本文關(guān)鍵詞：疫霉菌氮素代謝途徑與植物AGO4蛋白在其互作中的功能研究　出處：《南京農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 卵菌 大豆疫霉 氮素營養(yǎng) 營養(yǎng)吸收 致病性 ARGONAUTE4 抗病性

【摘要】：在植物與病原微生物的互作過程中,微生物要建立成功的寄生關(guān)系,不但要克服寄主的防御反應(yīng),還要能有效的從寄主體內(nèi)吸取自身所需要的營養(yǎng)物質(zhì),其中氮素營養(yǎng)的爭奪和利用是病原微生物與寄主間互作的關(guān)鍵因素之一。同時,寄主植物也進化出了復(fù)雜的防御系統(tǒng)來抵抗病原菌的入侵,其中植物Argonaute (AGO)蛋白是小分子RNA介導(dǎo)的基因沉默機制中效應(yīng)復(fù)合體的核心組分,對基因表達的重編程起重要作用。目前對疫霉菌侵染過程中的氮素營養(yǎng)利用方式以及AGO蛋白在抗病中的功能與作用機制還缺乏系統(tǒng)了解,本研究分別以大豆疫霉菌和模式植物擬南芥為實驗材料對這兩個科學(xué)問題進行了初步的研究,獲得的主要發(fā)現(xiàn)如下:全基因分析揭示了大豆疫霉中氮素代謝相關(guān)基因的進化特征:利用生物信息學(xué)技術(shù)對大豆疫霉的氮素營養(yǎng)吸收、氮素同化和氨基酸合成與代謝三個方面的基因進行了鑒定,并和其它病原卵菌和真菌進行了比較分析。在大豆疫霉中鑒定到274個與氮素代謝相關(guān)基因,相對于其它物種更為豐富,除了賴氨酸、苯丙氨酸和酪氨酸的合成途徑中部分基因是缺失的外,其它參與氮素代謝的基因在7個物種中都是保守存在的。參與GABA代謝的基因在大豆疫霉中是顯著擴張的,并且在侵染過程中是上調(diào)表達的。進一步分析發(fā)現(xiàn)在卵菌氨基酸合成途徑中存在大量的多功能酶,并且與植物和真菌在結(jié)構(gòu)組成上具有一定的差異。結(jié)果表明卵菌的氮素代謝與其它物種相似,但是在數(shù)量分布和基因結(jié)構(gòu)上存在一定的差異:大豆疫霉可以吸收和利用各種形式的氮素來合成除賴氨酸、苯丙氨酸和酪氨酸外的全部氨基酸,并且可能通過代謝寄主防衛(wèi)反應(yīng)的產(chǎn)物GABA和尿酸來作為抵抗寄主防衛(wèi)的一種策略�；诘貭I養(yǎng)吸收發(fā)現(xiàn)其參與大豆疫霉的致病過程:通過生物信息學(xué)分析,鑒定了大豆疫霉中參與氮素轉(zhuǎn)運的基因并與其它所檢測物種進行比較分析,在大豆疫霉中分別鑒定了 8個銨鹽轉(zhuǎn)運子、16個硝酸鹽/肽轉(zhuǎn)運子和78個氨基酸轉(zhuǎn)運子并且它們的數(shù)目都是顯著多于其它卵菌和真菌,表明轉(zhuǎn)運子基因在大豆疫霉中經(jīng)歷了顯著地擴張。其中Rh型銨鹽轉(zhuǎn)運子基因在卵菌中是特異的,與此相反的是,植物和真菌中普遍存在的寡肽轉(zhuǎn)運子在卵菌中沒有鑒定到同源基因。大豆疫霉的轉(zhuǎn)錄組數(shù)據(jù)顯示大部分轉(zhuǎn)運蛋白基因在其侵染過程中是上調(diào)表達的,并且在RT-PCR中得到了驗證。通過分析大豆疫霉在含有不同氮源的合成培養(yǎng)基上生長狀態(tài),發(fā)現(xiàn)大豆疫霉可以在以不同氮素形式為單一氮源的培養(yǎng)基上生長,并且不同氮素對其生長的影響是有差異的,例如半胱氨酸顯著抑制了其生長,而甲硫氨酸、谷氨酸、天冬酰胺等則有利于其生長。進一步研究發(fā)現(xiàn)大豆疫霉侵染的葉片中游離氨基酸的含量是增加的,其中谷氨酸最為顯著。此外,大豆疫霉的銨鹽轉(zhuǎn)運子PsRh1是定位質(zhì)膜上并且是受無機氮鹽誘導(dǎo)表達的�；诨虻奶禺愋院褪芗闹髡T導(dǎo)表達的特性,選擇銨鹽轉(zhuǎn)運子PsRh1和氨基酸轉(zhuǎn)運子PsCAT3通過PEG介導(dǎo)的沉默進一步研究其在致病中的功能,結(jié)果發(fā)現(xiàn)分別沉默這兩個基因后大豆疫霉的致病力明顯下降。這些結(jié)果表明大豆疫霉可以利用廣泛的氮源,并且在侵染過程中可能會通過干擾寄主的代謝來增加侵染組織中氨基酸的含量,同時氮素營養(yǎng)的吸收在其致病中可能扮演著重要的角色。卵菌特異的天冬氨酸轉(zhuǎn)氨酶PsAAT3在大豆疫霉致病過程中是需要的:通過生物信息學(xué)鑒定了大豆疫霉的天冬氨酸轉(zhuǎn)氨酶,發(fā)現(xiàn)大豆疫霉比所檢測的真菌編碼了數(shù)量更多的AATs。功能域分析發(fā)現(xiàn)其中一些AATs是卵菌所特有的,它們在N端分別包含了額外的預(yù)苯酸脫水酶功能域或苯酸脫氫酶功能域,甚至個別基因兩者兼有。PsAAT3是在侵染過程中誘導(dǎo)表達的,將其沉默后發(fā)現(xiàn),大豆疫霉的致病性下降并且影響其在不含N素培養(yǎng)基上的生長,這表明PsAAT3在侵染過程中參與到了大豆疫霉的致病和氮素利用。該結(jié)果表明大豆疫霉和其它病原卵菌有區(qū)別于其它物種的氨基酸代謝途徑并且PsAAT3是大豆疫霉致病所需的。ARGONAUTE4以不同的方式參與擬南芥對辣椒疫霉和棉花黃萎菌的抗性:為了研究AGO蛋白在植物免疫反應(yīng)中的作用,通過ago突變體的篩選我們發(fā)現(xiàn)擬南芥ago4-2突變體對辣椒疫霉和棉花黃萎菌的抗性具有顯著差異。ag04-2突變體顯著增強了對辣椒疫霉的抗性,但是其對棉花黃萎菌的抗性是減弱的�；钚匝鹾碗蓦召|(zhì)積累的細胞學(xué)觀察也證實了這一結(jié)果。此外,AG04在辣椒疫霉處理時是下調(diào)表達的,而黃萎菌則誘導(dǎo)其上調(diào)表達。進一步研究發(fā)現(xiàn),RdDM途徑中在AG04上游發(fā)揮功能的成員突變體如nrpe1、rdr2-2及dcl3-1和處于下游的成員突變體如drd1-6、nrpdl對辣椒疫霉的抗性是增強的,但是這些成員的突變體對棉花黃萎菌的抗性卻沒有發(fā)生改變。結(jié)果表明AG04可能是依賴于RNA介導(dǎo)的DNA甲基化途徑來負調(diào)控擬南芥對辣椒疫霉的抗性,而其參與正調(diào)控對黃萎菌的抗性可能是通過其它途徑來實現(xiàn)的。
[Abstract]:In the process of interaction between plant and pathogenic microorganisms, microorganisms to establish a parasitic relationship success, not only to overcome host defense responses, but also can effectively absorb the nutrients from the host's own needs, the nitrogen nutrition and utilization is one of the key factors for the pathogen and host interaction. At the same time, the host plant has evolved a complex intrusion defense system against pathogens, including plant Argonaute (AGO) protein is the core component of gene silencing effect of small molecule RNA mediated in complex, plays an important role in reprogramming of gene expression. The process of Phytophthora infection in nitrogen utilization and AGO protein in disease resistance function and mechanism is still a lack of systematic understanding, this study respectively to Phytophthora sojae and Arabidopsis thaliana as experimental materials were studied on the two scientific problems, mainly found as follows: whole genome analysis revealed the evolutionary characteristics of nitrogen metabolism of Soybean Phytophthora genes: Technology of nitrogen nutrition on Phytophthora absorption, three aspects of nitrogen assimilation and synthesis of amino acids and metabolic genes were identified by bioinformatics, and other pathogenic bacteria and fungi were analyzed. In Phytophthora identified 274 genes associated with nitrogen metabolism, compared with other species more abundant, in addition to part of the gene synthesis pathway of lysine, phenylalanine and tyrosine in the absence of the other is, involved in nitrogen metabolism genes are conserved in the 7 species. The genes involved in GABA metabolism are significantly expanded in Phytophthora sojae, and are up-regulated during the infection process. The further analysis shows that there are a large number of multifunctional enzymes in the amino acid synthesis pathway of oobacterium, and there are some differences in the structure composition of the plants and fungi. The results showed that the nitrogen metabolism of oomycetes and other similar species, but there are some differences in the quantity distribution and genetic structure: Phytophthora can absorb and use various forms of nitrogen to synthesize all amino acids except phenylalanine and tyrosine, lysine, and host defense responses through metabolic products GABA and uric acid as a kind of strategy against host defense. The absorption of nitrogen in Soybean Phytophthora pathogenesis based on bioinformatics analysis, identification of nitrogen transporters involved in Phytophthora sojae genes and compared with other tested species, in Phytophthora were identified in 8 ammonium transporters, 16 nitrate / peptide transporter and 78 amino acid transporter and the number of them are significantly more than other oomycetes and fungi showed that the transporter gene in P. sojae has experienced significant expansion. Among them, the Rh type ammonium transporter gene is specific in oosides. On the contrary, the oligopeptide transporters commonly found in plants and fungi do not identify homologous genes in oosides. The transcriptional data of Phytophthora sojae showed that most of the transporter genes were up-regulated during the infection process and were verified in RT-PCR. Through the analysis of the growth state of Phytophthora in synthetic medium containing different nitrogen sources, found that Phytophthora can grow in medium with different nitrogen forms as a single source of nitrogen, and nitrogen have different effect on their growth, such as cysteine significantly inhibited its growth, methionine, glutamic acid, and asparagine is beneficial to its growth. Further study found that the content of free amino acids in the leaves infected by Phytophthora sojae was increased, of which glutamic acid was the most significant. In addition, the ammonium salt transporter PsRh1 of Phytophthora sojae is located on the plasmalemma and is induced by inorganic nitrogen salts. Specific genes and host induced expression based on the characteristics of the choice of ammonium transporter PsRh1 and amino acid transporter PsCAT3 by PEG mediated silencing of the further research of the pathogenic function of the pathogenicity of Phytophthora were silencing of these two genes significantly decreased. These results indicate that Phytophthora sojae can utilize a wide range of nitrogen sources, and may interfere with host metabolism to increase the content of amino acids in infected tissues. Meanwhile, the absorption of nitrogen nutrition may play an important role in its pathogenesis. Oaspore specific aspartate aminotransferase PsAAT3 is needed in the pathogenesis of Phytophthora sojae. Aspartic aminotransferase from Phytophthora sojae is identified by bioinformatics. It is found that Phytophthora sojae has more AATs than the tested fungi. Functional domain analysis showed that some of AATs were specific to oosides. They contained additional N domain, or functional domains of benzoic acid dehydrogenase, and even some individual genes. PsAAT3 was induced to express in the process of infection. After silencing, it was found that the pathogenicity of Phytophthora sojae decreased and its growth was not on N medium. This indicates that PsAAT3 is involved in the pathogenesis and nitrogen utilization of Phytophthora sojae in the process of infection. The results show that Phytophthora soja and other pathogenic oobacteria are different from other species' amino acid metabolic pathways and PsAAT3 is needed for the pathogenesis of Phytophthora sojae. ARGONAUTE4 in different ways in Arabidopsis resistance to Phytophthora capsici and cotton Verticillium wilt: in order to study the role of AGO protein in plant immune response, by screening the ago mutants we found that Arabidopsis ago4-2 mutants resistant to Phytophthora capsici and Verticillium dahliae has significant difference. The ag04-2 mutant significantly enhanced the resistance to Phytophthora capsici, but its resistance to Verticillium wilt was weakened. Cytological observation on active oxygen and accumulation of callose also confirmed this result. In addition, the expression of AG04 was downregulated during the treatment of Phytophthora capsici.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.651
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本文編號：1342664