碩士 / 國立臺灣大學 / 微生物與生化學研究所 / 97 / Pleurotus ostreatus is the second largest edible mushrooms next to Agaricus bisporus. P. ostreatus is popular worldwide due to ease of and use of agricultural wastes as substrates. Establishing a stable transformation system of P. ostreatus may provide a tool for its heterologous expression and even oral vaccine production in the future. Agrobacterium tumefaciens has been known as “natural engineer”. There are many advantages of Agrobacterium tumefaciens mediated transformation (ATMT) over other transformation methods such as convenience and less expenssive. The transformed heterologous gene can be integrated into host genome by mostly single insertion, and consequently more stable.
In this study, we successfully developed the A.tumefaciens mediated transformation of Pleurotus ostreatus. Agrobacterium harboring plasmid with different promoters driving green fluorescent protein (GFP) and hygromycin phosphotransferase gene (hph) was co-cultivated with Pleurotus ostreatus. P. ostreatus gill tissues used as explants co-cultivating with A.tumefaciens AGL-1 and LBA4404 could get more transformants, with transformation efficiency around 80 ~ 90%. However, only a few of transformants were obtained when homogenized mycelia was used as starting material, and no transfomant was found using mycelia modified pellets (MMP). Fluorescent microscopic observation revealed that GFP can be driven by the califlower mosaic virus (CaMV) 35S promoter and Agaricus bisporus glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter, in which GFP tended to appear as aggresome-like fluorescent spot. When GFP gene with C terminal endoplasmic reticulum retention signal was driven by P. ostreatus homologous GPD promoters, the GFP also formed green fluorescent spots. In the other hand, the Flammulina velutipes GPD and P. ostreatus homologous GPD promoters without retention signal showed scattered green fluorescence. These results suggested that fused signal peptide led to GFP accumulation in P. ostreatu as aggregates. The highest amount of soluble GFP extracted from P. ostreatus detected by ELISA is 43.83 ng per gram total soluble protein. Besides, heterologous gene could be stably expressed after mitosis and meiosis.
| Identifer | oai:union.ndltd.org:TW/097NTU05381025 |
| Date | January 2009 |
| Creators | Yuan-Chen Tsai, 蔡媛禎 |
| Contributors | Ching-Tsan Huang, 黃慶璨 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 89 |
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