Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23946
標題: Photobacterium leiognathi sodCII 基因表現之調節機制分析
Regulation Mechanism Analysis of the sodCII Gene from Photobacterium leiognathi
作者: 陳惟君
Chen, Wei-Chun
關鍵字: Photobacterium leiognathi;海生螢光菌;sod;ferric uptake regulator;超氧歧化酵素
出版社: 生物化學研究所
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摘要: 
Photobacterium leiognathi ATCC 25521 sodCII 基因在一般生理環境下表現受調控。分析 sodCII 基因調節區域序列,利用片段剪切及偵測螢光表現可証明 sodCII 基因之負向調節機制:(1) 位於 P(II)-promoter 上游之Photobacterium leiognathi ATCC 25521 sodCII 基因在一般生理環境下表現受調控。分析 sodCII 基因調節區域序列,利用片段剪切及偵測螢光表現可証明 sodCII 基因之負向調節機制:(1) 位於 P(II)-promoter 上游之 ΣFur (ferric uptake regulator binding site);(2) 位於 P(I)-promoter 下游之對稱序列 ΣNE/O (negative element/operator)。研究結果顯示當 ΣNE/O 去除可使 P. leiognathi sodCII 表現,若同時去除 ΣFur,螢光值增強至 ~300 V/ml,若保留 ΣFur 僅去除下游 ΣNE/O 則表現為前者之一半,可証明 ΣNE/O 及 ΣFur 皆為 sodCII 基因之負向調節因子,且 ΣNE/O 的負向調節機制較強,將其對稱序列作 base mutation 發現可增強基因表現,得知為負向調節減弱造成表現增強。利用引子延伸反應分析 sodCII 基因之轉錄起始點,發現若調節區域 R&R 含 ΣFur 則由 P(I)-promoter 帶動表現,轉錄起始位置相對於 start codon 上游 -18 (C) 位置,若去除 ΣFur 則以 P(II)-promoter 帶動下游基因,轉錄起始位置相對於 start codon 上游 -43 (C) 位置。推測當 (Fur-Fe2+)2 protein 結合於 ΣFur 區域影響 RNA polymerase 結合於 sodCII 基因 promoter 之位置,由 P(II)-promoter 位移而結合於 P(I)-promoter 啟動基因表現。利用 M9 medium 培養發現可增強基因表現,亦証明鐵離子缺乏之環境 P(II)-promoter 之表現強度大於 P(I)-promoter。在 sodCII 低量表現以 P(I)-promoter 啟動表現,高度表現則以P(II)-promoter 為主; Fur protein 失活釋放負向調節機制而促使 sodCII 基因啟動。但加入過多鐵離子則可能造成細胞產生過量 superoxide 使相關調控機制啟動以保護細胞的損害。設計引子於 E. coli 中找出作為 sodCII 基因之負向調控因子 fur 基因,Fur protein 以 EMSA (electrophoretic mobility shift assay) 實驗分析證明 Fur protein 可結合於 sodCII 基因調節區域 ΣFur 位置。並利用 PCR 嘗試找出 P. leiognathi fur 基因,結果得到 5’ 端 ~270 bp fur 基因序列,經比對與 E. coli fur 基因具同源性,保留 Fur protein 與 DNA 作用之重要 domain,可印證 P. leiognathi 亦應含此調節機制調控 sodCII 基因表現。

The sodCII gene of Photobacterium leiognathi ATCC 25521 shows no expression dectected by Lux-bioassays in E. coli. The fact suggests that the sodCII gene might be repressed and regulated in general condition. Sequence analysis reveals that there are two negative regulation mechanisms involved in the sodCII regulation: (1) The ΣFur (ferric uptake regulator binding site) is resided upstream of the P(II)-promoter; (2) The ΣNE/O (negative element/operator) is located downstream of the P(I)-promoter. The R&R/DNA which deleted the ΣFur and the ΣNE/O negative element enabled to enhance the sodCII gene expression. It certified that ΣFur and ΣNE/O might act as the sodCII negative regulation factors, and ΣNE/O is the critical one. The result elicits that the sodCII gene expression increased with ΣNE/O base down mutation. The transcription initiation sites of the sodCII gene were identified by primer extension; transcription initiation site was shifted from the locus -18 nt (C) to -43 nt (C) relative to the start codon. The sodCII gene's expression was led by the P(I)-promoter instead by the P(II)-promoter when the R&R/DNA deleted ΣFur. The fact suggests Fur-Fe2+ protein bound ΣFur was able to block the gene expression. The sodCII gene expression increased when cells cultured in M9 medium. The result suggests that if Fe3+ were limited, Fur-Fe2+ protein could be released Fe2+ to be inactive Fur protein, the Fur-Fe2+ negative regulation of the sodCII gene will be dismissed. The E. coli fur gene was cloned, and Fur protein was purified to do Fur-R&R/DNA binding assays. EMSA shows that Fur protein enables to bind R&R (ΣFur)/DNA of P. leiognathi sodCII gene. Partial of P. leiognathi fur gene was cloned by PCR, the fact shows that the fur genes of P. leiognathi and E. coli are homologous. It implied that the Fur negative regulation could be functioned in P. leiognathi sodCII gene's regulation.
URI: http://hdl.handle.net/11455/23946
其他識別: U0005-2706200614065300
Appears in Collections:生物化學研究所

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