本文選題：根腫菌 + 擬南芥�。� 參考：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：由活體專性寄生病原菌根腫菌引起的十字花科根腫病是一種世界性分布的重要土傳病害,研究寄主與根腫菌的互作機理對防治作物根腫病具有重要理論和實踐意義。但目前根腫菌和寄主互作的研究多集中于轉錄組和蛋白組水平,涉及其代謝組方面的報道較少。本研究以擬南芥和油菜為材料,采用LC-MS分析技術,探討受根腫菌侵染后寄主在代謝組水平上的變化,主要研究結果如下:(1)優(yōu)化了根腫菌侵染寄主的接種方法。通過將含有根腫菌的罹病根組織進行組織培養(yǎng),獲得較為純化的、單一的根腫菌接種體,然后將提取出的根腫菌孢子液與擬南芥種子混合后涂布1/2MS培養(yǎng)基共培養(yǎng),有效克服了非根腫菌污染問題,建立了一種無菌條件下的根腫菌接種體系。顯微觀察顯示根腫菌在愈傷組織中能夠進行繁殖,在1/2MS培養(yǎng)基上根腫菌對擬南芥可以完成侵染循環(huán),證明了建立的接種體系具有可行性。(2)分析了擬南芥根受根腫菌侵染后期代謝產(chǎn)物的含量變化。采用優(yōu)化的根腫菌接種體系,通過LC-MS技術分析了根腫菌侵染后期對擬南芥代謝的影響,發(fā)現(xiàn)類黃酮、木質素、萜類、甾體皂苷、油菜素內(nèi)酯和磷脂等物質的含量在根腫侵染后期的擬南芥中顯著上升,說明這些物質可能在根腫菌侵染晚期時寄主與根腫菌的互作中起重要作用。差異代謝物的代謝通路富集分析顯示,擬南芥脂肪酸、氨基糖和色氨酸代謝發(fā)生了顯著變化。(3)基于LC-MS的代謝組學方法研究了油菜在根腫菌侵染早期的代謝物差異。通過PLS-DA、OPLS-DA多變量統(tǒng)計方法結合T-test、Two-way ANOVA等單變量統(tǒng)計方法進行分類判別和潛在生物標志物的篩選。結果表明接種根腫菌1 d、2 d和5 d等不同時間點,油菜根代謝產(chǎn)物均產(chǎn)生了明顯變化,且油菜根受根腫菌侵染1 d時,代謝物差異最顯著,表明寄主與根腫菌的早期識別和互作中油菜應答反應強烈。鑒定出的生物標志物包括木質素、萜類、生物堿、類黃酮等植物抗生物和非生物脅迫相關物質,與生長素合成相關的吲哚三乙醛肟,與精氨酸和芳香族氨基酸的合成和代謝相關的2-氧代精氨酸、4-胍基丁酸和吲哚以及磷脂類物質等。研究結果表明寄主在識別根腫菌初期啟動初級代謝和次級代謝反應,促進抗病物質的合成以應對根腫菌的入侵,磷脂類物質也可能參與早期侵染時寄主與根腫菌的互作中,同時根腫菌侵染早期也可誘導寄主生長素等相關激素的表達;研究結果還表明類黃酮等抗病相關次級代謝產(chǎn)物的含量和種類在根腫菌侵染過程中是動態(tài)變化的,說明它們在寄主與根腫菌互作中可能扮演更為復雜的角色。差異代謝物的代謝通路富集分析顯示,根腫菌早期侵染對寄主氨基酸和糖類代謝、生物堿合成、細胞色素P450代謝等代謝途徑產(chǎn)生了顯著影響。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S435.654

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本文編號：1985853