水稻类受体激酶调控纹枯病抗性的分子机理及应用研究

Rice sheath blight is one of the most important diseases of rice. Rice resistance to sheath blight (SB) is typically controlled by quantitative trait loci. So far there are no reports on cloning of rice genes with high SB resistance. Based on theory of basal disease resistance, we used transcriptomic analysis and RNA interference (RNAi) transgenic approach to investigate 186 rice genes that were responsive to SB infection and potentially related to the pathogen-associated molecular pattern (PAMP）-triggered immunity (PTI). RNA transgenic rice plants with significant knockdown expression were generated for 109 of the SB-responsive genes. RNAi rice plants of 50 candidate genes were inoculated with R. solani, and the SB resistance gene OsSBR1 encoding a receptor-like kinase was identified. This project will focus on functional characterization of the OsSBR1 gene. We will perform transcriptomic and proteome analysis of the OsSBR1 knockout rice plants and screen OsSBR1-interactive proteins to unveil the molecular mechanism of OsSBR1-mediated SB resistance. Moreover, we will continue SB inoculation of other transgenic rice populations to screen more SB resistance genes. Genetic interaction between OsSBR1 and other rice genes with substantial level of SB resistance will be investigated by transgenic modification of their gene expression and test of the multi-gene modification transgenic plants. The availability of gene combinations with additive resistance effect will contribute to the pyramiding breeding of SB resistance QTLs in rice.

水稻纹枯病是水稻最重要的病害之一。水稻纹枯病抗性属于典型的数量遗传性状，目前尚未有高抗纹枯病水稻基因克隆的报道。我们根据植物基础抗病反应的理论，将转录组分析与RNAi转基因技术相结合，研究了186个表现纹枯病应答反应、并可能与PTI免疫途径相关的水稻基因。获得了109个水稻基因具有显著抑制表达效果的RNAi转基因水稻材料。对其中50个基因的水稻材料进行抗纹枯病接种鉴定，筛选获得了编码类受体激酶的抗纹枯病基因OsSBR1。本项目将重点研究OsSBR1的基因功能，通过其基因敲除植株的转录组和蛋白质组分析、OsSBR1互作蛋白筛选，解析OsSBR1抗纹枯病分子机理。此外，利用上述RNAi转基因水稻群体，继续筛选抗纹枯病基因，结合转基因技术，同时调控OsSBR1和其它抗纹枯病基因的表达水平，研究其遗传互作，筛选具有加性抗病效应的基因组合，为抗纹枯病QTL聚合育种建立研究基础。

OsSBR1是前期研究中通过转录组分析筛选获得的编码重金属结构域相关蛋白的水稻纹枯病抗性相关基因。本项目对OsSBR1基因及编码蛋白进行研究。与未转化受体水稻比较，OsSBR1基因编辑突变株系、RNAi抑制表达株系对纹枯病菌表现抗病，过表达株系则感病，说明OsSBR1负调控水稻纹枯病抗性。从纹枯菌侵染24h的OsSBR1突变株系和未转化水稻的组织，检测到纹枯菌转录物并鉴定出266个纹枯菌差异表达基因，其中多个基因与纹枯菌致病性相关，因此抗感水稻使纹枯菌基因呈差异表达，从而验证了OsSBR1调控纹枯病抗性的结果。几丁质和鞭毛蛋白处理OsSBR1突变体，导致活性氧（ROS）含量显著上升，说明OsSBR1可能通过与PTI和基础抗病反应来调控水稻抗病性。对纹枯菌侵染的OsSBR1突变株系及未转化水稻进行比较转录组分析，空白处理和接种处理分别有851个和2341个差异基因，包括茉莉酸信号途径相关基因、乙烯生物合成途径相关基因以及乙烯转录因子。根据GO和KEGG富集分析结果，差异表达基因在光合作用、氧化还原过程、信号转导途径、代谢途径、次级代谢生物合成通路有显著富集。在接种纹枯菌条件下，OsOSBR1-RNAi株系和未转化水稻的茉莉酸和脱落酸含量均呈升高趋势，但RNAi株系上述激素的含量低于未转化水稻。根据RNAi株系和未转化水稻接种样品的代谢组分析结果，水杨酸在RNAi株系中显著上调。因此，植物激素参与OsSBR1对水稻纹枯病抗性的调控。根据酵母双杂交和双分子荧光互补实验结果，OsSBR1与已发表的调控稻瘟病抗性的OsGF14b和OsGF14f水稻蛋白发生互作；用4个稻瘟病菌系接种水稻，OsSBR1-RNAi株系比未转化水稻显著抗病，说明OsSBR1还调控水稻稻瘟病抗性。本项目通过基因编辑和RNAi转基因方法创制的水稻抗病种质材料，可望在水稻纹枯病和稻瘟病的抗性育种中得到应用。