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研究生:黃奕鼎
研究生(外文):Yi-Ding Huang
論文名稱:腸炎弧菌93內的轉錄因子RfaH之功能分析
論文名稱(外文):Functional analysis of Vibrio parahaemolyticus 93 transcription factor RfaH
指導教授:林光慧
指導教授(外文):Guang-Huey Lin
學位類別:碩士
校院名稱:慈濟大學
系所名稱:微生物學免疫學暨生物化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:80
中文關鍵詞:腸炎弧菌
外文關鍵詞:RfaH
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腸炎弧菌是造成腹瀉與敗血症的主要病源菌。本實驗的研究目的是了解為何腸炎弧菌可以抵抗胃酸到達小腸而造成人類疾病,在本篇研究中以pH為5的酸性培養基篩選腸炎弧菌93跳躍子突變菌株庫,並利用隨機黏附聚合酶鏈鎖反應放大並選殖被跳躍子破壞的基因。在序列分析後,我們進行核苷酸比對找到與耐酸性相關的基因rfaH,同時我們也針對此基因進行突變株、互補株、大量表現菌株的建構。比較野生株與突變菌株的生長曲線並無顯著差異,顯示rfaH並不影響突變株的存活能力,當野生株的rfaH互補入突變株時,突變株對酸的耐受性回復,證實rfaH對酸耐受性的重要性。同時也觀察到,突變株形成生物膜的能力略低於野生株而大量表現菌株的生物膜形成能力則明顯高於野生株。此外,觀察細菌菌落形態時發現,突變株表面光滑無皺摺產生,而互補株與大量表現菌株的表面則是有觀察到明顯的皺摺。此外,RfaH會專一的與12 bp的ops (operon polarity suppressor)序列結合後抑制轉錄終止,因此我們將此序列與腸炎弧菌全基因體進行比對,分別在三個基因組的啟動子序列與五個基因內找到ops。當rfaH在酸性環境時,其參與酯多醣 (LPS)生合成的酵素相關基因表現量會增加(VPA1403、VP0890),同時也會增加產生胺基酸asparagine基因(VPA1717)的表現量,另一方面會去抑制能量代謝相關基因的表現量(VPA0117),以節省能量消耗。由此實驗可以得到結論,RfaH會幫助V. parahaemolyticus 93抵抗酸性環境。
Vibrio parahaemolyticus is a pathogen that causes gastroenteritis and septicemia. The aim of this research is to elucidate the mechanism by which V. parahaemolyticus resists stomach acid, allowing the organism reaches the small intestine to cause diseases. This study screens a V. parahaemolyticus mutant library that had been established by transposon mutagenesis for the mutants that are resistant to acid using a pH 5 TSB3S medium. The screening identifies an acid-resistant mutant that contains a transposon inserted in rfaH. Meanwhile, the sensitivity to acid is restored after a wild-type rfaH gene is introduced into the mutant, verifying the importance of rfaH in acid resistance. This study also finds that the rfaH mutation does not affect bacterial growth as the growth rate of the mutant strain is comparable to that of the wild-type strain. Additionally, not only does the rfaH mutation abolish biofilm formation, but also overexpression of RfaH in the wild-type strain increases the capacity of the bacteria to produce biofilms and also gives the colonies a rugose phenotype. RfaH is a transcription factor that binds to a specific sequence, ops, to prevent transcription termination. Analyzing the genome sequence of V. parahaemolyticus reveals the promoters of 3 operons and 5 genes contain ops. Additionally, rfaH appears to activate the transcription of asparagine (VPA1717) and LPS synthesis (VPA1403, VP0890) operons under acidic conditions. The expression of rfaH also represses the genes (VPA0117) that are involved in energy production. The study concluds that RfaH confers resistance of V. parahaemolyticus 93 to acid.
目錄
摘要 iv
Abstract v
前言 1
腸炎弧菌 (Vibrio parahaemolyticus) 2
跳躍子及其應用 3
RfaH的角色與功能 4
生物膜 (biofilms) 6
總結 7
材料與方法 8
一、材料 8
二、實驗方法 9
結果 20
一、腸炎弧菌突變庫篩選 20
二、腸炎弧菌rfaH序列分析 20
三、腸炎弧菌rfaH突變株、互補株及大量表現菌株之建構 22
四、腸炎弧菌各菌株型態觀察與生理特性測試 24
五、各菌株在環境壓力下轉錄階層的表現情況 26
討論 28
一、RfaH在腸炎弧菌參與菌落皺摺型態轉變 28
二、RfaH在腸炎弧菌參與生物膜的生成 29
三、RfaH在腸炎弧菌遭受環境壓力時的存活反應 30
四、RfaH在腸炎弧菌遭受環境壓力時轉錄階層的調控 31
參考資料 67
參考資料

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