臺灣博碩士論文加值系統

農桿菌是一植物病原細菌，其可感染大多數的雙子葉植物，產生腫瘤的病徵；腫瘤的產生係由於農桿菌會將其T-DNA (Transferred DNA) 切下並送至植物基因組中，而將植物細胞轉形為腫瘤。當植物細胞受傷時會釋出酚類化合物（例如acetosyringone，AS），進而誘導農桿菌ㄧ巨大質體Ti (tumor-inducing) 質體之毒性基因 (vir genes) 的表現，其表現係由VirA/VirG two-component 系統所調控，故稱為vir regulon。 vir regulon中之virB操縱子包含 (virB1~virB11)會轉譯出11個VirB蛋白質，與VirD4組成第四型分泌系統，負責T-DNA及其他基質的轉移。農桿菌培養於含AS之酸性AB-MES minimal培養基中可有效地誘導毒性基因在轉錄層級之表現，但在酸性rich培養基中是否可有效地誘導其研究結果卻不一致，而對於毒性基因在後轉錄層級的調控也未有系統性的分析。所以，本論文利用已知適合毒性基因誘導的酸性AB-MES minimal培養基，以及選用酸性的523 rich培養基當作控制組，針對virB毒性基因受AS誘導表現之轉錄、轉譯及蛋白質穩定狀態進行探討，期了解virB基因是否在不同培養基中受到不同層級的調控。首先，利用轉錄融合分析毒性基因啟動子的活性，發現virB、virD及virE啟動子在AB-MES及523培養基皆可有效地進行轉錄，但在AB-MES中較523培養基在AS誘導前期有較高的表現；進一步針對virB毒性基因之表現再使用西方雜合反應分析VirB蛋白質穩定狀態，發現不同的VirB蛋白質在不同培養基中累積的狀況不同，可將其概分為三群。第一群包含VirB2、VirB7及VirB9，此群蛋白質在AS誘導前期（16小時前）在AB-MES中之累積量較高於其在523中之累積量；第二群包含VirB1、VirB4、VirB5、VirB8及VirB11，此群蛋白質在AS誘導後期 （24小時後） 在AB-MES中之累積量較高於523中之累積量；第三群包含VirB3及VirB10，此群蛋白質在AB-MES中之累積量遠高於其在523中之累積量。再透過轉譯融合分析VirB蛋白質轉譯效率，與virB啟動子活性比較，發現轉譯融合菌株除了VirB1-GFP無法偵測到GFP螢光值，其餘VirB2-GFP、VirB3-GFP及VirB10-GFP皆能有效地被AS誘導表現，推測當農桿菌523培養基受AS誘導時，可有效率地轉錄及轉譯出VirB蛋白質，但不同之VirB蛋白質則有不同的穩定度。利用IncQ質體RSF1010經由VirB/D4在農桿菌間之轉移試驗，發現RSF1010在523培養基的轉移效率極低，為在AB-MES培養基之0.41%，推測在523培養基RSF1010之低轉移效率可能係由於部分VirB蛋白質之低累積量，進而無法組合成有功能之T4SS來進行DNA之轉移。

Agrobacterium tumefaciens is a plant pathogenic bacterium, the causal agent of crown gall diseases on wide range of dicotyledons. The tumors are caused by transferring T-DNA (Transferred DNA) from bacterium into the host plant genome. A. tumefaciens is capable of sensing the plant-released signal molecules such as sugars and phenolic compounds (e.g. acetosyringone，AS) to activate the expression of virulence (vir) genes encoded by the tumor-inducing (Ti) plasmid. Among these, the virB operon encoding 11 VirB proteins and VirD4 comprises a type IV secretion system (T4SS) to transfer T-DNA and effectors from bacteria into the host plant cells. AS-induced vir gene expression is regulated at transcriptional level via VirA/VirG with maximal activity in acidic AB-MES minimal medium. However, it is not clear whether vir genes can be efficiently induced by AS when grown in rich medium or are regulated at posttranscriptional levels. In this study, we aim to understand whether virB genes are expressed and regulated differently when A. tumefaciens is grown in different culture media for AS induction. The virB gene expression was analyzed at transcriptional/translational levels and protein steady state when induced by AS in both acidic AB-MES minimal medium and acidic 523 rich medium. By transcriptional fusion to gfp (green fluorescent protein), the promoter activities of virB, virD, and virE are efficiently induced when grown in either AB-MES or 523 media although the promoter activities are higher in AB-MES medium than in 523 medium at early time points. To further investigate virB gene expression at protein levels, Western blotting and translational fusions were carried out. By Western blot analysis, three groups of VirB proteins are classified based on their protein accumulation patterns in both AB-MES and 523 cultures. VirB2, VirB7, and VirB9 belong to group I as they accumulate at higher levels at early time points (up to 16 hr) in AB-MES in comparison to those in 523. The second group of proteins include VirB1, VirB4, VirB5, VirB8, and VirB11 as they accumulate at higher levels at late time points (after 24 hr) in AB-MES in comparison to those in 523. The third group includes VirB3 and VirB10 as they accumulate at very low levels in 523, in contrast to those at much higher levels in AB-MES. Interestingly, several VirB-GFP translational fusions result in efficient AS-induced activity in both AB-MES and 523 media. By VirB/D4-mediated IncQ plasmid RSF1010 transfer between A. tumefaciens strains, only very little transfer events are detected when conjugation was performed on 523. In conclusions, our data suggest that virB is transcribed and translated efficiently when A. tumefaciens was induced by AS in both AB-MES and 523 media. However, while all tested VirB proteins accumulated stably when grown in AB-MES medium, certain VirB proteins are less stable when grown in 523 medium, which may lead to the failure in assembly of the functional T4SS for DNA transfer.

口試委員會審定書....................................I
誌謝...............................................II
中文摘要..........................................III
英文摘要............................................V
表目錄...........................................VIII
圖目錄.............................................IX
壹、前言............................................1
一、農桿菌簡介...................................1
二、農桿菌研究的重要里程碑.......................1
三、農桿菌感染植物的分子機制.....................3
四、農桿菌毒性基因的表現及調控之研究.............8
貳、研究目的.......................................11
参、研究材料與方法.................................12
肆、研究結果.......................................24
一、農桿菌在不同培養基其virB基因表現的分析......24
二、 IncQ質體 RSF1010的轉移效率試驗.............30
伍、討論...........................................31
參考文獻...........................................36
附錄...............................................70
附錄圖.............................................75

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