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研究生:陳怡君
研究生(外文):Yi Jung Chen
論文名稱:受PhotobacteriumleiognathiLuxZ蛋白調控增強之特定基因的選殖與功能分析
論文名稱(外文):Cloning and Functional Analysis of the Specific Genes that Enhanced by the LuxZ from Photobacterium leiognathi
指導教授:林瑞文林瑞文引用關係
指導教授(外文):Juey Wen Lin
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
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:2001
畢業學年度:89
語文別:中文
論文頁數:73
中文關鍵詞:螢光基因luxZ 基因glxI 基因
外文關鍵詞:lux operonluxZ geneglxI gene
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海生螢光菌 Photobacterium leiognathi的螢光基因組成以 lux operon 形式存在。將完整的 P. leiognathi PL741 lux operon 轉形至 E. coli,螢光表現明顯降低,顯示螢光機制受到調控。前人構築 P. leiognathi ATCC 25521 genome library,以胞內互補性螢光表現分析的方法,在 E. coli 中篩選到可增強 PL741 螢光表現的基因 luxZ ,但對 ATCC 25521 的 lux operon 螢光表現則無影響。 luxZ 基因主導 LuxZ 蛋白的形成,推測 LuxZ 可能為一 GntR family的調節蛋白,其確切功能與相關機制仍未知。本實驗研究目的為選殖可受 LuxZ 蛋白調控的特定基因,以了解 LuxZ 蛋白在 ATCC 25521中的功能與調控機制。目前篩選到一受 LuxZ 蛋白增強的 DNA 片段,將 此嵌入片段定序及分析比對後,知此 DNA 片段大小為 2221 bp,分別主導一完整的預測蛋白 Ufo 及部份 Glyoxalase I 蛋白,基因排列順序為 ←WT-ufo-R&R-glxI→,兩基因轉錄方向相反,其間有相距 196 bp 的調控區 R&R,經起動子與轉錄終止子分析結果顯示,調控區 R&R 具左右兩向的起動子功能,potential hairpin loop WT 為一個強終止子,且緊連 WT 約 60 bp 的 promoter-like 序列也具起動子功能。 利用引子延伸法找到 ufo 與 glxI 兩基因的轉錄起始點分別位於其 start codon 上游 95 base 之 A (1861th bp) 與 215 base 之 C (1739th bp);不同大小調節區片段的螢光分析結果發現LuxZ 蛋白具增強 ufo 與 glxI 兩基因表現的特性;蛋白表現分析的結果顯示 pHI1 可在 maxicell 中表現較多量的 LuxZ 蛋白;凝膠遲滯分析的結果不能確定此調節區 DNA片段可與LuxZ 蛋白結合。推測LuxZ 蛋白其結合區位於兩基因上游的 SAS/LuxZ-R 和 SAS/LuxZ-L 區域 (具 ATNNNNNTATNNA 保留序列);此外 P. leiognathi PL741 lux operon 的調控區中存在此類似保留序列,但 ATCC 25521 lux operon中則無,表示此序列的存在可能和 LuxZ 蛋白增強基因表現的機制有關,仍待研究證明。

The lux operon of Photobacterium leiognathi PL741 showed dim in E. coli. It suggests that the specific regulatory gene(s) is required for the the lux operon of P. leiognathi. P. leiognathi ATCC 25521 genome library was constructed in E. coli to select the specific regulatory gene(s) by in trans complementation bioluminoassays in vivo. The luxZ gene was cloned. The encoded LuxZ protein apparently enables to enhance the gene expression of P. leiognathi PL741 lux operon in E. coli, but not function for ATCC 25521 lux operon. LuxZ is like the regulatory protein belonging to the GntR family, but the function of LuxZ is not clearly known. The specific gene(s) were selected from P. leiognathi ATCC 25521 genome library that enhanced by the luxZ gene. pHI13 in one of the bright clones was selected. The nucleotide sequence of the P. leiognathi genome DNA fragment in pHI13 has been determined, and the encoded proteins were deduced. The gene order of the genes is ←WT-ufo-R&R-glxI→. The regulatory region R&R includes two divergent promoter systems, PR-promoter system for the glxI, and PL-promoter system for the ufo gene. Functional analysis elucidates that the PR- and PL-promoter systems both are able to lead the gene expression and apparently enhanced by the LuxZ. Although there are no evidence to support LuxR-R&R binding. It seems that the conserved sequences SAS/LuxZ-R and SAS/LuxZ-L (ATNNNNNTATNNA) resided downstrem of the PR- and PL-promoter are the specific sequences recognized by the LuxZ to enhance the gene expression. The similar SAS/LuxZ sequence also resided in the regulatory region of the P. leiognathi PL741 lux operon, but not in the ATCC 25521 lux operon. It suggests that the conserved SAS/LuxZ sequence could be recognized by the LuxZ to enhance the gene expression.

中文摘要………………………………………………. ii
英文摘要………………………………………………… iii
前言……………………………………………………… 1
材料與方法……………………………………………. 7
結果與討論……………………………………………. 19
一 P. leiognathi ATCC 25521受 LuxZ 增強螢光表現之基因選殖.. 19
(一) 相容質體之構築………………………………………… 19
(二) 受 LuxZ 蛋白增強的特定基因之選殖…………… 22
二 受 LuxZ 蛋白調控增強的 DNA 片段之定序與分析…… 24
(一) 選殖的 P. leiognathi ATCC 25521 genomic DNA序列的定序 24
(二) 序列比對及分析…………………………………………. 24
三 轉錄終止子與調節區的功能分析……………… 28
(一) ufo 基因轉錄終止子的功能分析………………… 32
(二) 基因調控區 R&R 的起動子功能分析…………………. 35
(三) ufo 與 glxI 的轉錄起始位置分析……………… 39
(四)受 LuxZ 蛋白的調控位置分析…………………… 39
四 基因 luxZ 在不同質體上於 Escherichia coli 中的蛋白表現分析………46
五 LuxZ 蛋白的凝膠遲滯分析……………………………….. 48
總結…………………………………………………………… 55
參考文獻……………………………………………………... 60
附錄………………………………………………………….....68

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