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Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes

  • Kim, Jong-Kun (Graduate School of Biotechnology & Information Technology, Hankyong National University) ;
  • Park, Young-Jin (National Academy of Agricultural Science, Rural Development Administration, NAAS) ;
  • Kong, Won-Sik (National Institute of Horticultural and Herbal Science, Development of Herbal Crop Research, RDA) ;
  • Kang, Hee-Wan (Graduate School of Biotechnology & Information Technology, Hankyong National University)
  • Received : 2010.08.13
  • Accepted : 2010.10.19
  • Published : 2010.12.31

Abstract

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/${\mu}g$ of DNA in $1{\times}10^7$ 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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