疫霉菌氮素代谢途径与植物AGO4蛋白在其互作中的功能研究

发布时间：2017-12-27 18:19

  本文关键词：疫霉菌氮素代谢途径与植物AGO4蛋白在其互作中的功能研究　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 卵菌 大豆疫霉 氮素营养 营养吸收 致病性 ARGONAUTE4 抗病性

【摘要】：在植物与病原微生物的互作过程中,微生物要建立成功的寄生关系,不但要克服寄主的防御反应,还要能有效的从寄主体内吸取自身所需要的营养物质,其中氮素营养的争夺和利用是病原微生物与寄主间互作的关键因素之一。同时,寄主植物也进化出了复杂的防御系统来抵抗病原菌的入侵,其中植物Argonaute (AGO)蛋白是小分子RNA介导的基因沉默机制中效应复合体的核心组分,对基因表达的重编程起重要作用。目前对疫霉菌侵染过程中的氮素营养利用方式以及AGO蛋白在抗病中的功能与作用机制还缺乏系统了解,本研究分别以大豆疫霉菌和模式植物拟南芥为实验材料对这两个科学问题进行了初步的研究,获得的主要发现如下:全基因分析揭示了大豆疫霉中氮素代谢相关基因的进化特征:利用生物信息学技术对大豆疫霉的氮素营养吸收、氮素同化和氨基酸合成与代谢三个方面的基因进行了鉴定,并和其它病原卵菌和真菌进行了比较分析。在大豆疫霉中鉴定到274个与氮素代谢相关基因,相对于其它物种更为丰富,除了赖氨酸、苯丙氨酸和酪氨酸的合成途径中部分基因是缺失的外,其它参与氮素代谢的基因在7个物种中都是保守存在的。参与GABA代谢的基因在大豆疫霉中是显著扩张的,并且在侵染过程中是上调表达的。进一步分析发现在卵菌氨基酸合成途径中存在大量的多功能酶,并且与植物和真菌在结构组成上具有一定的差异。结果表明卵菌的氮素代谢与其它物种相似,但是在数量分布和基因结构上存在一定的差异:大豆疫霉可以吸收和利用各种形式的氮素来合成除赖氨酸、苯丙氨酸和酪氨酸外的全部氨基酸,并且可能通过代谢寄主防卫反应的产物GABA和尿酸来作为抵抗寄主防卫的一种策略。基于氮素营养吸收发现其参与大豆疫霉的致病过程:通过生物信息学分析,鉴定了大豆疫霉中参与氮素转运的基因并与其它所检测物种进行比较分析,在大豆疫霉中分别鉴定了 8个铵盐转运子、16个硝酸盐/肽转运子和78个氨基酸转运子并且它们的数目都是显著多于其它卵菌和真菌,表明转运子基因在大豆疫霉中经历了显著地扩张。其中Rh型铵盐转运子基因在卵菌中是特异的,与此相反的是,植物和真菌中普遍存在的寡肽转运子在卵菌中没有鉴定到同源基因。大豆疫霉的转录组数据显示大部分转运蛋白基因在其侵染过程中是上调表达的,并且在RT-PCR中得到了验证。通过分析大豆疫霉在含有不同氮源的合成培养基上生长状态,发现大豆疫霉可以在以不同氮素形式为单一氮源的培养基上生长,并且不同氮素对其生长的影响是有差异的,例如半胱氨酸显著抑制了其生长,而甲硫氨酸、谷氨酸、天冬酰胺等则有利于其生长。进一步研究发现大豆疫霉侵染的叶片中游离氨基酸的含量是增加的,其中谷氨酸最为显著。此外,大豆疫霉的铵盐转运子PsRh1是定位质膜上并且是受无机氮盐诱导表达的。基于基因的特异性和受寄主诱导表达的特性,选择铵盐转运子PsRh1和氨基酸转运子PsCAT3通过PEG介导的沉默进一步研究其在致病中的功能,结果发现分别沉默这两个基因后大豆疫霉的致病力明显下降。这些结果表明大豆疫霉可以利用广泛的氮源,并且在侵染过程中可能会通过干扰寄主的代谢来增加侵染组织中氨基酸的含量,同时氮素营养的吸收在其致病中可能扮演着重要的角色。卵菌特异的天冬氨酸转氨酶PsAAT3在大豆疫霉致病过程中是需要的:通过生物信息学鉴定了大豆疫霉的天冬氨酸转氨酶,发现大豆疫霉比所检测的真菌编码了数量更多的AATs。功能域分析发现其中一些AATs是卵菌所特有的,它们在N端分别包含了额外的预苯酸脱水酶功能域或苯酸脱氢酶功能域,甚至个别基因两者兼有。PsAAT3是在侵染过程中诱导表达的,将其沉默后发现,大豆疫霉的致病性下降并且影响其在不含N素培养基上的生长,这表明PsAAT3在侵染过程中参与到了大豆疫霉的致病和氮素利用。该结果表明大豆疫霉和其它病原卵菌有区别于其它物种的氨基酸代谢途径并且PsAAT3是大豆疫霉致病所需的。ARGONAUTE4以不同的方式参与拟南芥对辣椒疫霉和棉花黄萎菌的抗性:为了研究AGO蛋白在植物免疫反应中的作用,通过ago突变体的筛选我们发现拟南芥ago4-2突变体对辣椒疫霉和棉花黄萎菌的抗性具有显著差异。ag04-2突变体显著增强了对辣椒疫霉的抗性,但是其对棉花黄萎菌的抗性是减弱的。活性氧和胼胝质积累的细胞学观察也证实了这一结果。此外,AG04在辣椒疫霉处理时是下调表达的,而黄萎菌则诱导其上调表达。进一步研究发现,RdDM途径中在AG04上游发挥功能的成员突变体如nrpe1、rdr2-2及dcl3-1和处于下游的成员突变体如drd1-6、nrpdl对辣椒疫霉的抗性是增强的,但是这些成员的突变体对棉花黄萎菌的抗性却没有发生改变。结果表明AG04可能是依赖于RNA介导的DNA甲基化途径来负调控拟南芥对辣椒疫霉的抗性,而其参与正调控对黄萎菌的抗性可能是通过其它途径来实现的。
[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.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.651
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