灰葡萄孢菌隔膜蛋白septin的生物学功能解析

发布时间：2018-03-24 17:19

本文选题：灰葡萄孢菌　切入点：隔膜蛋白septin　出处：《吉林大学》2017年硕士论文

【摘要】：灰葡萄孢菌是一种全球重要的植物病原真菌,属于典型的死体营养型,寄主范围十分广泛,能够侵染多达1400种植物。该菌不仅能在植物生长期侵染,也能在果蔬储藏期侵染发病,危害广泛,损失严重。目前灰葡萄孢菌作为一种模式植物病原备受关注,但关于其寄主侵染过程的分子机理仍不清楚。在过往的研究中发现,灰葡萄孢菌需要形成特定的侵染结构:附着胞或侵染垫,才能正常侵入寄主,进一步分泌多种致病因子杀死寄主从而获取营养。此外,隔膜蛋白septin作为真核生物中一类高度保守的蛋白家族,参与细胞骨架,细胞周期,细胞分泌等多项生命过程并发挥重要功能。在真菌中septin蛋白家族具有四个核心成员:Sep3、Sep4、Sep5、Sep6,其中Sep4是最小的蛋白。本研究重点解析新型细胞骨架隔膜蛋白septin如何在灰葡萄孢菌生长发育发挥功能,并参与寄主侵染过程,主要研究结果如下:(1)通过生物信息学分析,检索到灰葡萄孢菌中四个核心septins组分:BcSep3、BcSep4、BcSep5、BcSep6;(2)利用农杆菌介导转化,同源重组基因替换的方法,筛选获得灰葡萄孢菌四个核心septin基因敲除突变体ΔBcsep3,ΔBcsep4,ΔBcsep5,ΔBcsep6菌株。通过致病性分析,结果表明核心septins组分是灰葡萄孢菌致病所必需的;(3)通过野生型与突变体表型差异分析,发现BcSep4是灰葡萄孢菌侵染结构发育所必需的;另外,BcSep4还在灰葡萄孢菌无性繁殖、芽管发育、柔性生长、逆境响应、胞内物质运输等过程中发挥重要功能;(4)通过GFP融合亚细胞定位,证明BcSep4可能通过参与脂膜微曲面形成,调控灰葡萄孢菌生长发育和侵染结构形成过程;(5)利用二亚苯基碘(diphenylene iodonium,DPI)和环磷酸腺苷(cyclic adenosine monophosphate,cAMP)处理,发现灰葡萄孢菌BcSep4在侵染结构形成过程中受活性氧ROS信号传导调控,并与cAMP信号传导有关;(6)通过对四个核心septin基因缺失突变体菌株ΔBcsep3、ΔBcsep4、ΔBcsep5、ΔBcsep6生物表型分析,发现四个核心septin组分都是灰葡萄孢菌侵染结构形成所必需的;(7)对四个核心septin组分分别进行亚细胞定位,结果表明BcSep3、BcSep4、BcSep5、BcSep6在胞内能够以不同的自我组装分配模式,影响细胞极性,共同调控灰葡萄孢菌生长发育和侵染结构形成。本研究首次解析灰葡萄孢菌septin的生物学功能,让我们对灰葡萄孢菌的生长发育和分子致病机理有了更深入的认识。由于植物中没有septin存在,因此未来可能作为潜在的防治靶标应用于农业防治中。
[Abstract]:Grapevine cinerea is one of the most important plant pathogenic fungi in the world. It belongs to the typical dead body nutrition type and can infect up to 1400 species of plants in a wide range of hosts. It can also infect fruits and vegetables during storage period, causing extensive damage and serious losses. At present, Grapevine Aspergillus, as a model plant pathogen, has attracted much attention, but the molecular mechanism of its host infection process is still unclear. Grapevine fungus needs to form a specific infection structure: appressorium or infection pad in order to normally invade the host, further secreting a variety of pathogenic factors to kill the host and obtain nutrition. As a family of highly conserved proteins in eukaryotes, membrane protein septin is involved in cytoskeleton and cell cycle. In fungi, the septin protein family has four core members: Sep3C Sep4N Sep5N Sep6, in which Sep4 is the smallest protein. This study focuses on how the novel cytoskeleton membrane protein septin is present in the grapevine grays of Staphylococcus gravis. The main purpose of this study is to investigate the role of septin in the development of a novel cytoskeleton membrane protein septin. The growth and development of grape spores play a role, The main results were as follows: (1) by bioinformatics analysis, we found four core septins components of Grapevine Aspergicus: BcSep3, BcSep4CSep4BcSep5BcSep5BcSep6 / 2) by Agrobacterium tumefaciens mediated transformation and homologous recombination gene replacement. Four core septin gene knockout mutants 螖 Bcsep3, 螖 Bcsep4, 螖 Bcsep5, 螖 Bcsep6 were obtained. The results of pathogenicity analysis showed that the core septins components were essential for virulence of grapevine. It was found that BcSep4 was necessary for the development of the infective structure of Grapevine Aspergillus, and that BcSep4 was also used in asexual reproduction, bud tube development, flexible growth and stress response of Grapevine. Through GFP fusion subcellular localization, it is proved that BcSep4 may be involved in the formation of lipid membrane microsurfaces. In order to regulate the growth and development of grapevine and the formation of infection structure, we found that BcSep4 was regulated by active oxygen species (ROS) signal transduction during the process of infection structure formation by using diphenyl iodiphenylene iodoniumn (DPI) and cyclic adenosine monophosphate (cAMP), which were treated with diphenyl iodiphenylene and cyclic adenosine monophosphate monophosphate (cAMP). Four core septin gene deletion mutants 螖 Bcsep3, 螖 Bcsep4, 螖 Bcsep5, 螖 Bcsep6 were analyzed. It was found that the four core septin components were subcellular localization of the four core septin components, which were necessary for the formation of infection structure of grapevine. The results showed that BcSep3, BcSep4, BcSep4 and BcSep5BcSep6 could affect cell polarity in different self-assembly patterns. In this study, the biological function of septin was analyzed for the first time. As there is no septin in plants, it may be used as a potential target for agricultural control in the future.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.44

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