非生物發光性的創傷弧菌 (Vibrio vulnificus CKM-1) 含有一個NADPH依賴性的藍螢光蛋白基因。為進一步探討藍螢光蛋白基因在植物科學研究中的應用，本研究利用三個不同啟動子，RbcS、Adh與DR5，分別調控藍螢光蛋白基因在不同組織與環境刺激因子下的表現，並將藍螢光蛋白累積於不同的細胞位置，例如細胞質、胞外、內質網、葉綠體與粒線體等。受RbcS與DR5啟動子調控的藍螢光蛋白暫時性表現之菸草葉片，在細胞質、胞外、內質網可觀察到藍螢光訊號，但在葉綠體與粒線體的藍螢光訊號則較微弱。受Adh與DR5啟動子調控的藍螢光蛋白暫時性表現之蝴蝶蘭花瓣，經H2O2與NAA處理後，藍螢光蛋白在細胞質與胞外的訊號較強，但在其他胞器的訊號則較微弱。若將藍螢光蛋白與家禽里奧病毒的鞘蛋白C端序列重組，發現重組蛋白的mRNA表現量較多，藍螢光也較強。含有藍螢光蛋白基因的轉殖阿拉伯芥，受Adh 啟動子調控的mBFP送至細胞質或胞外，在缺氧處理下，根部有較強的藍螢光訊號。在掃描式共軛焦顯微鏡下觀察阿拉伯芥轉殖株的原生質體，在細胞質、內質網與葉綠體可觀察到藍螢光訊號，其中葉綠體自發性螢光可與藍螢光座落於同一位置；但受RbcS啟動子調控mBFP送至粒線體的轉殖阿拉伯芥，其藍螢光蛋白訊號並未與粒線體重疊。 此一來自創傷弧菌的藍螢光蛋白可做為報導基因應用於植物學的研究。然而若能進一步修正mBFP的序列，將會更適合應用於植物科學與科技的研究。

The BFPvv, a NADPH-dependent blue fluorescent protein, was identified from non-bioluminescent pathogenic bacteria, Vibrio vulnificus CKM-1. The BFPvvD9 (mBFP) is a version of BFPvv which the fluorescent intensity was greatly improved by random mutagenesis and DNA shuffling technology. To explore the possibility for the application of mBFP in plants, plant nuclear expression vectors were constructed which the expression of mBFP gene was drived by three different promoters, e.g. tissue-specific(RbcS), hypoxia(Adh) or auxin(DR5) inducible promoters, respectively. In addition, the mBFP protein was targeted to different cellular compartments such as cytosol, extra-cellular matrix, ER, chloroplast and mitochondria. When the mBFP gene drived by RbcS or DR5 promoters was transiently expressed and proteins were targeted to cytosol, extracellular matrix, chloroplast or mitochondria in the tobacco leaves by Agro-infiltration, the blue fluorescence could be observed under fluorescent microscope. Although the mBFP gene driven by Adh or DR5 promoters was transiently inducible with 1 mM H2O2 or 50 μM NAA, respectively, for 24 hr in the flower of moth orchid, the intensity of blue fluorescence is relatively weak. When the mBFP was fused with C-terminal of avian reovirus σC protein, the recombinant fusion protein (mBFP-mS1C) show the enhanced intensity of fluorescence which was co-related with the accumulation of more abundant of RNA transcripts as compared with control. Furthermore, transgenic Arabidopsis overexpressing mBFP lines were generated by floral dip method, and the mBFP fluorescence can be observed in the cytosol, ER, and chloroplasts of protoplasts. However, the blue fluorescence couldn’t be detected in the mitochondria. In general, the mBFP can be used as reporter gene in plants, but further modification of mBFP sequences will make it more ideal for the plant research and plant biotechnology.

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