臺灣博碩士論文加值系統

貳、中文摘要
革蘭氏陰性菌 Xanthomonas campestris pv. Campestris 為十字花科蔬菜黑腐菌的植物病原菌。由於它會分泌大量的胞外多醣 (exopolysaccharide,簡稱 EPS)，因此使得菌外觀呈現黃色黏溼狀，工業上稱此胞外多醣為 Xanthan gum。Xanthan gum 有許多用途，可利用於鑽探石油時的潤滑、食品加工、農業上以及工業上。此菌所產生的胞外多醣與其致病性有關。
生物為了適應不同的環境變化，會發展出多種不同的因應方法。細菌當面對環境的轉變，會藉著RNA 聚合酶和所含的不同 σ 因子及相關的調控蛋白來活化或關閉一群受其調控的基因。鞭毛形成的過程是非常複雜的，鞭毛的形成是非常複雜和被幾個  因子與轉錄調控者所調節。

本研究探討在 Xanthomonas campestris pv. campestris 中 fleN 所扮演的功能與角色，於是將此基因利用 insertional mutation方法，取得 Xc17 的 fleN 突變株。本研究結果發現，fleN 突變株在電子顯微鏡下呈現多鞭毛 (三根) 之現象，在運動性測試方面，fleN 破壞後呈現不具運動之現象，並且在致病能力與胞外酵素分泌並沒有影響。在β-半乳醣苷酶活性測試下， FleN 負調控鞭毛基因 fliE (鞭毛 MS 環桿狀交界蛋白基因)、fliL (鞭毛運送蛋白基因)、fliQ (鞭毛運輸蛋白基因)、flgB (鞭毛基底的桿狀物近端結構蛋白基因)、flgG (鞭毛基底的桿狀物遠端結構蛋白基因) 及 flhF (鞭毛生合成基因)，但對 fliC (鞭毛蛋白) 及非鞭毛基因 pilA1 (纖毛合成蛋白) 並沒有影響。

Abstract
Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot disease in cruciferous plants. This bacterium synthesizes great amounts of xanthan gum rendering the colonies mucoid. Xanthan gum has many industrial applications, such as oil drilling, food, agriculture and industry. Production of xanthan gum is also refuired for the virulence of this bacterium.

To adapt to the diverse environmental changes, bacteria develop various responsive mechanisms. Bacteria are able to activate or switch off specific sets of genes as they face changing environmental conditions. This can be achieved through the activities of RNA polymerases containing alternate sigma factors and their cognate regulatory proteins. Biogenesis of flagellum is very complicate and is delicately regulated by several sigma factors and transcription regulators.

Xcc fleN mutants were constructed by insertional mutagenesis. Mutation in fleN does not interfere with the pathogenicity and exocellular enzymes production and growth of the cell. Transcriptional fusion assay indicated that fleN Electron microscopy photography revealed that fleN mutants are hyper-flagellated but immotile, fleN regulates expression of six flagellar genes, including fliE, fliL, fliQ, flgB, flgG and flhF, in a negative manner. However, fleN is not necessery for the expression of the major flagellin gene fliC, nor for the type IV pilin gene pilA1.

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目錄
壹、縮寫字對照表 1
Abstract 3
貳、中文摘要 4
參、前言 5
肆、材料與方法 10
A. 材料 10
一、菌種、噬菌體、及質體 10
二、一般藥品 10
三、酵素 10
四、培養基 10
五、試劑與緩衝溶液 11
B. 方法 13
一、細菌之培養與保存 13
二、DNA 之製備 13
三、洋菜膠體電泳分析 14
四、聚合酶鏈鎖反應 15
五、質體之選殖 (cloning) 15
六、細菌之轉形作用 16
七、噬菌體 L7 的培養 18
八、噬菌體 Lf 的培養 19
九、噬菌體濃度 ( titer ) 測定 19
十、運動性 ( Motility) 測試 19
十一、胞外酵素分泌測試 19
十二、生長曲線 20
十三、點測試法 ( Spot test ) 20
十四、病原性測試 ( Pathogenicity ) 21
十五、電子顯微鏡觀測 (Electron microscope, EM) 21
十六、β-半乳醣苷酶活性 ( β-galactosidase ) 測試 21
伍、結果與討論 23


一、 Xc17 之 fleN突變株，Xc17fleN::Gm1與 Xc17fleN::Gm2之構築與特性分析 23
(一) 質體 pBB-fleN之構築 23
(二) 同源互換之構築 24
(三) Xc17 野生株及 Xc17fleN::Gm1與Xc17fleN::Gm2 突變株之特性分析 24
1. Xc17fleN::Gm1突變株在電子顯微鏡觀察之表現分析 24
2. Xc17 野生株及 Xc17fleN::Gm1與Xc17fleN::Gm2突變株在運動性測試之表現分析 25
3. Xc17 野生株及 Xc17fleN::Gm1與Xc17fleN::Gm2突變株胞外酵素分泌測試之表現分析 25
4. Xc17 野生株及 Xc17fleN::Gm1 與 Xc17fleN::Gm2 突變株點測試法之表現分析 27
5. Xc17 野生株及 Xc17fleN::Gm1 突變株致病性之測試分析 27
二、 鞭毛基因 fliE, fliL, fliQ, flgB, flgG, fliC, flhF 與非鞭毛 (纖毛) 基因 pilA1 (pilE) 啟動子 (promoter) 區域之選殖、與啟動子區域 -galactosidase 活性之分析 28
(一) 鞭毛基因與非鞭毛基因啟動子區域之選殖 28
(二) 鞭毛基因啟動子區域 -galactosidase 活性之分析 29
1. fliE 啟動子區域 -galactosidase 活性之分析 29
2. fliL 啟動子區域 -galactosidase 活性之分析 30
3. fliQ 啟動子區域 -galactosidase 活性之分析 31
4. flgB 啟動子區域 -galactosidase 活性之分析 32
5. flgG 啟動子區域 -galactosidase 活性之分析 33
6. flhF 啟動子區域 -galactosidase 活性之分析 34
7. fliC 啟動子區域 -galactosidase 活性之分析 36
(三) 非鞭毛 (纖毛) 基因啟動子區域 -galactosidase 活性之分析 37
1. pilA1(pilE) 啟動子區域 -galactosidase 活性之分析 37
陸、圖表 39
柒、參考文獻 79

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