ESB1基因在植物对灰霉病抗性中的分子机制研究

Botrytis cinerea causes severe disease both in the field and in postharvest situations, resulting in enormous economic losses. To seek resistant genes, and grow disease resistant crops is the most effective and environment-friendly strategy for protecting the crop from grey mould disease. In our previous research, we screened mutants from a T-DNA insertion-mutagenized Arabidopsis population. A mutant with enhanced susceptibility to Botrytis was identified, named esb1. The further analysis showed that esb1 mutant was not only susceptibility to Botryti stress, but also impaired tolerance to water deficit and increased salinity. The insertion of T-DNA suppresses the expression of ESB1 gene in esb1 mutant according to RT-PCR analysis. DNA sequence analysis indicates that ESB1 encods a hypothetical protein. And its function remains unclear. The result of transformation ESB1 gene to the mutant showed that ESB1 gene could recovery its susceptibility to Botrytis mutant phenotype into the phenotype same as the wild type. This result showed that ESB1 gene is essential for resistance to gray mould disease. Those mentioned results indicated that ESB1 gene probably involved in signal transduction of plant response to biotic and abiotic stresses. And this project plan to conduct the following research based on the above experimental data: 1. Analysis the expressional pattern of ESB1 gene, in order to determine the period and location of ESB1 gene working during interaction between plant and pathogen. 2. Explore the panorama of how ESB1 gene mediates expression of related genes in response to infection of Botrytis. 3. Find the signaling pathway components which interaction with ESB1 gene and identifying which pathway that ESB1 involved. Our work will provide insights into the molecular mechanisms of Arabidopsis defense against pathogen and facilitates the development of resistant crops to control this devastating pathogen.

灰霉菌对农作物生长和储运均有重大危害，寻找具有抗性的基因并培育抗性作物甚为重要。本项目前期研究已从拟南芥的T-DNA插入群体中筛选到一个对灰霉菌及干旱和盐渍均敏感的突变体，命名为esb1。RT-PCR分析表明T-DNA插入突变导致ESB1基因转录抑制。DNA序列分析表明该基因编码一个功能未知的推定蛋白。导入野生型基因，能够互补灰霉病敏感的突变性状，证明该基因是灰霉病抗性所必需的。前期研究结果表明ESB1基因可能参与了植物对生物和非生物胁迫反应的信号转导。本项目拟在上述基础上，利用分子生物学和生理学技术开展以下研究：1.分析该基因的表达模式，确定该基因在植物与灰霉菌互作中发挥作用的时期和部位。2.研究该基因对灰霉菌反应相关基因表达调控的全景图像。3.寻找与其互作的其它组分，探索其参与的信号转导途径。本研究能深入认识ESB1基因调控抗灰霉病的分子机制，对于利用基因工程提高作物抗病性具有意义。

灰葡萄孢菌（Botrytis cinerea）被认为是世界上重要的植物致病真菌，给农业生产造成严重的经济损失。目前常规的防治效果有限，因此，寻找抗性基因，通过转基因技术培育抗性品种是防治新方向。我们从拟南芥中筛选到一个对灰葡萄孢菌敏感的突变体，命名为enhance susceptibility botrytis1（esb1），基因命名为ESB1（At4g39690），该基因编码一个推定蛋白。本项目订购了该基因的另外三个T-DNA插入突变体，构建了esb1的遗传互补和ESB1超量表达植物。对这三种类型的植物进行灰葡萄孢菌、干旱、高盐和氧化胁迫，进一步证实了ESB1的抗性功能。研究表达模式，发现ESB1在拟南芥中组成型表达，且贯穿整个生育期。但在根尖几乎不表达，而病菌和高盐则能够诱导该基因在根尖表达，qRT-PCR分析也得到相似的结果。将ESB1与GFP蛋白融合，观察到ESB1定位于细胞核，且N端1-219氨基酸具有定位作用。基因芯片表达谱分析表明，ESB1的突变影响了大量胁迫响应基因的表达，其中JA信号途径被显著聚类。用qRT-PCR方法，检测了SA、JA、ET和NO激素信号通路缺陷部分突变体中ESB1基因的表达，也检测了esb1突变体中各个激素通路相关基因的表达情况。结果表明，ESB1可能主要通过JA/ET激素介导的途径参与植物的抗性反应。本项目还开展了对灰葡萄孢菌分泌蛋白的纯化和性质鉴定，结果表明该蛋白为漆酶，对植物产生毒性。同时，发现灰葡萄孢菌的分泌蛋白能诱导拟南芥悬浮细胞和叶片发生细胞程序性死亡（PCD），证实了腐生性病原物也诱导寄主细胞发生PCD。且该过程伴有膜损伤、DNA和蛋白质裂解等。并存在活性氧、半胱氨酸蛋白酶和线粒体途径介导的PCD，提示蛋白诱导拟南芥发生的PCD在信号通路上与防卫反应有部分交叉重叠。本项目还获得了番茄的ESB1转基因植株。