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研究生:劉安琪
研究生(外文):An-Chi Liu
論文名稱:農桿菌受dimethylsulfoxide抑制蛋白質ActC之研究
論文名稱(外文):A putative tricarboxylate transporter, ActC, is down regulated by dimethyl sulfoxide in Agrobacterium tumefaciens
指導教授:許濤許濤引用關係賴爾珉
指導教授(外文):Todd HsuErh-Min Lai
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:116
中文關鍵詞:抑制檸檬酸轉送蛋白農桿菌
外文關鍵詞:ActCAgrobacteriumcitratedimethyl sulfoxiderepresstransporter
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農桿菌(Agrobacterium tumefaciens)為土壤植物病原菌,會感染許多真雙子葉植物造成腫瘤病(crown gall disease),其致病機制主要是利用VirA/VirG two-component system感知植物受傷組織釋放之酚類化合物以誘導一群Vir蛋白質,進而將tumor-inducing (Ti) plasmid上的一段transferred DNA (T-DNA)轉入植物所造成。本實驗室先前利用二維電泳配合質譜儀之方法,在農桿菌中鑑定到11個被酚類化合物acetosyringone (AS)誘導的蛋白質。此外,我們也發現一個未知蛋白質在AS處理後表現量會降低。本論文針對此抑制蛋白質進行其基因調節及功能探討,由於此蛋白質與tricarboxylate (citrate) transporter protein C (TctC)有相似性,故命名為ActC (Agrobacterium citrate transporter C)。針對ActC蛋白質表現之調控分析,構築一表現glutathione S-transferase (GST)ActC蛋白質的質體,大量表現及純化後免疫兔子取得抗血清。經由西方墨點法,意外發現此蛋白質的表現或累積是受到溶解AS之有機溶劑dimethyl sulfoxide (DMSO)所抑制,同時也證明此抑制與Ti plasmid不相關。進一步利用不同濃度之DMSO添加測試,發現ActC蛋白質之抑制程度受劑量所調控,隨濃度增加(0.5 ~10 mM)而有漸趨明顯之抑制效果。但更高濃度之DMSO (10~ 100 mM)對ActC之抑制程度相同。同時構築一actC啟動子融合綠螢光蛋白質報導基因,實驗結果推測actC基因在transcriptional level即受到DMSO之抑制。我們也發現,不論在蛋白質或啟動子層級,actC之表現在不處理DMSO時在log phase期間會隨著細菌生長而增加,推測actC基因可能與細菌生長有關。同時,本論文也探討是否ActC在農桿菌致病上之重要性。根據馬鈴薯腫瘤定量分析之結果發現在農桿菌中過度表現ActC會造成腫瘤產生率的下降。但是刪除actCBA operon之突變株,其腫瘤產生率則是與野生株相同。本論文證明actC表現與細菌生長有關且受DMSO所抑制,其過度表現會降低農桿菌之制病力。
Agrobacterium tumefaciens is a soil-borne plant pathogen that causes crown gall disease in various eudicotyledonous plants. A. tumefaciens utilizes the VirA/VirG two-component system to sense the phenolic compounds such as acetosyringone (AS), leading to the transfer of transferred DNA (T-DNA) from tumor-inducing (Ti) plasmid into plant cells. In the efforts to identify AS-induced proteins by proteomics approaches, we also discovered an unknown protein that is repressed by AS treatment. This protein is named as ActC (Agrobaterium citrate transporter C ) due to its amino acid sequence homology to that of tricarboxylate transporter protein C (TctC) in Salmonella typhimurium. In this study, GST-TctC fusion protein was overexpressed and purified to generate an antibody. Immunoblotting demonstrated that ActC protein level is decreased with the presence of dimethyl sulfoxide (DMSO), the solvent used to dissolve AS. ActC protein level is decreased with the increase of DMSO concentration, while the growth of A. tumefaciens was unaffected. In parallel, we generated transcriptional fusion to analyze the transcription of actC. While the actC promoter activity remains at the similar level after DMSO treatment at 19℃ for 5 days, the promoter activity increases along with the Agrobacterium growth during the log phase in the absence of DMSO. Likewise, the expression of actC also increases during the log phase in the absence of DMSO as the cell number increases. To determine whether ActC play a crucial role on Agrobacterium virulence, the over-expressed and deletion strains were generated and analyzed by quantitative tumor assay using potato tuber discs. The data showed that the efficiency of tumor formation is slightly reduced when ActC is over-expressed. However, no effects on the tumorigenesis efficiency on potato discs can be observed when the actCBA operon was deleted. In conclusions, the expression of actC is repressed by DMSO and related to bacterial growth. Overexpression of ActC result in the decrease of A. tumefaciens tumorigenesis efficiency.
目錄
中文摘要…………………………………………………………………I
英文摘要………………………………………………………………III
壹、 前言
一、 農桿菌 ……………………………………………………………1
二、dimethyl sulfoxide (DMSO)……………………………………14
三、Tricarboxylate transporter …………………………………17
貳、研究緣起 …………………………………………………………22
參、實驗材料及方法 …………………………………………………23
肆、結果 ………………………………………………………………44
伍、討論 ………………………………………………………………54
參考文獻 ………………………………………………………………61
圖表 ……………………………………………………………………77
附錄 ……………………………………………………………………98
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