基于基因表达谱与代谢谱解析NO缓解黄瓜碱胁迫的生理机制

A great progress has been made on the research of NO role in plants adapting to abiotic stress. However, there is few investigations about NO alleviating alkali stress on plants. Our early experiment indicated that exogenous NO could reverse the adverse symptom of cucumber induced by alkali stress, but the mechanism of molecular and metabolism is short of detailed exploration. In the present study, RNA-Seq and GS-MS method are used to get the gene expression profiles and metabolomic profiles of cucumber leaves and roots, and based on the combination of the two profiles, the candidate genes and metabolites regulated by NO under alakli stress are prelimilarily selected. Then Western blot is used to make further analysis for candidate genes, and HPLC and CE are used to make further analysis for candidate metabolites. At the same time, the content and distribution of nutrient elements in cucumber plants are measured.Through the combined assay, the key genes and metabolites playing important role in the process of NO alleviating alkali stress on cucumber are determined.At last, transgene method is applied to make sure that some key candidate genes really take part in the cucumber tolerance to alkali stress.For some key candidate metabolites, exogenous application method is used to check their funciton in increasing the cucumber tolerance to alkali stress. Based on the above results, it is expected to make clear the gene regulation pathway and metabolomic basis of NO alleviating alkali stress on cucumber. Furthermore, a great number of candidate genes and metabolites responsing alkali stress and NO can be obtained, which provides a basis for the future further study.

NO调控植物对非生物胁迫适应性方面的研究已取得较大进展，但关于其缓解植物碱胁迫的研究报道很少。本课题组的前期试验表明外源NO可以显著缓解黄瓜碱胁迫症状，而对其分子机制和物质代谢基础缺乏深入了解。本项目拟通过RNA-Seq和GC-MS技术获得黄瓜叶片与根系的基因表达谱和代谢谱，将二谱结合初步筛选出碱胁迫下黄瓜中受NO调控的候选基因和代谢物。然后分别采用Western杂交和HPLC、CE等方法对候选基因的蛋白表达和候选代谢物的含量与分布做进一步分析，结合营养元素分布等生理指标，找出受NO调控且在缓解黄瓜碱胁迫中发挥功能的关键基因和代谢物。再通过转基因和外源物质添加处理等试验分别对筛选的关键基因和代谢物在调控黄瓜适应碱胁迫方面的功能进行验证。在此基础上，系统地探明NO缓解黄瓜碱胁迫的基因调控途径和物质代谢基础。同时，可以获得一批对碱胁迫和NO响应的候选基因和代谢物，为后续的深入研究奠定基础。

首先采用基因表达谱与代谢谱相结合的方法，研究了外源 NO 对碱胁迫下黄瓜基因表达和物质代谢的影响，共发现724个差异表达基因，其中上调基因477条，下调基因247条，差异基因多与激素信号传导、糖酵解途径、三羧酸循环以及抗氧化系统有关。外源NO还调控了水杨酸、色氨酸(IAA和褪黑素合成前体)、6-脱氧CT(油菜素内酯合成前体)、S-腺苷甲硫氨酸(多胺和乙烯合成前体)、多胺等一系列与激素代谢相关的物质积累。在组学分析的基础上，后续研究选择了水杨酸、表油菜素内酯、IAA、多胺等作为外源物质，研究了其对黄瓜、番茄等蔬菜作物碱胁迫的缓解效应，结果表明这4种物质均可显著缓解黄瓜、番茄等蔬菜作物的碱胁迫，其作用均与提高了碱胁迫下的抗氧化能力和离子平衡有关，并且IAA和多胺对碱胁迫的缓解与NO有密切的互作效应。另外，从基因表达谱中选择了2个与氮信号相关基因S-腺苷甲硫氨酸合成酶(SAMS)和谷胱甘肽亚硝基还原酶(GSNOR)作为目标基因，研究了2个基因超表达或抑制表达对番茄耐碱性的影响，发现超表达SAMS显著提高了番茄的耐碱性，并且其作用的发挥主要是促进了多胺的合成，并且依赖于活性氧(ROS)信号，激发了离子转运与信号传导相关基因的表达，促进了有机酸分泌，降低了高pH胁迫。采用超表达SAMS番茄植株作为砧木嫁接野生番茄可促进地上部多胺的积累，显著延长碱胁迫的生长期限，提高番茄的产量。过表达GSNOR基因显著提高了番茄的耐碱性，而抑制该基因表达，显著降低了番茄的耐碱性。进一步研究表明，过表达GSNOR对番茄耐碱性的提高并不是由于其调控了离子平衡，而是调控了ROS和活性氮(RNS) 平衡，抑制了碱胁迫诱导的细胞程序化死亡。