臺灣博碩士論文加值系統

摘 要
Vibrio fischeri ATCC 7744 lux regulon 主導其螢光表現。 lux regulon 包含負責調節的 luxR、luxI 基因與負責表現螢光的 luxC、D、A、B、E 等結構基因，菌體中， LuxI 蛋白催化合成 autoinducer (AI)， AI 與 LuxR 形成 LuxR-AI 複合物 (LuxR-AI complex)，並結合 lux regulon 調節區域 R&R 而增強 lux regulon 表現。細胞內互補性螢光表現分析篩得受 LuxR-AI 調節而增強表現之 cya、thi 基因， cya 基因產物為 adenylate cyclase，其催化 ATP 形成 3’-5’ cAMP； thi 基因產物為 thiamin biosynthesis protein，與 thiamin 之生成有關。 cya 與 pbg 基因轉錄方向相反，兩基因間 256 bp 為調節區域 R&R，具雙向啟動子功能，啟動子活性分析顯示兩基因之啟動子活性及表現時機相仿，基因 cya 啟動子可受 LuxR-AI調節增強表現， pbg 基因則無此受調節現象。分析 cya 及 pbg 基因調節區域，於啟動子上游皆具類似 LuxR-AI 結合位 SUAS/RI (LuxR-AI complex binding site)，引子延伸法分析顯示 cya 基因轉錄起始位於其 start codon 上游 29 bp 之 A。蛋白表現分析的結果確實有 LuxR 的生成，蛋白凝膠遲滯分析證實 LuxR-AI 結合於 lux regulon 調節區域 R&R 及基因 cya 調節區域 R&R，同時顯示 LuxR 必需在 AI 存在下始有結合特定 DNA 之活性，蛋白表現顯示， AI 對 LuxR 可能具負向作用機制，使 LuxR 穩定性下降。而 LuxR 與 AI 結合之直接證據則尚待更深入的分析。

Abstract
The genes concerned with bioluminescence formed the lux regulon in V. fischeri ATCC 7744. The lux regulon contains regulatory genes luxR, luxI responsible for regulation, and luxCDABE genes are the structural genes responsible for biolumine-scence. LuxI protein catalyzes the autoinducer AI synthesis. The AI and LuxR protein formed LuxR-AI complex to enhance bioluminescence of V. fischeri. The gene library was constructed to clone the specific genes that under regulation of LuxR-AI by in trans complementation bioluminoassays in vivo. The cya and thi genes were isolated from the selected clones, which apparently enhanced by LuxR-AI complex. The cya gene encodes the adenylate cyclase, which converts ATP to 3’-5’ cAMP. The thi gene encodes the thiamin biosynthesis protein, which is response for thiamin biosynthesis. The conserved sequence NNNTGT~N8-12~ACANNN resided in R&R of cya and thi genes might be recognized by the LuxR-AI complex to enhance the gene expression. Function analysis reveals that the cya gene is regulated by LuxR-AI complex. Protein assays confirm that the LuxR protein encoded by the gene and AI play an important role in the LuxR protein stability. Gel mobility shift assays show that LuxR-AI complex enables to bind the regulatory region DNA fragments.

目 錄
中文摘要…………………………………………………………………………. ii
英文摘要……………………………………………………………………….… iii
前言……………………………………………………………………………….. 1
材料與方法………………………………………………………………………
結果與討論………………………………………………………………………
一 V. fischeri 基因庫之構築與可受 LuxR-AI 複合物調節而增強表現之基因
選殖……………………………………………………………………………
(一) 細胞內互補性螢光表現分析之控制組質體構築…………………….
(二) 受 LuxR-AI 複合物調節而增強表現之特定基因選殖……………...
二 受 LuxR-AI 複合物調節而增強表現之基因定序及分析…………………..
(一) 選殖之 V. fischeri ATCC 7744 genomic DNA 的基因定序………….
(二) 基因序列比對及分析…………………………………………………..
(三) 選殖基因來源之分析…………………………………………………..
(四) LuxR-AI 複合物影響選殖基因之分析………………………………..
三 選殖基因調節區域的功能分析………………………………………………..
(一) 基因之調節區域分析…………………………………………………..…
(二) 基因 cya 調節區域功能分析……………………………………………
(三) 基因 cya轉錄起始分析………………………………………………….
四 LuxR-AI 複合物於 E. coli 中的表現分析…………………………………....
(一) 利用 Maxicell 的方法表現 LuxR-AI 複合物………………………….
(二) 利用 T7 expression system 表現 LuxR-AI 複合物…………………….
五 蛋白凝膠遲滯分析……………………………………………………………..
(一) 利用 Maxicell 製備蛋白進行凝膠遲滯分析……………………………
(二) 利用 T7 expresion system 製備蛋白進行凝膠遲滯分析……………….
六 LuxR 蛋白穩定性分析…………………………………………………………
總結………………………………………………………………………………….
參考文獻……………………………………………………………………………
附錄………………………………………………………………………………….

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