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KMID : 1160220100380040331
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Mycobiology
2010 Volume.38 No. 4 p.331 ~ p.335
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Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes
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Kim Jong-Kun
Park Young-Jin
Kong Won-Sik
Kang Hee-Wan
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Abstract
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In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/μg of DNA in 1 × 107 protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.
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KEYWORD
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Electroporation, Flammulina velutipes, Flammutoxin gene, Hygromycin B gene, Protoplast
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