Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21692
標題: Cloning and Functional Analysis of the Genes that Enhance Bioluminescence of Photobacterium leiognathi lux Operon
可調節 Photobacterium leiognathi lux Operon 螢光表現的基因選殖與分析
作者: 孫芳君
Sun, FangChun
關鍵字: 基因選殖;Gene Cloning;細胞內互補螢光分析;調節區域與轉錄終止子;調節機制;In trans complementation biolumino assays in vivo;Regulator region and transcription terninator;regulation mechanisms
出版社: 分子生物學研究所
摘要: 
Plasmid pL741 carries a ~15 kb HindIII partial digested Photobacterium leiog-nathi genomic DNA in pACYC 184. However, the P. leiognathi lux operon cloned in pL741 shows dim bioluminescence in E. coli; it suggested that the lux operon required regulatory gene(s) to induce or enhance the gene expression. In trans complementa-tion bioluminoassays in vivo were used to construct P. leiognathi genome library for cloning the specific genes, which enable to enhance bioluminescence of the lux operon. pXY5 and pXY6 were isolated from two of the selected bright clones. Nucleotide sequences of the P. leiognathi genes carried on pXY5 and pXY6 were determined, and the genes were identified. The gene orders of the genes are -ufoI-ufoII-WTRR&R-gltS-WTI-WTIIR and torA-R&R-torCR on pXY5 and pXY6, respectively (R&R: regulatory region; W: transcription terminatior). Apparently ufoII, torA, torC, gltS genes are not the specific regulatory genes of the lux operon; but functional analyses show that ufoII, torA, torC genes enable to enhance biolumine-scence. It elicits that not only the specific regulatory genes enable to enhance bioluminescence of the lux operon, also many other genes enable to do. However, ufoII, torA, torC genes can enhance bioluminescence of the lux operon in E. coli, but the enhance mechanisms are not clear yet.

Photobacterium leiognathi lux operon 在 E. coli 的螢光表現微弱,顯示可能需要調節基因或因子的參與調節機制。利用細胞內互補螢光分析的方法篩選可調節或增強螢光表現的基因,以了解其調節機制。由互補螢光分析結果選取二個具有 ~2.7 kb 及 ~1.5 kb P. leiognathi genomic DNA 的質體 pXY5、pXY6,二者可使螢光表現增強約 ~1.3 倍。DNA序列分析顯示 pXY5 所包含 DNA 片段全長為 2,744 bp 的序列,其基因排列順序為 -ufoI-ufoII-WTRR&R-gltS-WTI-WTIIR (R&R 為調節區域﹔W 為轉錄終止子)。ufoII 及 gltS 基因為完整的基因。pXY6所包含DNA片段全長為 1,568 bp 的序列,其基因排列順序為 torA-R&R-torCR,torA 及 torC 基因轉錄方向相反且為不完整的基因。序列分析顯示 GltS 蛋白為一 glutamate permease。ORFII 蛋白則為一 pI 等電點為 10.16 未知的蛋白。torA 基因可主導 TMAO reductase 的形成,torC 基因可主導 cytochrome C 的形成。依據調節區域及轉錄終止子的功能分析,確認 potential hairpin loop WT 為 ufoII 基因的 transcription terminator,而 gltS 基因之調節區域其啟動子可能需要 regulator protein 或 enhancer binding protein 的協助才能啟動,以引導基因的表現。Potential hairpin loops WTI-WTII 為 gltS 基因的 transcription terminator。 ufoII, gltS, torA, torC 基因以 maxicell 蛋白質分析及螢光表現分析,確認產生 ~14 kDa 之 ORFII 和 ~35 kDa GltS 的產物,torA 及 torC 基因產物可能不穩定,沒能確認。此實驗篩選所得之 ufoII, gltS, torA, torC 基因應不是 P. leiognathi lux operon 的調節基因 (regulatory genes),但實驗的數據,可以肯定此等基因與 lux operon 在 in trans complementation 時確可加強螢光的表現,應是間接影響的結果。其中,GltS 蛋白大量表現可能使得菌體細胞膜受損,影響到菌體正常細胞生長狀況,使得螢光表現下降。ufoII, torA 及 torC 基因在 lac-promotor 的帶動下可增強 lux operon 的螢光表現,但其參與螢光表現的基因調節機制尚不清楚。
URI: http://hdl.handle.net/11455/21692
Appears in Collections:分子生物學研究所

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