L-谷氨酸对果实抗性的诱导作用及其相关机理研究

发布时间：2018-07-20 14:13

【摘要】：在果实采后贮运实践中,由真菌病害引起的果实腐烂会造成经济上的巨大损失。尽管化学杀菌剂仍然是控制采后病害的主要防治方法,但在健康环保的理念指导下,其使用种类和范围愈发严格和局限。通过化学、物理或者生物手段激发果实内在的抗性机制防御病害已成为果实采后病害防治的新概念和新手段。作为生物体内最重要的氨基酸之一,L-谷氨酸及其钠盐被广泛应用于实际生活中的各个领域,具有安全环保,价格低廉,容易获得且使用简单等特点。L-谷氨酸在植物代谢中占据极为重要的地位,不仅参与合成多种与增强植物抗逆性相关的代谢物质,且在非生物胁迫下的逆境响应中发挥重要作用。然而目前有关它在植物病害防御反应中的作用鲜有报道。本论文以L-谷氨酸作为外源诱导因子,探讨其对果实采后病害的防治效果,并通过基因、蛋白、物质等多个水平和角度系统分析研究了 L-谷氨酸对果实的抗性反应机制。研究结果不仅为开发基于L-谷氨酸的新型保鲜剂提供理论依据,而且可为研究植物抗性提供新的视角和策略。主要研究结果如下:(1)L-谷氨酸可以通过诱导果实(梨、番茄、柑橘)自身的抗性来抵御或延缓其采后病害的发展,且抑制效力与诱导时间(24h以上)、处理浓度(100mg U1以上)等因素密切相关。L-谷氨酸不论是以采前喷洒还是采后处理的方式均可有效提高果实对采后病原菌的抵御能力,在果实的防腐保鲜领域有广阔的应用前景。(2)L-谷氨酸作为γ-氨基丁酸(GABA)前体物质,激活了番茄果实体内GABA支路上与GABA合成和代谢相关关键基因的表达;而用外源GABA处理可有效抵御番茄果实腐生型病原菌Alternariaalternate的侵染,其机理与激活果实体内GABA支路有关。GABA支路的激活一方面可能与三羧酸(TCA)循环路径的加强为防御反应提供更多的能量有关;另一方面可能是阻止了活性氧的积累减少了细胞死亡从而不利于腐生型病原菌的侵染。因此,L-谷氨酸对番茄果实抗性的诱导机制可能与GABA支路密切相关。(3)呼吸跃变型果实番茄经L-谷氨酸处理后,其体内的主要氮代谢路径谷氨酰胺合成酶/谷氨酸合成酶(GS/GOGAT)循环上的GS被激活,同时,与碳代谢有关的糖酵解路径上编码己糖激酶和丙酮酸激酶的基因和TCA循环中的苹果酸脱氢酶和琥珀酸脱氢酶均被强烈诱导表达。也就是说,L-谷氨酸对A.alternata的抗性机制可能与碳氮代谢路径的激活有关。(4)番茄果实经L-谷氨酸处理后,与水杨酸合成和信号路径相关的基因,如PAL、NPR1、TGA1、TGA2、WRKY70和PR基因,其转录水平明显上调;乙烯合成路径上的两个关键乙烯合成酶基因ACS、ACO和乙烯受体ETR3、ETR4的转录表达被强烈抑制;茉莉酸合成路径上的LOX1、AOS2、AOC呈下调表达趋势,其信号转导路径上的茉莉酸受体COI1,正调控转录因子MYC2和茉莉酸诱导的蛋白酶抑制剂PI-Ⅱ的转录水平下降的同时,负调控转录因子JAZ1则有一定的上调趋势。由上可知,L-谷氨酸对番茄果实采后黑斑病的抗性机制可能依赖于水杨酸的合成和信号传导路径;同时谷氨酸对乙烯/茉莉酸合成与信号路径存在一定的抑制作用。(5)同重同位素相对与绝对定量(iTRAQ)试验结果显示,经L-谷氨酸处理后的番茄果实相对于对照组有97个蛋白显著上调表达,42个蛋白显著下调表达,且这些差异蛋白显著富集于植物与病原菌互作、苯丙烷类合成、能量代谢(氧化磷酸化和多糖类分解路径)、脂肪酸代谢等与植物抗性反应密切相关的代谢路径上。(6)通过气相色谱质谱联用(GC-MS)技术分析了谷氨酸对番茄果实物质含量的变化,结果显示,L-谷氨酸可能激活了果实的能量代谢、氨基酸代谢和水杨酸信号路径,同时对乙烯/茉莉酸合成路径有一定的抑制效果。(7)对于呼吸非跃变型果实柑橘转录表达谱的试验结果表明,在未接种病原菌Penicillium digitatum条件下,经L-谷氨酸处理后的柑橘果实相对于对照组有623个基因上调表达,647个下调表达;在接种病原菌的条件下,L-谷氨酸处理组则有234个上调基因,193个下调基因。这些差异基因涉及碳代谢,氨基酸代谢,植物激素代谢和次级代谢等多条与果实抗性密切相关的路径。综上所述,L-谷氨酸可以通过诱导果实抗性有效抵御果实的病害,其机理可能与激活GABA支路、碳氮代谢和水杨酸路径,同时抑制乙烯/茉莉酸路径有关。
[Abstract]:In the practice of postharvest storage and transportation of fruit, fruit decay caused by fungal diseases will cause great economic loss. Although chemical fungicides are still the main control methods for controlling postharvest diseases, under the guidance of the concept of health and environmental protection, the types and limits of their use and scope are more stringent and limited. The inherent resistance mechanism of the fruit has become a new concept and new means for the prevention and control of postharvest diseases of the fruit. As one of the most important amino acids in the organism, L- glutamic acid and its sodium salt are widely used in various fields of real life. It has the characteristics of safe and environmental protection, low price, easy access and simple use of.L- glutamic acid. Plant metabolism occupies a very important position, not only to participate in the synthesis of a variety of metabolic substances related to stress resistance, but also plays an important role in the response to abiotic stress. However, there are few reports on its role in the plant disease defense response. This paper uses L- glutamic acid as a exogenous inducer. The prevention and control effect on postharvest diseases of fruit was discussed, and the resistance mechanism of L- glutamic acid to fruit was studied through multiple levels and angles of genes, proteins and substances. The results not only provide a theoretical basis for the development of a new type of fresh preservative based on L- glutamic acid, but also provide a new perspective and strategy for the study of plant resistance. The main results are as follows: (1) L- glutamic acid can resist or delay the development of postharvest diseases by inducing the resistance of the fruit (pear, tomato, citrus) itself, and the inhibition effect and induction time (above 24h), the treatment concentration (100mg U1) and other factors closely related to.L- glutamic acid, whether by pre harvest spraying or after postharvest treatment It can effectively improve the resistance of fruit to Postharvest Pathogens, and has broad application prospects in the field of fruit preservation and preservation. (2) L- glutamic acid as a precursor of gamma aminobutyric acid (GABA) activates the expression of key genes related to GABA synthesis and metabolism in the GABA branch of tomato fruit, and the exogenous GABA treatment can effectively resist tomatoes. The infection of the fruit saprophytic pathogen Alternariaalternate, its mechanism and activation of the.GABA branch related to the activation of the GABA branch in the fruit of the fruit may be related to the strengthening of the three carboxylic acid (TCA) cycle path to provide more energy for the defense response; on the other hand, it may prevent the accumulation of active oxygen and reduce cell death. The induction mechanism of L- glutamic acid on tomato fruit resistance may be closely related to the GABA branch. (3) after L- glutamic acid treatment, the main nitrogen metabolism pathway of the respiratory climacteric fruit tomato is activated by the GS on the cycle of glutamine synthetase / glutamic synthetase (GS/ GOGAT). At the same time, it is associated with carbon metabolism. The genes encoding hexokinase and pyruvate kinase in the glycolysis pathway and the malate dehydrogenase and succinic dehydrogenase in the TCA cycle are strongly induced. That is to say, the resistance mechanism of L- glutamic acid to A.alternata may be related to the activation of carbon and nitrogen metabolism pathway. (4) the fruit of tomato is treated with Salicylic acid after L- glutamic acid treatment. Genes related to the pathway of signaling, such as PAL, NPR1, TGA1, TGA2, WRKY70 and PR genes, are significantly up-regulated, and the two key ethylene synthetase genes on the ethylene synthesis pathway, ACS, ACO and ethylene receptor ETR3, are strongly suppressed in the transcriptional expression of ETR4; the LOX1 on the jasmonic acid synthesis pathway is downregulated and its signal turns Jasmonate receptor COI1 on the guide path, the transcriptional level of the transcriptional factor MYC2 and the jasmonic acid induced protease inhibitor PI- II, while the negative regulatory transcription factor JAZ1 has a certain upward trend. It is known that the resistance mechanism of L- glutamic acid to the Postharvest black spot of tomato fruit may be dependent on the synthesis and letter of salicylic acid. There was a certain inhibitory effect of glutamic acid on the synthesis of ethylene / jasmonic acid and signal pathway. (5) the results of the relative and absolute quantitative (iTRAQ) test of the same heavy isotopes showed that 97 proteins in the tomato fruit treated with glutamic acid were significantly expressed in comparison with the control group, and the 42 proteins were significantly down expressed, and these differences were different. The protein was significantly enriched in the interaction of plant and pathogenic bacteria, synthesis of phenylpropane, energy metabolism (oxidation phosphorylation and polysaccharide decomposition pathway), fatty acid metabolism and other metabolic pathways closely related to plant resistance. (6) the results of the changes in the material content of tomato fruit by GC / MS (GC-MS) were analyzed. The results showed that L- glutamic acid may activate the energy metabolism of fruit, amino acid metabolism and the pathway of salicylic acid signal, and have certain inhibitory effects on the synthesis pathway of ethylene / jasmonic acid. (7) the results of the transcription of Citrus transcriptional expression of non climacteric fruit show that L- glutamic acid under the condition of unplanted pathogen Penicillium digitatum There were 623 up-regulated genes and 647 down-regulated genes in the citrus fruits compared with the control group. Under the condition of inoculation, the L- glutamic acid treatment group had 234 up-regulated genes and 193 down-regulation genes. These genes involved carbon metabolism, amino acid metabolism, plant irritable Sudache and secondary metabolism, which were closely related to the fruit resistance. In summary, L- glutamic acid can effectively resist fruit disease by inducing fruit resistance. The mechanism may be related to the activation of the GABA branch, carbon and nitrogen metabolism and salicylic acid pathway, and the inhibition of the ethylene / jasmonate pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS255.3

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