马铃薯青枯病防卫基因的二次上调表达与系统获得抗性的关系研究

Potato is the fourth most important food crop in China while the bacterial wilt disease caused by Ralstonia solanacearum inflicts very serious losses everywhere potatoes are grown. It is urgent to understand the molecular mechanism of the interaction between potato and pathogen for the molecular breeding work on resistance against the pathogen. In the previous study, We identified several important defense genes by transcriptomical analysis, and found that some of these genes had a secondary upregulation expression during the interaction between potato and R. solanacearum. The purpose of this project is to further verify the secondary defense characteristics of several defense genes whose expression could be elicited by R. solanacearum and abiotic elicitor such as salicylic acid (SA) and piperaconic acid (Pip). The study would be focused on the relationship among the upregulation of these defense genes and the niche specificity of the bacteria and the Systemic acquired resistance (SAR) in potato system. Firstly, three experiments, the elicitor treatment, the signal substance accumulation, transcriptional coactivition of the defense gene and the defensive regulatory protein NPR1, will be carried out. Secondly, we will analyze the temporal and spatial association of the primary and secondary upregulation of these defense genes, in which the niche construction involved. Finally, we will identify the presence of SAR by Inoculation of Bacteria on the transgenic plant and transient expression of the defense genes in tobacco. The relationship may be derived to provide theoretical basis and new ideas for the effective use of defense genes to improve the resistance of bacterial wilt in potato.

马铃薯是我国第四大粮食作物，青枯病是严重危害其生产的病害之一。培育抗性品种是防治青枯病的有效途径，当前急需了解抗病分子机制，为育种工作提供理论基础。我们前期结合病原生态位模型，在转录组水平上筛选出几类重要防卫基因，并且发现其中一些基因在马铃薯与青枯菌互作的过程中存在“二次上调表达”的现象。本项目拟结合生物激发子即青枯菌和非生物激发子水杨酸（SA）和哌可酸（Pip）的诱导，深入验证多个防卫基因的上调特性；着重解析这些基因表达的上调特性与青枯菌生态位的特异性以及马铃薯系统抗性三者之间的关系。通过分析激发子诱导的防卫基因表达、信号物质积累、防卫基因与防御调节蛋白NPR1的转录共激活状态，解析这些基因的初次上调和二次上调与青枯菌生态位的时空关联，继而通过转基因烟草瞬时表达和外源接种病原物来鉴定其与马铃薯系统获得抗性（SAR）的关系。研究结果将为有效利用防卫基因来改良青枯病抗性提供理论依据。

结合上一个项目中病原生态位模型，在转录组水平上筛选出3个重要防卫基因，针对其中一些基因在马铃薯与青枯菌互作的过程中存在“二次上调表达”的现象，本项目结合生物激发子即青枯菌和非生物激发子的诱导，着重解析这些基因表达的上调特性与青枯菌生态位的特异性以及马铃薯系统抗性三者之间的关系。主要内容包括通过分析激发子诱导的防卫基因表达、信号物质积累、防卫基因与防御调节蛋白NPR1的转录共激活状态，解析这些基因的初次上调和二次上调与青枯菌生态位的时空关联；继而通过转基因过表达和VIGS介导的烟草瞬时表达和外源接种病原物来鉴定其与马铃薯系统获得抗性（SAR）的关系；并通过酵母单双杂交分析基因上游转录激活调控和基因产物互作对象来确定其分子互作。重要结果有（1）克隆分析了30个基因的结构特征与表达模式，确证了部分基因在青枯病发病过程中的二次上调特征；（2）阐明了StMBF1c的二次上调表达的功能机制：发现StMBF1c通过自身二次上调表达应答青枯菌并作为正向调节子协同有关信号分子和抗性标志基因参与马铃薯青枯病抗性，其中涉及ETI、PTI和与StTPS5蛋白的互作；这种二次上调属于两次独立的诱导上调；（3）据我们所知，首次报道并初步解释了二次上调与获得抗性的关系：这些基因表达的二次上调特性与青枯菌生态位的特异性以及马铃薯系统获得抗性三者之间的关系表现在与病程发展的时间轴相关。也就是青枯病菌的种群动态结果表明二次上调的时间点与病原菌在茎中繁殖的最高浓度的时间相吻合。存在不同的效应因子，分别在两个时间点激发依次通过PTI和ETI激发了寄主植物马铃薯的系统获得抗性；（4）发现上述基因表达过程中存在与枢纽效应子RipAE和RipO1有关联，暗示基因产物可能是其靶对象。上述结果将为有效利用防卫基因来改良马铃薯青枯病抗性提供理论依据。另外提出了一个关于RipTALI-1激活的基因功能及应用的新问题。