Application of a blue fluorescent protein from Vibrio vulnificus for plant research

• Main Authors: Jin-MinTu, 涂晉敏
• Other Authors: Ching-Chun Chang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/51604590231793366963

碩士 === 國立成功大學 === 熱帶植物科學研究所 === 102 === 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.