Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes.
- Author:
Jong Kun KIM
1
;
Young Jin PARK
;
Won Sik KONG
;
Hee Wan KANG
Author Information
1. Graduate School of Biotechnology & Information Technology, Hankyong National University, Ansung 456-749, Korea. kanghw2@hknu.ac.kr
- Publication Type:Brief Communication
- Keywords:
Electroporation;
Flammulina velutipes;
Flammutoxin gene;
Hygromycin B gene;
Protoplast
- MeSH:
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
- From:Mycobiology
2010;38(4):331-335
- CountryRepublic of Korea
- Language:English
-
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.
