番茄微小核酸miR156与JAZs耦合抗灰霉病机制研究

The defence response mediated by JA signal is the main weapon against Botrytis cinerea infection in tomato, while the signal pathway is gradually suppressed with the decrease of the content of miR156. The association between the decline of miR156 and the ubiquitination degradation process of protein containing Jasmonate ZIM-domain (JAZ) is the key point of the JA signal pathway blocking. In the present project, iTRAQ-based quantitative proteomics and Western blotting were used to research the response ability of SlJAZ family proteins for extra-MeJA treatment in tomato at different mature degree, and the trend of miR156-target protein SlSPLs accumulation. Meanwhile, exploring mechanism behind SlSPL interacts with SlJAZ, and the SlSPL represses SlJAZ ubiquitination in tomato fruit with the method of Co-immunoprecipitation and Y2H technology. What's more, we explore the differences of secondary metabolites during fruit growth and maturation, and the regulatory effect of fruit sugar metabolism on miR156, as a verification mechanism for correlation analysis to construct the dynamic gene network regulated by miR156 and SlJAZs. In conclusion, the present results were aimed to clarify the molecular mechanism behind the decline of disease resistance to Botrytis cinerea in tomato fruit, developing the biochemical marker system of sugar metabolism-miR156-JAZs-rot risk factors, provides a theoretical basis for green and modern fruit and vegetable preservation system.

茉莉酸信号通路介导的寄主防卫反应，是番茄果实抵御灰霉病菌侵染的主要途径；但该信号通路随miR156含量衰减而逐渐失敏，相关机制目前尚不清楚。本项目针对JA途径关键抑制因子JAZs泛素化降解是否受到miR156衰减过程抑制这一科学问题，利用蛋白质组学、免疫印迹技、免疫共沉淀与Y2H技术，探索不同成熟体系中JA信号源对JAZs泛素化过程的调控能力以及miR156靶蛋白SPLs的累积趋势，研究SPL与JAZ蛋白互作及其阻遏JAZ泛素化降解的分子机制，进而从表观遗传学层面阐明番茄果实抗灰霉病衰退的分子机理。同时，利用代谢组学方法探索果实次生代谢产物差异性响应，解析果实糖代谢对miR156的调控效应，构建miR156与JAZs动态负调控抗病基因网络模型，探索果实糖代谢→miR156→JAZs→抗病物质→腐败危险系数生化标记体系，为绿色、现代化果蔬保鲜体系提供理论依据。

本项目围绕“鲜活果蔬成熟过程中抗病能力衰退导致采后腐烂现象高发”这一科学问题开展探索，以茉莉酸信号途径为切入点，结合采后病理学、生理学以及组学技术手段，研究了番茄果实三个成熟进度（青熟MG、红熟RR、过熟ORR）与果实抗灰霉菌病能力、内源激素含量、miR156表达量等因素之间的关联关系，明确番茄果实抗灰霉病能力随成熟度增高而降低，且果实对茉莉酸处理的响应能力不断衰退。利用转录组学和荧光定量PCR等技术发现采后成熟过程中茉莉酸合成通路中AOC、AOS和OPR3等相关编码基因表达量显著降低；年龄分子标记miR156表达量逐渐减少、1个SBP（SPL-CNR）编码基因表达显著增加以及现有已知的JAZ编码基因全部下调表达。结合蛋白质组学技术，研究发现生物胁迫条件下发现SPLs、ERF等蛋白显著积累，而仅发现1个JA信号途径下游转录因子SlJAZ3差异变化。利用SRM/MRM多反应监测技术，发现成熟前后茉莉酸含量显著下降92.83%。综合以上研究结果，本项目首次明确番茄果实茉莉酸合成与信号通路“失活”是导致其采后抗病能力衰退的主要原因之一。此外，本项目利用高能碰撞诱导裂解QE质谱技术对果蔬免疫诱抗因子进行了筛选，结合发酵回补法富集制备了一种寡糖保鲜剂，可有效提高番茄果实机械损伤的自愈能力，并明显抑制番茄果实的软化，抑制率达到65%，靶标基因推测为果胶酶编码基因SlPG2和扩展蛋白编码基因SlEXP1。项目成果已在Food Chem、JAFC、Carbohydr Polym 和PBT等国内外高水平刊物上发表论文6篇（中科院一区期刊5篇、EI1篇，均为第一标注），获授权国际发明专利1项、申请国内发明专利2项，培养研究生2名。