草疫霉互作系统结合转基因技术，明确了其在植物与疫霉菌互作中的起积极作用，即GmHsf-25过表达可使植物抵御疫霉菌侵染的能力提高；通过免疫沉淀试验结合质谱分析筛选挖掘GmHsf-25候选互作蛋白，发现多个蛋白参与细胞内氧化反应；通过不同植物中关键信号通路分析、结合大豆-大豆疫霉互作DGE数据、启动子区元件分析挖掘调控网络相关基因，发现GmHsf-25调控网络涉及多条抗病信号途径，并且与胞内活性氧的合成和清除密切相关。本研究初步探明GmHsf-25的生物学功能及其调控网络模型，阐明大豆GmHsf-25在响应疫霉菌侵染过程的转录调控机理，为设计高效抗病育种方案和植物疫病防控策略提供理论依据。