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dc.contributorChieh-Chen Huangen_US
dc.contributorErh-Min Laien_US
dc.contributor.advisorHau-Hsuan Hwangen_US
dc.contributor.authorLee, Ying-Lingen_US
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dc.description.abstract土壤農桿菌(Agrobacterium tumefaciens)為植物病原菌,其感染過程是利用VirA/VirG兩蛋白質所構成的two-component系統所啟動及調節。此系統可辨認自植物根部傷口所分泌出的酚類化合物(例如:乙醯丁香酮, AS),並誘導表現位在Ti 質體上的vir基因(virulence genes),進而使得T-DNA傳遞進入植物細胞,最後嵌入植物基因體中表現。野生種農桿菌的T-DNA上含有iaaH和iaaM及ipt基因,iaaH和iaaM基因會使受感染的植物細胞,產生過多的吲哚乙酸(Indoleacetic acid﹐IAA),而ipt基因則促使寄主細胞,產生過多的細胞分裂素(cytokinin),以致植物產生冠癭狀腫瘤。在nopaline系的農桿菌Ti質體上,含有一與ipt基因同源的tzs基因,其表現亦受乙醯丁香酮和VirA/VirG two-component系統所調節。tzs基因轉譯合成反玉米素合成蛋白質(Tzs: trans-zeatin synthesis protein),可使農桿菌在乙醯丁香酮誘導下產生並分泌反玉米素(trans-zeatin)。目前研究已知Tzs蛋白質在AS誘導下,會被運送到土壤農桿菌的細胞膜上。故本研究針對Tzs蛋白質是否參與土壤農桿菌與植物交互作用,及其於感染過程中,所扮演的角色做進一步分析。本研究結果顯示當農桿菌的tzs基因突變後,會造成其在與阿拉伯芥切根共培養時生長遲緩,同時造成其阿拉伯芥根部細胞後產生腫瘤和短暫表現T-DNA效率亦下降。且經由基因及化學互補實驗得知,推測tzs突變株因缺乏細胞分裂素而導致其感染力下降。並發現當使用低濃度的野生種農桿菌感染阿拉伯芥時,外加細胞分裂素可增加其短暫表現T-DNA的效率與細菌的生長。另外,當土壤農桿菌的tzs基因突變後,會造成其於19℃或25℃酸性的AB-MES培養基中,並加入乙醯丁香酮誘導培養下生長遲緩,並使得其VirB2、VirD2和VirE2蛋白質的表現量比野生株高。由本研究結果可知農桿菌的tzs基因或反玉米素的分泌,於農桿菌感染植過過程中,可能扮演著重要的角色。zh_TW
dc.description.abstractAgrobacterium tumefaciens is a pathogen and is capable of infecting many kinds of plants and few other eukaryotic species. When plant wounded sites secrete phenolic compounds, such as acetosyringone (AS), and under acidic conditions, the VirA/VirG two-component system induces virulence gene expressions from the Ti plasmid (tumor-inducing plasmid), and transfers the transferred DNA (T-DNA) into plant cells, and finally integrated into plant genomes. In the wild type Agrobacterium, its T-DNA region contains iaaH, iaaM, and ipt genes. The iaaH and iaaM genes lead to the productions of indoleacetic acid (IAA) and the ipt gene leads to the productions of cytokinin in the infected plant cells, which causes crown gall diseases in plants. In the nopaline type Ti plasmid, there is a tzs gene which shares the sequence similarity with the ipt gene. The tzs gene expression is induced by acetosyringone and regulated by the VirA/VirG two-component system. The tzs gene encoded the trans-zeatin synthesizing protein (Tzs) can promote trans-zeatin productions and secretions in Agrobacterium. In order to explore further the importance and the roles of the tzs gene, we utilized the tzs deletion and frame-shift mutants to determine if the tzs gene is involved in the Agrobacterium virulence. Our data demonstrated that tzs deletion and frame-shift mutants showed growth defects and reduced the stable and transient transformation efficiencies when infected Arabidopsis roots. Data from genetic and chemical complementation tests showed that reduced infection efficiencies can be complemented by exogenous cytokinins. Additionally, supplements of cytokinin during infections slightly enhanced the growth and infection ability of lower concentrations of the wild-type Agrobacterium. Our data also revealed that tzs mutants showed growth defects when grown in acidic AB-MES medium with AS at either 19℃ or 25℃, and caused higher VirB2, VirD2, and VirE2 protein expressions compared to the wild type. Taken together, our data suggest that the tzs gene and/or trans-zeatin secretions may play an important role during infections.en_US
dc.description.tableofcontents中文摘要..............................................i 英文摘要..............................................ii 目錄..................................................iii 圖目錄................................................v 表目錄................................................vii 壹、前言. .............................................1 一、土壤農桿菌感染植物的過程..........................1 二、土壤農桿菌的群數感應(qurom-sensing)系統...........4 三、細胞分裂素........................................5 四、tzs基因相關之前人研究.............................7 五、本研究之目的......................................8 貳、材料與方法........................................10 一、菌種和質體........................................10 二、培養基成分........................................13 (一) 523培養基........................................13 (二) B5固態培養基.....................................13 (三) MS固態培養基.....................................13 (四) CIM (callus induction medium)固態培養基..........13 (五) AB-MES液態培養基.................................13 三、土壤農桿菌的培養..................................14 四、阿拉伯芥植物的培養................................14 五、土壤農桿菌腫瘤性狀分析 (Tumorigenesis assays).....14 六、土壤農桿菌短暫表現T-DNA之性狀分析(Transient transformation assays)................................15 七、土壤農桿菌於與阿拉伯芥切根共培養時生長狀態分析....15 八、土壤農桿菌於AB-MES液態培養基的生長狀態與致病蛋白質表現分析....................................................16 (一) 土壤農桿菌生長狀態測定...........................16 (二) 細菌蛋白質萃取...................................16 (三) 蛋白質濃度測定...................................16 (四) 蛋白質電泳分析 (SDS-polyacrylamide gel analyses).17 (五) 西方轉漬法 (Western blot analysis)...............17 參、結果..............................................19 一、土壤農桿菌tzs基因缺失(deletion)突變和框移(frame-shift)突變,造成其感染阿拉伯芥切根後產生腫瘤的效率下降........19 二、土壤農桿菌tzs基因缺失突變和框移突變,造成其感染阿拉伯芥根部後短暫表現T-DNA的效率下降.........................19 三、土壤農桿菌tzs基因框移突變株,可經基因互補實驗(genetic complementation test)增加其短暫表現T-DNA的效率........20 四、土壤農桿菌tzs基因缺失突變株和框移突變株,可經化學互補實驗(chemical complementation test)增加其短暫表現T-DNA的效率....................................................21 五、土壤農桿菌tzs基因缺失突變株和框移突變株,可經化學互補實驗(chemical complementation test)增加其產生腫瘤的效率.22 六、細胞分裂素可增加低濃度(105 cells/ml和106 cells/ml)野生株土壤桿菌感染阿拉伯芥切根後的短暫表現T-DNA效率.........23 七、土壤農桿菌tzs基因突變後,會造成其於MS培養基上及與阿拉伯芥切根共培養時生長遲緩及細菌累積生長量減少............24 八、土壤農桿菌tzs基因框移(frame-shift)突變株,可經基因互補實驗增加其於MS培養基上及與阿拉伯芥切根共培養時的生長及細菌累積生長量................................................26 九、細胞分裂素可增加低濃度(106 cells/ml)野生株土壤農桿菌於與阿拉伯芥切根共培養時的生長及細菌累積生長量............26 十、土壤農桿菌tzs基因突變後,會造成其於19℃或25℃在酸性AB-MES培養基中,並加入乙醯丁香酮誘導性培養下生長遲緩.....27 十一、tzs基因突變後使得VirB2、VirD2和VirE2蛋白質累積量比野生種高..................................................28 肆、討論..............................................31 伍、參考文獻..........................................36zh_TW
dc.subjectAgrobacterium tumefaciensen_US
dc.subjectvirulence geneen_US
dc.subjecttrans-zeatin synthesis proteinen_US
dc.subjecttzs geneen_US
dc.titleFunctional studies of the Tzs protein in Agrobacterium tumefaciens virulence and growthen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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