本文選題：灰葡萄孢菌　切入點：隔膜蛋白septin　出處：《吉林大學》2017年碩士論文

【摘要】：灰葡萄孢菌是一種全球重要的植物病原真菌,屬于典型的死體營養(yǎng)型,寄主范圍十分廣泛,能夠侵染多達1400種植物。該菌不僅能在植物生長期侵染,也能在果蔬儲藏期侵染發(fā)病,危害廣泛,損失嚴重。目前灰葡萄孢菌作為一種模式植物病原備受關注,但關于其寄主侵染過程的分子機理仍不清楚。在過往的研究中發(fā)現(xiàn),灰葡萄孢菌需要形成特定的侵染結(jié)構(gòu):附著胞或侵染墊,才能正常侵入寄主,進一步分泌多種致病因子殺死寄主從而獲取營養(yǎng)。此外,隔膜蛋白septin作為真核生物中一類高度保守的蛋白家族,參與細胞骨架,細胞周期,細胞分泌等多項生命過程并發(fā)揮重要功能。在真菌中septin蛋白家族具有四個核心成員:Sep3、Sep4、Sep5、Sep6,其中Sep4是最小的蛋白。本研究重點解析新型細胞骨架隔膜蛋白septin如何在灰葡萄孢菌生長發(fā)育發(fā)揮功能,并參與寄主侵染過程,主要研究結(jié)果如下:(1)通過生物信息學分析,檢索到灰葡萄孢菌中四個核心septins組分:BcSep3、BcSep4、BcSep5、BcSep6;(2)利用農(nóng)桿菌介導轉(zhuǎn)化,同源重組基因替換的方法,篩選獲得灰葡萄孢菌四個核心septin基因敲除突變體ΔBcsep3,ΔBcsep4,ΔBcsep5,ΔBcsep6菌株。通過致病性分析,結(jié)果表明核心septins組分是灰葡萄孢菌致病所必需的;(3)通過野生型與突變體表型差異分析,發(fā)現(xiàn)BcSep4是灰葡萄孢菌侵染結(jié)構(gòu)發(fā)育所必需的;另外,BcSep4還在灰葡萄孢菌無性繁殖、芽管發(fā)育、柔性生長、逆境響應、胞內(nèi)物質(zhì)運輸?shù)冗^程中發(fā)揮重要功能;(4)通過GFP融合亞細胞定位,證明BcSep4可能通過參與脂膜微曲面形成,調(diào)控灰葡萄孢菌生長發(fā)育和侵染結(jié)構(gòu)形成過程;(5)利用二亞苯基碘(diphenylene iodonium,DPI)和環(huán)磷酸腺苷(cyclic adenosine monophosphate,cAMP)處理,發(fā)現(xiàn)灰葡萄孢菌BcSep4在侵染結(jié)構(gòu)形成過程中受活性氧ROS信號傳導調(diào)控,并與cAMP信號傳導有關;(6)通過對四個核心septin基因缺失突變體菌株ΔBcsep3、ΔBcsep4、ΔBcsep5、ΔBcsep6生物表型分析,發(fā)現(xiàn)四個核心septin組分都是灰葡萄孢菌侵染結(jié)構(gòu)形成所必需的;(7)對四個核心septin組分分別進行亞細胞定位,結(jié)果表明BcSep3、BcSep4、BcSep5、BcSep6在胞內(nèi)能夠以不同的自我組裝分配模式,影響細胞極性,共同調(diào)控灰葡萄孢菌生長發(fā)育和侵染結(jié)構(gòu)形成。本研究首次解析灰葡萄孢菌septin的生物學功能,讓我們對灰葡萄孢菌的生長發(fā)育和分子致病機理有了更深入的認識。由于植物中沒有septin存在,因此未來可能作為潛在的防治靶標應用于農(nóng)業(yè)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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S432.44

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本文編號：1659269