转录因子ERFs在生防酵母诱导番茄果实抗病中的调控机理研究

Fresh fruit is an essential component of our daily diet due to its nutrition- and minerals-rich. However, losses caused by various impact factors accounted for nearly 20-25 percent of the total production in China, led to a huge economic loss. Postharvest disease pathogens are one of the major factors, and various researchers had put their attention on killing pathogen fungus and reducing its invasion chances. Little is known about how the activator of defense related genes, transcription factor ERFs, improves fruit resistance against postharvest diseases. Then, the aim of the proposed project is to investigate the roles of ERFs between biological control yeast and Lycopersicum esculentum, and the network among ERFs and defense genes. Specifically, the objectives of this project were to (1) Characterize the proteome of Lycopersicum esculentum treated by biocontrol yeast Cryptococcus laurentii, and finding the over expressed ERFs, defense related proteins and their functional motifs within database PLACE; (2) Indentify the ERFs controlled DNA fragment with methods of ‘ChIP-sequencing’, defining the binding motifs between ERFs and the promoters, and mapping these promoters to defense related genes within the genome of tomato fruit; (3) Varify the biological roles of ERFs in Lycopersicum esculentum through ‘knocking-down’ their expression with methods of RNAi. The results of this project not only will lay a foundation on how ERFs activated disease resistance in postharvest tomato fruit, and increase knowledge how the ways REFs involved in the low cost defense mechanism, but also is critical for prolonging postharvest fruit storage and developing new safely disease control strategy.

新鲜水果营养丰富，富含大量维生素、矿物质和膳食纤维，但其采后腐烂十分严重，在我国平均损失率高达20%至25%，经济损失巨大。由于病原菌是引起新鲜果实腐烂的主要原因，以往果实病害控制的研究多集中于抑制病原菌生存与阻遏病原菌侵染等方面。对果实抗病改善及其分子机制的研究报道较为有限。果实抗病基因转录的主要启动元件，ERFs，在果实采后防御机制中的作用尚不清楚。为明确该类转录因子在生防菌改善番茄果实抗病中的作用机制，本项目拟通过iTRAQ、GFP定位和Chip-Seq技术并结合生物信息学分析，在蛋白表达、转录因子与启动子互作层面上系统研究抗病相关ERFs和其抗病基因调控网络及抗病启动子互作机制，并通过RNA干扰技术探索ERFs在番茄果实抗病中的生物学功能。研究结果不仅对明确番茄果实抗病改善机制和采后低损耗储藏机制具有重要的理论意义，而且还将为延长新鲜水果贮藏保鲜和开发新型病害防治技术提供新的信息。

番茄是世界范围内最为重要的果蔬作物和模式植物之一，作为呼吸跃变型果实，采前潜伏侵染和采后交叉感染的真菌病害是造成其采后腐烂的主要原因，目前利用物理、化学和生物激发子诱导果实中潜在的抗性机制被认为是防治果实采后病害的新型、可持续手段，可有效增强果实病害防御能力并显著降低番茄果实采后病害发生率。此外，番茄基因组中大量的乙烯响应因子（ERFs）被报道在果实采后逆境胁迫中起着关键的调控作用，由此研究其在番茄果实采后抗病中的调控作用也十分必要。.本项目以罗伦隐球酵母（Cryptococcus laurentii）与番茄为研究材料，通过高通量（iTRAQ）蛋白组学技术和二代转录组测序技术筛选到信号通路上的关键调控因子为乙烯响应因子Pti5，结合RACE技术对Pti5蛋白进行CDS区域克隆，利用蛋白异源表达体系体外得到了Pti5活性蛋白，证明其在体外具有DNA结合活性，并运用EMSA技术验证了Pti5与抗性基因PR5的结合；最后利用VIGS技术成功构建了SlPti5基因沉默番茄植株pTRV2：Pti5，SlPti5基因沉默显著影响番茄植株前期的生长并减弱了番茄植株叶片对B. cinerea的抗性，pTRV2: Pti5番茄植株叶片抗病能力减弱与ROS积累、抗病相关酶活性减弱、ET/JA介导的信号通路及病程相关蛋白基因的表达密切相关。此外，我们利用VIGS侵染技术真空渗透处理番茄果实并成功得到了离体的pTRV2:Pti5番茄果实，SlPti5基因沉默显著增强了番茄果实对B. cinerea侵染的敏感性，同时也减弱了C. laurentii对番茄果实的诱导抗性，乙烯响应因子Pti5可能是通过平衡ET-和JA-介导的信号通路中相关基因的表达来促进番茄发挥对B. cinerea侵染的积极免疫应答。.本项目通过高通量（iTRAQ） 蛋白组学技术、转录组测序技术、RACE技术、病毒诱导的基因沉默技术（VIGS）、生理学、病理学、分子生物学等研究方法全面揭示乙烯响应因子Pti5在拮抗酵母C. laurentii诱导番茄采后抗性中的调控作用，为增强果实采后病害抵御能力，指导果实采后贮藏环境控制策略，延长果实贮藏物流与商品货架期和开发新型病害控制技术提供基础信息。.