Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23869
標題: Photobacterium leiognathi 調節基因 luxZ 功能與調節機制分析
Functional anaiysis of the regulatory luxZ gene from Photobaterium leiognathi
作者: 陳玉峰
關鍵字: Photobacterium leiognathi;海生螢光菌;luxZ;gel retardation;luxZ;凝膠遲滯分析
出版社: 生物化學研究所
摘要: 
Photobacterium leiognathi PL741 lux operon 轉形至 E. coli 中,螢光表現明顯降低,顯示應有特殊基因參與螢光調節機制。前之研究選殖到 P. leiognathi ATCC 25521 luxZ 基因,生成調節蛋白 LuxZ (Mr ~25.8 kDa),胺基酸序列與 GntR 調節蛋白 FadR subfamily 相似,可增強 PL741 lux operon 於 E. coli 中之螢光表現。分析 [luxZ]R&R 發現 luxZ 基因主要由 P*-promoter 帶動表現,[luxZ]R&R 可能藉由形成 mRNA secondary structure 或受 trans-acting factors binding UAS/OI-AS/OII 自我調節。螢光分析顯示 LuxZ 可增強 PL741 [lux]R&R(PR),及 V. fischeri lux regulon [lux]R&R(PL) 表現,但對 ATCC 25521 [lux]R&R(PR) 調節能力微弱,另選殖可受 LuxZ 調節之 ATCC 25521 基因,得到一 DNA 片段 -lig-R&R-orf2→,LuxZ 可調節 [orf2]R&R 及 orf2 內含的次調節之 intern promoter,且 LuxZ 存在可能影響 [orf2]R&R mRNA 二級結構構形,並造成 orf2 轉錄起始點位移。Glucose repression 得知 [orf2]R&R 亦受 cAMP-CRP 調節,探討 LuxZ 與 cAMP-CRP 在 PL741 [lux]R&R(PR) 及 ATCC 25521 [orf2]R&R 調節區域中彼此之互動,顯示兩者調節能力各自獨立,共存具加成調節效果;但不同調節區域由於 cAMP-CRP, LuxZ 可結合、辨認的 DNA-binding 位置 conserved 程度不同而顯現出不同程度的調節。調節蛋白 LuxZ N 端序列具高保留helix-turn-helix (H-T-H) motif,應有 DNA 結合能力,由 LuxZ-DNA binding assays 顯示 LuxZ 可藉由辨認 A-T rich, 具 repeat 形式之特定 DNA 序列與其所增強之調節區域直接結合,LuxZ 調節能力強弱應與該調節區域中是否具有 LuxZ 可辨認之結合序列、序列保留度及位置恰當與否等重要因素有關。luxZ 為 stationary phase 表現之基因;調節蛋白 LuxZ 調節之基因亦多為 stationary phase 表現,推測 LuxZ 調節功能可能與 stress- resistant 相關,待進一步研究。

Photobacterium leiognathi PL741 lux operon cloned in E. coli showed dim; it suggests that the specific regulatory gene(s) is required for P. leiognathi lux operon induction. P. leiognathi ATCC 25521 luxZ gene was cloned, by in trans complementation Lux-assays in vivo, it encoded the LuxZ protein which enables to enhance the PL741 lux operon in E. coli. LuxZ, a regulatory protein belonging to the FadR sub-family of GntR family, has a Mr ~25.8 kDa. Analysis of the [luxZ]R&R shows that the major promoter for the luxZ transcription is P*, and the [luxZ]R&R is possible to autoregulate by the secondary mRNA structures or trans-acting factors binding the UAS/OI,AS/OII sequences. Bioassays of the luxZ gene's enhancement revealed that the LuxZ can enhance bioluminescence of PL741 lux operon and V. fischeri lux regulon [lux]R&R(PL), but functions weak for ATCC 25521 [luxZ]R&R(PR). In addition, the specific gene(s) which enhanced by the luxZ were selected from P. leiognathi ATCC 25521 genomic library. The LuxZ can regulate the [orf2]R&R and the intern promoter resided in the orf2 gene. Besides, the LuxZ can shift the orf2 transcription initiation sites by unknown mechanism. Glucose repression shows that the [orf2]R&R is also regulated by cAMP-CRP. Functional analysis of the cAMP-CRP and LuxZ enhancements elucidates that (1) the enhancements in PL741 [luxZ]R&R are independent, and additive. (2) The conservation of the cAMP-CRP and LuxZ conserved DNA binding sites determines the function of enhancement. The DNA-binding assays display that the LuxZ enhancement is related to the specific conserved DNA sequences (AT rich DNA repeats) and the convenient binding sites for RNA polymerase to transcript. The luxZ gene was induced at stationary phase, it suggested that the function of the LuxZ was concerned with stress-resistant.
URI: http://hdl.handle.net/11455/23869
Appears in Collections:生物化學研究所

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