Mycobiology.
2010 Dec;38(4):331-335.
Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes
- Affiliations
-
- 1Graduate School of Biotechnology & Information Technology, Hankyong National University, Ansung 456-749, Korea. kanghw2@hknu.ac.kr
- 2Institute of Genetic Engineering, Hankyong National University, Ansung 456-749, Korea.
- 3National Academy of Agricultural Science, Rural Development Administration, NAAS, Suwon 441-707, Korea.
- 4National Institute of Horticultural and Herbal Science, Development of Herbal Crop Research, RDA, Suwon 441-707, Korea.
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/microg of DNA in 1 x 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.
MeSH Terms
-
Agaricales
Blotting, Southern
Chimera
Cinnamates
Coat Protein Complex I
DNA
Electroporation
Flammulina
Fungal Proteins
Genome
Hygromycin B
Mycotoxins
Oxidoreductases
Plasmids
Polymerase Chain Reaction
Protoplasts
Recombination, Genetic
Sprains and Strains
Cinnamates
Coat Protein Complex I
DNA
Fungal Proteins
Hygromycin B
Mycotoxins
Oxidoreductases
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