- 學位論文
東方弧菌螢光基因之選殖與應用
Cloning and Application of lux Genes from Vibrio orientalis
摘要
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。
並列摘要
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.