寄主与根肿菌互作的代谢组学研究

发布时间：2018-06-06 08:09

本文选题：根肿菌 + 拟南芥　；参考：《华中农业大学》2017年硕士论文

【摘要】：由活体专性寄生病原菌根肿菌引起的十字花科根肿病是一种世界性分布的重要土传病害,研究寄主与根肿菌的互作机理对防治作物根肿病具有重要理论和实践意义。但目前根肿菌和寄主互作的研究多集中于转录组和蛋白组水平,涉及其代谢组方面的报道较少。本研究以拟南芥和油菜为材料,采用LC-MS分析技术,探讨受根肿菌侵染后寄主在代谢组水平上的变化,主要研究结果如下:(1)优化了根肿菌侵染寄主的接种方法。通过将含有根肿菌的罹病根组织进行组织培养,获得较为纯化的、单一的根肿菌接种体,然后将提取出的根肿菌孢子液与拟南芥种子混合后涂布1/2MS培养基共培养,有效克服了非根肿菌污染问题,建立了一种无菌条件下的根肿菌接种体系。显微观察显示根肿菌在愈伤组织中能够进行繁殖,在1/2MS培养基上根肿菌对拟南芥可以完成侵染循环,证明了建立的接种体系具有可行性。(2)分析了拟南芥根受根肿菌侵染后期代谢产物的含量变化。采用优化的根肿菌接种体系,通过LC-MS技术分析了根肿菌侵染后期对拟南芥代谢的影响,发现类黄酮、木质素、萜类、甾体皂苷、油菜素内酯和磷脂等物质的含量在根肿侵染后期的拟南芥中显著上升,说明这些物质可能在根肿菌侵染晚期时寄主与根肿菌的互作中起重要作用。差异代谢物的代谢通路富集分析显示,拟南芥脂肪酸、氨基糖和色氨酸代谢发生了显著变化。(3)基于LC-MS的代谢组学方法研究了油菜在根肿菌侵染早期的代谢物差异。通过PLS-DA、OPLS-DA多变量统计方法结合T-test、Two-way ANOVA等单变量统计方法进行分类判别和潜在生物标志物的筛选。结果表明接种根肿菌1 d、2 d和5 d等不同时间点,油菜根代谢产物均产生了明显变化,且油菜根受根肿菌侵染1 d时,代谢物差异最显著,表明寄主与根肿菌的早期识别和互作中油菜应答反应强烈。鉴定出的生物标志物包括木质素、萜类、生物碱、类黄酮等植物抗生物和非生物胁迫相关物质,与生长素合成相关的吲哚三乙醛肟,与精氨酸和芳香族氨基酸的合成和代谢相关的2-氧代精氨酸、4-胍基丁酸和吲哚以及磷脂类物质等。研究结果表明寄主在识别根肿菌初期启动初级代谢和次级代谢反应,促进抗病物质的合成以应对根肿菌的入侵,磷脂类物质也可能参与早期侵染时寄主与根肿菌的互作中,同时根肿菌侵染早期也可诱导寄主生长素等相关激素的表达;研究结果还表明类黄酮等抗病相关次级代谢产物的含量和种类在根肿菌侵染过程中是动态变化的,说明它们在寄主与根肿菌互作中可能扮演更为复杂的角色。差异代谢物的代谢通路富集分析显示,根肿菌早期侵染对寄主氨基酸和糖类代谢、生物碱合成、细胞色素P450代谢等代谢途径产生了显著影响。
[Abstract]:Cruciferae rhizomegaly caused by in vivo specific parasitic pathogen is an important soil-borne disease in the world. It is of great theoretical and practical significance to study the interaction mechanism between host and rhizomegaly. However, most of the studies on the interaction between rhizomegaly and host are focused on the transcriptional and proteomic levels, and there are few reports on the metabolism of rhizomegaly. In this study, Arabidopsis thaliana and Brassica napus were used as materials, and LC-MS analysis technique was used to study the changes of host in metabolic group level after infection by rhizomegaly. The main results were as follows: 1) the inoculation method of rhizomegaly infection host was optimized. A single rhizomegaly inoculum was obtained by tissue culture of diseased root tissue containing rhizomegaly bacteria, and then mixed with the seeds of Arabidopsis thaliana and coated with 1/2MS medium. The problem of non-rhizomegaly contamination was overcome effectively and a inoculation system was established under aseptic condition. Microscopic observation showed that rhizomegaly could propagate in callus, and the infection cycle of Arabidopsis thaliana could be completed by rhizomegaly on 1/2MS medium. It was proved that the established inoculation system was feasible) and the content of metabolites in Arabidopsis thaliana root was analyzed in the later stage of infection by rhizomegaly bacteria. The effects of rhizomegaly inoculation system on Arabidopsis thaliana metabolism were analyzed by LC-MS technique. Flavonoids, lignin, terpenes, steroidal saponins were found. The contents of Brassinolide and phospholipid increased significantly in Arabidopsis thaliana in the late stage of root-swelling infection, indicating that these substances may play an important role in the interaction between host and rhizomonas in the late stage of rhizomegaly infection. The metabolic pathway enrichment analysis of differential metabolites showed that the metabolism of fatty acids, amino sugar and tryptophan in Arabidopsis thaliana had changed significantly. The multivariable statistical method of PLS-DA-OPLS-DA and the single-variable statistical method such as T-test-Two-way ANOVA were used to classify and discriminate and screen potential biomarkers. The results showed that at different time points, such as 1 day, 2 d and 5 d, the metabolites of rape root changed obviously, and when the root of rape was infected by the fungus for 1 day, the difference of metabolites was the most significant. The results indicated that the rapeseed response was strong in the early recognition and interaction of host and rhizomegaly. The identified biomarkers include lignin, terpenes, alkaloids, flavonoids and other plant biostress-related and abiotic stress-related substances, and indoletriacetaldehyde oxime related to auxin synthesis. The synthesis and metabolism of arginine and aromatic amino acids are related to 2-oxo-arginine 4-guanidine butyric acid, indole, phospholipid and so on. The results showed that the host initiated primary metabolism and secondary metabolic reaction in the early stage of identification of rhizomegaly and promoted the synthesis of disease-resistant substances to deal with the invasion of rhizomegaly. Phospholipids may also be involved in the interaction between host and rhizomegaly during early infection. At the same time, the expression of host auxin and other related hormones could also be induced in the early stage of rhizomegaly infection. The results also showed that the content and species of secondary metabolites related to disease resistance, such as flavonoids, changed dynamically during the process of rhizomegaly infection. These results suggest that they may play a more complex role in the interaction between host and rhizomegaly. The analysis of metabolic pathway enrichment of differential metabolites showed that the early infection of rhizomegaly had a significant effect on host amino acid and carbohydrate metabolism, alkaloid synthesis, cytochrome P450 metabolism and other metabolic pathways.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.654

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