香蕉野生种Pahang对枯萎病菌4号生理小种热带型的抗侵染机制研究

Bananas (Musa spp.) are very important tropical and subtropical crops. Currently, China and the global banana plantations are facing a very serious epidemic Panama disease (Fusarium oxysporum f.sp. cubense Tropical Race 4:TR4) challenge. The disease results in its devastating in banana production, a direct threat to the income of local farmers and the industry to survive. Therefore, studies on banana TR4 resistance related issues are urgently required in terms of both theoretical and application implications. On the basis of ongoing cooperation with Bioversity International, a rapid in vitro vegetative propagation system of highly resistant to TR4 wild species Musa acuminata Pahang has been developed in our research team. This project is proposed on using this highly resistant Pahang as starting material. TR4 with a green fluorescent protein (GFP) labeling will be used to monitor the process of Musa acuminata Pahang anti-blight pathogen infection. Differences in the level of cells and tissues between resistant and susceptible materials will be investigated. Also the quantification of dynamic changes of endogenous phytohormones such as jasmonic acid and salicylic acid will be analyzed in time course between plant-pathogen interactions. Using isotopic labeling relative and absolute quantitation (isobaric tags for relative and absolute quantitation (iTRAQ), anti-blight pathogen infection-related resistant proteins will be screened, identified and isolated. Afterwards candidate genes related to disease resistance will be identified and cloned. Furthermore, quantitative qRT-PCR will be applied for validation of candidate genes related to differential expression patterns upon disease challenge. At the end, the mechanism of high disease resistance of banana Pahang to TR4 will be unraveled. The outcome will provide us a solid theoretical foundation and key materials for potential application in banana TR4 resistant breeding and its effective way for disease control.

香蕉是非常重要的热带和亚热带作物。目前我国乃至全球香蕉种植业面临香蕉枯萎病菌4号生理小种热带型（TR4）非常严峻的挑战,直接威胁到蕉农的收入和产业的稳定发展,因此开展香蕉抗枯萎病的研究具有十分重要的理论与应用意义。本项目组从现有与国际生物多样性中心合作的基础上，建立了高抗TR4的香蕉野生种Musa acuminata Pahang的组培培养快速繁殖体系。本申请以Pahang为材料,利用绿色荧光蛋白标记的TR4研究抗感材料在细胞、组织水平的抗侵染差异;测定病原菌与寄主互作后内源激素茉莉酸和水杨酸的动态变化;采用同位素标记相对和绝对定量蛋白组学（iTRAQ)技术筛选和鉴定抗枯萎病病菌侵染相关的抗性蛋白，获得与抗病相关的关键基因；利用实时荧光定量PCR技术对相关基因表达模式进行验证,初步阐明高抗枯萎病TR4香蕉材料的抗病机理。研究结果将为香蕉枯萎病抗病育种和防控提供理论依据和关键材料。

香蕉是许多发展中国家重要的经济作物，也是一些热带和亚热带地区的主要粮食作物。然而，全球香蕉产业的发展受到香蕉枯萎病菌4号生理小种热带型（Foc TR4）的严重危害。筛选可靠的Foc TR4抗性材料，并探索其潜在的防御机制对于香蕉未来的抗病育种至关重要。二倍体香蕉野生种Pahang高抗Foc TR4，且双单倍体基因组已完成测序。但是截止目前，Pahang在细胞组织学和分子水平方面的抗侵染机理研究至今仍未开展。通过项目实施，顺利完成细胞组织学、转录组学和蛋白质组学研究目标。将抗感供试材料接种菌株，发现根部感染过程相似，但是Pahang球茎内菌丝的生长明显延后。表明Pahang抑制病菌在球茎内的增殖是其抗枯萎病的机制之一。根据Foc TR4在抗感材料球茎内的侵染进程以及病情指数差异，通过转录组学分析共筛选6319个差异表达基因（DEGs），其中Pahang较高表达DEGs 3867个。通过DEGs筛选、PIE分析以及RT-qPCR验证获得了17个潜在的抗病相关基因，将为香蕉抗病育种提供优异的基因资源。另外，在供试香蕉的木质部汁液中共检测到1036种蛋白质，其中一些蛋白质参与“信号转导”，“环境适应性”，“次生代谢产物的生物合成”和“脂质代谢”，表明木质部汁液中含有防御相关蛋白质。一共鉴定了129种差异表达蛋白（DEPs）。 在这些DEPs中，超敏反应蛋白1（HIRP1），E3泛素连接酶（E3）可能在Pahang对TR4的响应中起负向作用，而查尔酮异构酶（CHI）是富含甘氨酸的RNA结合蛋白（GRP），羧酸酯酶（CXE）和GDSL脂肪酶（GLIP）可能在Pahang防御TR4响应中发挥正向积极作用。.对来自不同国家的Foc TR4菌株进行全基因组测序之后通过特定SNP分析，首次划分了大湄公河次区域、印度次区域和中东地区三个区域存在3种不同演化类型的Foc TR4菌株。分析了病原菌演化机制，提出了防止香蕉枯萎病传播蔓延的应对策略。.本项目通过研究野生种Pahang对香蕉枯萎病的抗病机制，筛选和鉴定香蕉枯萎病菌侵染的相关蛋白和基因。明确相关抗病基因的调控表达差异，为下一步抗病基因的功能验证奠定基础，为今后香蕉抗病育种和枯萎病防控提供理论依据。本项目实施过程中总共发表研究论文7篇和获得3件授权专利。