WRKY转录因子在岷江百合应对尖孢镰刀菌防卫反应中的功能研究

A pathogenic fungus, Fusarium oxysporum f.sp. lilii, causes wilt disease in lilies, which endangers both the fresh cut flower and bulb productions worldwide. Lilium regale Wilson is a local lily species of China, moreover, it is a kind of rare wild plant resource with a high level of resistance against F. oxysporum. Therefore, L. regale was widely used as material in lily breeding for disease resistance, however, the molecular mechanism on resistant defense response of L. regale is still unclear, especially for the regulation mechanism of L. regale resistance to F. oxysporum. The WRKY transcription factors are key regulators of plant defense response, and the gene family of WRKY transcription factor will be isolated from L. regale in order to elucidate the molecular mechanism of WRKY transcription factors in defense response to F. oxysporum. Then the expression pattern and trans-activation of WRKY genes will be analyzed, and the regulation of pathogenesis-related protein gene expression by WRKY transcription factors will be studied through transient expression assay. In addition, the interacting proteins of WRKY transcription factors will be fished and confirmed using yeast two-hybrid system and bimolecular fluorescence complementation assay, and the function of L. regale WRKY genes during response to F. oxysporum infection will be obtained using the reverse genetics techniques. Furthermore, the molecular defense network regulated by L. regale WRKY transcription factors will be revealed by gene-chip analysis. The present project aims to elucidate the molecular mechanism on regulation of resistant defense response in L. regale, and implement of this project will be helpful in further understanding the regulation of defense response in L. regale and resistant mechanism of L. regale against F. oxysporum at the molecular level. Besides, the WRKY genes from L. regale will be very useful for developing novel lily cultivars and resources with strong resistance to F. oxysporum.

由病原真菌尖孢镰刀菌引起的百合枯萎病是危害百合鲜切花生产及种球繁殖的主要病害，严重制约了我国百合产业的持续发展。我国的百合特有种岷江百合是高抗尖孢镰刀菌的珍稀野生资源，已应用于百合抗病育种，然而岷江百合抗病防卫反应的基础理论研究相对滞后，尤其是岷江百合应对尖孢镰刀菌入侵的抗性调控机制仍知之甚少。WRKY转录因子是植物抗病防卫反应的重要调控因子，本项目拟克隆岷江百合WRKY转录因子基因家族，分析其表达特性及反式激活的能力，研究其对病程相关蛋白基因转录活性的调控，分离与其相互作用的蛋白质，验证岷江百合WRKY转录因子在抵抗尖孢镰刀菌侵染过程中的作用，并解析其调控的分子防卫网络，最终阐明岷江百合WRKY转录因子响应尖孢镰刀菌侵染的分子机理。本项目的实施有助于深入认识岷江百合抗病防卫反应的调控机理及其抗尖孢镰刀菌侵染的分子机制，为利用岷江百合培育抗性百合新品种和新材料提供理论和物质基础。

由病原真菌尖孢镰刀菌引起的百合枯萎病是危害百合鲜切花生产及种球繁殖的主要病害，而我国的百合特有种岷江百合高抗尖孢镰刀菌。WRKY转录因子是植物抗病防卫反应的重要调控因子，为了揭示WRKY转录因子在岷江百合抗尖孢镰刀菌防卫反应中的作用，本项目通过岷江百合受尖孢镰刀菌侵染过程的转录组测序分析，得到了35个WRKY转录因子基因。聚类分析将岷江百合WRKY分为I、II和III三大类，第II类又进一步分为五个亚类。水杨酸、茉莉酸甲酯、乙烯利和过氧化氢四种信号分子处理和尖孢镰刀菌侵染均能上调一些LrWRKY基因的表达。LrWRKY1定位于植物细胞核，其原核重组蛋白能结合包含w-box的DNA序列，在酵母细胞中表达的LrWRKY1反式激活岷江百合枯萎病抗性基因LrPR10-5的表达，并且LrWRKY1过表达增强了转基因烟草对尖孢镰刀菌的抗性。此外，细胞核定位蛋白LrWRKY2在烟草中过表达增强了对尖孢镰刀菌的抗性，同时一些茉莉酸生物合成、病程相关蛋白、超氧化物歧化酶基因的表达水平明显上升。相反地，在岷江百合鳞片中瞬时表达LrWRKY2的RNAi片段，降低了对尖孢镰刀菌的抗性。LrWRKY2原核重组蛋白特异结合抗病基因LrCHI2的启动子序列，并激活LrCHI2启动子的转录，可见LrWRKY2通过正调控几丁质酶基因LrCHI2的表达介导岷江百合对尖孢镰刀菌的防卫反应。LrWRKY3也是一个细胞核定位蛋白，LrWRKY3转基因烟草对尖孢镰刀菌的抗性高于野生型烟草，且一些茉莉酸生物合成、水杨酸信号转导和抗病相关基因在LrWRKY3转基因烟草株系中的转录水平明显上升，然而，瞬时表达LrWRKY3 RNAi载体的岷江百合鳞片对尖孢镰刀菌表现出更高的敏感性。此外，从岷江百合中分离鉴定了一个枯萎病抗病基因LrDef1，并发现LrWRKY3通过调控防御素基因LrDef1的表达参与岷江百合对尖孢镰刀菌的防卫反应。还通过酵母双杂交获得了45个可能与LrWRKY3转录因子互作的蛋白。本项目揭示了岷江百合与枯萎病菌分子互作过程的正调节因子WRKY的转录调控机制，有助于深入认识岷江百合抗病防卫反应的调控机理及其抗尖孢镰刀菌侵染的分子机制。