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研究生:劉又維
研究生(外文):Yu-Wei leu
論文名稱:探討十字花科黑腐病菌中flhA與flhB基因之功能
論文名稱(外文):Functionl study of flhA and flhB genes in Xanthomonas campestris pv.campestris
指導教授:胡若梅
指導教授(外文):Rouh-Mei Hu
學位類別:碩士
校院名稱:亞洲大學
系所名稱:生物科技學系碩士班
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:2008
畢業學年度:96
語文別:中文
論文頁數:99
外文關鍵詞:Yu-Wei leuRouh-Mei HuflhA and flhB
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Xanthomonas campestris pv. campestris ( Xcc ) 為革蘭氏陰性菌,具單極性單鞭毛,且能感染十字花科植物而導致黑腐病。根據文獻所載鞭毛細菌與第三類型分泌系統(T3SS)關係密切,T3SS 對許多病源體的致病毒素相當重要,包括 Xcc。在相似序列中,我們在 Xcc 查尋超過 40 幾個基因為鞭毛的合成相關。rpoN、fleQ、fliA、flgM、fleN、flhF 等6個基因,在我們實驗室已證明為調控許多鞭毛基因的表現。本研究中,我們報告FlhA與FlhB的功能於相似性的T3SS 蛋白InvA與SpsS 於過去報導Salmonlla中為鞭毛構造的輸出。 Xcc flhA 與 flhB 突變株經崁入突變後分析其表現型。 flhA 或 flhB 突變株具致病性,但沒有運動的能力。兩個突變株在FleQ 與 RpoN 蛋白的表現量為正常。flhA突變株在 FliA 蛋白的表現量稍有減少,但 flhB 突變株沒有。兩個突變株分別不能合成主要的鞭毛蛋白 FliC。 fliC 基因的啟動子是包含一個供選擇的 sigma 因子RNA 聚合酶轉錄與fliA基因編碼。FlgM 蛋白是一個多重 sigma 因子抑制 FliA 。成熟鞭毛的 T3SS 從細胞質釋放 FlgM 可恢復 FliA 的功能。因為 FlhA 與 FlhB 在鞭毛 T3SS 為不可缺的成分, flhA 與 flhB 突變株在 FliC 蛋白的表現量可能是由細胞質的 FlgM 蛋白堆積所抑制。
第二部分的研究使用酵母菌雙雜交系統所測試 RpoN、 FleQ、 FleN、 FlhF、 FlgM、 FliA 和 NtrC 蛋白之間的交互作用,結果推測 RpoN2 和 FlhF; RpoN2和 FleQ; RpoN2 和 NtrC 之間有交互作用。
Xanthomonas campestris pv. campestris ( Xcc ) is a Gram negative, rod-shaped bacterium, and is a causative agent of black rot disease in cruciferous plants. According to previously reports, bacterial flagella and the type III secretion systems (TTSS) are highly related. The TTSS is essential for the virulence in many pathogens, including Xcc. Based on sequence homology, our search in Xcc revealed that more than 40 genes might be involved in the flagellar biogenesis. Six genes, rpoN, fleQ, fliA, flgM, fleN and flhF, have been demonstrated in our laboratory to be involved in the regulation of expression of many flagellar genes. In this work, we report the function of FlhA and FlhB which are homologs of TTSS proteins InvA and SpaS reported to be involved in the exportation of flagellar structure in Salmonlla. Xcc flhA and flhB mutants were obtained by insertional mutagenesis and were used for phenotypical assays. The flhA and flhB mutants are virulent, but immotile. Both mutants expressed a normal quantity of FleQ and RpoN protein. The expression of FliA protein was slightly decreased in an flhA mutant but not in an flhB mutant. Both mutants cannot synthesis the major flagellin protein FliC. Induction of plasmid-born wild-type flhA or flhB genes can successfully complement the motility defect, and restore the biosynthesis of FliC protein in flhA and flhB mutants, respectively. The promoter of fliC gene was transcribed by RNA polymerases containing an alternative sigma factor encoded by fliA gene. The FlgM protein is an anti-sigma factor acts against FliA. A mature flagellar TTSS expels FlgM from the cytoplasm and recover the function of FliA. As FlhA and FlhB are essential components of flagellar TTSS, the expression of fliC in flhA and flhB mutants might be blocked by an accumulation of cytoplasmic FlgM protein.

The second part of this work is to use a yeast two-hybrid system to detect the protein-protein interaction between RpoN, FleQ, FleN, FlhF, FlgM, FliA and NtrC. The results suggested that RpoN2 and FlhF; RpoN2 and FleQ; RpoN2 and NtrC can
interact with each other.
目錄
縮寫字對照表-1
英文摘要-3
中文摘要-5
壹、 前言-6
貳、 文獻回顧-8
參、 材料與方法-14
一、 材料-14
(一) 菌株、質體及噬菌體-14
(二) 藥品-14
(三) 培養基-14
1. 培養液(Broth-14
2. 培養基(Plate-15
3. 抗生素與其它藥品使用濃度-16
4. 試劑與緩衝溶液-16
二、 方法-20
(一) 細菌之培養與保存-20
(二) 質體 DNA 之抽取-20
(三) 聚合酶鎖反應(Polymerase chain reaction;PCR)-21
(四) 洋菜膠體電泳分析(Agarose gel electrophoresis)-22
(五) 質體的選殖(Cloning)-22
(六) 勝任細胞之製備(Competent cell)-23
(七) 轉形作用(Transformation)-23
(八) 複製噬菌體 (Amplification of bacteriophage) -24
(九) 噬菌體濃度測試 (Phage titer assay) -25
(十) 溶菌斑測試法(Spot test)-25
(十一) 生長曲線測定(Growth curve)-26
(十二) 運動性測試(Motility assay)-26
(十三) 蛋白質凝膠電泳分析(SDS-PAGE)-27
(十四) 西方墨點法(Western blot)-28
(十五) 病原性測試(Pathogenecity)-29
(十六) 胞外多醣產量(EPS assay)-29
(十七) 胞外酵素分析(Extracellular enzyme)-30
(十八) 酵母菌勝任細胞之製備(Competent cell)-31
(十九) 酵母菌轉形作用(Yeast transformation)-31
(二十) 酵母菌活性濾膜分析法(Colony-lift filter assay)-32
肆、 結果與討論-33
一、 Xcc 的 flhA 與 flhB 基因之特性-33
(一) flhA 基因之分析-33
1. flhA 突變株之構築-33
2. flhA 互補株之構築-34
(二) flhB 基因之分析-34
1. flhB 突變株之構築-35
2. flhB 互補株之構築-36
二、 flhA與flhB突變後並不影響Xcc對噬菌體的敏感性-36
三、 生長曲線分析-37
四、 運動性分析-38
五、 電子顯微鏡鏡檢顯示flhA與flhB突變後不產生鞭毛-38
六、 flhA、flhB 基因在Xcc部分鞭毛基因之關係-39
(一) FleQ 蛋白偵測-39
(二) RpoN2 蛋白偵測-39
(三) FliA 蛋白偵測-40
(四) FliC 蛋白偵測-40
(五) PilA 蛋白偵測-41
七、 flhA突變會降低致病能力,但flhB突變後則否-42
八、 flhA與flhB突變不影響胞外多醣的產量-43
九、 flhA與flhB突變不影響胞外酵素的分解能力-43
十、 蛋白與蛋白交互作用分析-44
(一) 酵母菌雙雜交系統-44
1. 質體之構築-44
2. 濾膜呈色分析-47
十一、 結論-48
柒、 參考文獻-90
研討會論文發表-93
柒、 參考文獻
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