臺灣博碩士論文加值系統

具有群體感應 (quorum-sensing) 系统的細菌可相互交換一種自動誘導子 (autoinducer) 來傳遞彼此間的信息；當信號分子累積到一定濃度時則會改變細菌特定基因的表現，例如生物膜的形成、生物螢光的現象、致病基因的表現及孢子的形成等。東方弧菌 (Vibrio orientalis) 是一種海洋螢光細菌，目前對於其螢光表現及調控機制仍不甚瞭解，推測其螢光表現可能同樣受到群體感應系统的調控。本研究利用 NCBI 中的資料庫比對東方弧菌螢光操縱組 (lux operon) 之基因，發現其同樣具有 luxCDABE 等五個主要的結構基因；luxA 和luxB 基因分別可主導螢光酵素的合成，luxC, luxD及 luxE 基因則分別可主導脂肪醯基還原酵素、醯基轉移酵素及醯基蛋白質合成酵素的合成，這三個酵素可共同組成脂肪酸醯基還原酵素複合體，提供螢光酵素催化反應所需的受質。除此之外，其他位於螢光操縱組的基因則因不同螢光菌而有所差異。在本研究中，我們設計專一性引子以選殖出完整的東方弧菌螢光操縱組序列，並以 luxA 和luxB 螢光酵素基因作為報導基因並選殖至載體上，以構築成為報導載體，可用於偵測啟動子與終止子的活性。針對東方弧菌螢光操縱組的上游序列進行潛在啟動子區域的預測與分析，找出可能參與活化和抑制基因表達的調控元件；並以連續式刪除 DNA 片段的方式分段選殖上游序列，利用啟動子報導載體進行基因表現的分析。另外，選殖螢光操縱組中可能是 ρ 因子非依賴性轉錄终止子的 DNA 序列，並以終止子報導載體進行基因轉錄抑制的分析。本研究之結果可同時提供東方弧菌螢光基因調控的有用資訊以及可用於監測各種生物基因之潛在啟動子和終止子在活性上的有用工具。

Communication among bacteria with quorum-sensing system is accomplished through the exchange of signaling molecules called autoinducers. When the density of autoinducers accumulated to a certain concentration, bacteria responded by specialized gene expression such as genes involved in biofilm formation, bioluminescence, virulence and sporulation. Currently it still remains unclear about the light-emitting and regulatory mechanism of the marine luminous bacteria V. orientalis. It is speculated that the expression of bioluminescence genes in V. orientalis is also regulated by the quorum sensing system. In this study, the lux genes of V. orientalis are identified with the same five major structural genes luxCDABE in contrast to other luminous bacteria. The synthesis of luciferase is mediated by luxA and luxB genes, and that of fatty acyl reductase, acyl transferase, and acyl protein synthase, comprising the fatty acyl reductase complex for providing the substrate for luciferase, are mediated by luxC, luxD, and luxE genes, respectively. Moreover genes of lux operon are found varied in different luminous bacteria. Among these, the luxAB genes could be used as reporter genes for monitoring promoter and terminator activities after cloning into the vectors for the construction of reporter vectors. Upstream sequences of V. orientalis lux operon are analyzed to predict the potential promoter region and the regulatory elements involved in the activation and inhibition of gene expression. Serial deleted DNA fragments from upstream sequences are cloned and analyzed by the promoter reporter vector for this purpose. Possible DNA sequences of rho-independent terminators are also cloned and analyzed by the terminator reporter vector. The results of this study would provide both useful information for the investigation of lux genes regulation in V. orientalis and facile tools for the monitoring of potential promoter and terminator activities from genes of various organisms.

中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖目錄 X
表目錄 XIII
第一章 緒論 1
1.1 前言 1
1.2 研究動機與目的 3
第二章 文獻回顧 4
2.1 生物冷光 4
2.1.1 生物冷光 4
2.1.2 生物冷光的作用 4
2.1.3 發光機制 5
2.2 螢火蟲 6
2.2.1 螢火蟲的發光構造 6
2.2.2 螢光素之結構 7
2.2.3 發光原理 8
2.3 水母 (Aequorea victoria) 9
2.3.1 水母素 (Aeoquorin) 和綠螢光蛋白 (GFP) 的發現 9
2.3.2 水母素之發光原理 10
2.3.3 GFP之結構 11
2.3.4 GFP發光原理 12
2.4 螢光菌 13
2.4.1 螢光菌之種類 13
2.4.2 海洋中螢光菌的生活方式 14
2.4.3 螢光細菌之生物特性 14
2.4.4 細菌之發光原理 15
2.4.5 細菌之螢光基因 16
2.5 群體感應系統 (Quorum-Sensing, QS) 17
2.5.1 群體感應系統 17
2.5.2 細菌群體感應系統中的自動誘導子 (Autoinducers , AI) 17
2.5.3 不同類型的群體感應系統機制 18
2.6 生物冷光的應用 22
2.7 報導基因 24
第三章 材料與方法 26
3.1 實驗材料 26
3.1.1 藥品 26
3.1.2 實驗儀器 26
3.1.3 菌株 27
3.1.4 載體 27
3.1.5 培養基 28
3.1.6 DNA 分析用緩衝溶液 29
3.1.7 聚合酶連鎖反應之引子 29
3.1.8 酵素 33
3.1.9 Molecular weight markers 33
3.2 實驗方法 34
3.2.1 實驗架構 34
3.2.2 菌種之培養與保存 34
3.2.3 東方弧菌固態培養發光條件 35
3.2.4 東方弧菌螢光強度與生長曲線之測定 35
3.2.5 東方弧菌 DNA 之抽取 35
3.2.6 DNA 電泳 (Electrophoresis) 36
3.2.7 東方弧菌 lux operon 引子 (primers) 之設計 37
3.2.8 以 PCR 方式選殖 lux operon 基因 37
3.2.9 DNA 片段之切膠純化 38
3.2.10 DNA 接合反應 38
3.2.11 勝任細胞之製作 38
3.2.12 細胞轉形作用 39
3.2.13 小量質體 (Plasmid) 的抽取 39
3.2.14 限制酶剪切 40
3.2.15 DNA 定序 (DNA sequencing) 40
3.2.16 序列之電腦分析 40
3.2.17 luxAB 報導載體之構築 41
3.2.18 lux operon 啟動子選殖 43
3.2.19 東方弧菌 lux operon 啟動子之分析 44
3.2.20 lux operon 終止子之選殖 45
3.2.21 統計分析方法 46
第四章 結果與討論 47
4.1 東方弧菌固態培養基發光條件 47
4.2 東方弧菌細菌密度與螢光強度之影響 51
4.3 NaCl 濃度對東方弧菌螢光強度與生長曲線之影響 53
4.4 東方弧菌 lux operon 基因之選殖 59
4.5 東方弧菌 lux operon 基因之定序 64
4.6 東方弧菌 lux operon 與其它螢光菌比較 76
4.7 東方弧菌螢光基因之胺基酸序列與分析 79
4.8 啟動子報導載體之構築 86
4.9 東方弧菌 lux operon 啟動子序列之分析與引子設計 91
4.10 東方弧菌 lux operon 啟動子序列於報導載體上之構築 94
4.11 東方弧菌 lux operon 啟動子序列之活性分析 98
4.12 啟動子報導載體之靈敏度分析 102
4.13 終止子報導載體之構築 107
4.14 東方弧菌 lux operon 終止子序列之分析與引子設計 112
4.10 東方弧菌 lux operon 終止子序列於報導載體上之構築 115
第五章 結論 117
參考文獻 118
附錄 126
作者簡介 129

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