Browse > Article
http://dx.doi.org/10.4489/MYCO.2010.38.4.331

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)
Publication Information
Mycobiology / v.38, no.4, 2010 , pp. 331-335 More about this Journal
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.
Keywords
Electroporation; Flammulina velutipes; Flammutoxin gene; Hygromycin B gene; Protoplast;
Citations & Related Records
Times Cited By KSCI : 2  (Citation Analysis)
연도 인용수 순위
1 Li G, Li R, Liu Q, Wang Q, Chen M, Li B. A highly efficient polyethylene glycol-mediated transformation method for mushrooms. FEMS Microbiol Lett 2006;256:203-8.   DOI   ScienceOn
2 Cho JH, Lee SE, Chang WB, Cha JS. Agrobacterium-mediated transformations of the winter mushroom, Flammulina velutipes. Mycobiology 2006;34:104-7.   DOI   ScienceOn
3 Adachi K, Nelson GH, Peoples KA, Frank SA, Montenegro-Chamorro MV, DeZwaan TM, Ramamurthy L, Shuster JR, Hamer L, Tanzer MM. Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO. Curr Genet 2002;42:123-7.   DOI
4 Cho Y, Davis JW, Kim KH, Wang J, Sun QH, Cramer RA Jr, Lawrence CB. A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs. Mol Plant Microbe Interact 2006;19:7-15.   DOI   ScienceOn
5 Tomita T, Ishikawa D, Noguchi T, Katayama E, Hashimoto Y. Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell. Biochem J 1998;333 (Pt 1):129-37.   DOI
6 Tomita T, Mizumachi Y, Chong K, Ogawa K, Konishi N, Sugawara-Tomita N, Dohmae N, Hashimoto Y, Takio K. Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes: maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation. J Biol Chem 2004;279:54161-72.   DOI   ScienceOn
7 Kuo CY, Chou SY, Huang CT. Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes. Appl Microbiol Biotechnol 2004;65:593-9.
8 Hirano T, Sato T, Okawa K, Kanda K, Yaegashi K, Enei H. Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Lentinus edodes. Biosci Biotechnol Biochem 1999;63:1223-7.   DOI   ScienceOn
9 Tanesaka E, Kinugawa K, Okabe K, Kitamura Y, Ogawa M, Yoshida M. Electrophoretic karyotype of Flammulina velutipes and its variation among monokaryotic progenies. Mycoscience 2003;44:67-9.   DOI
10 Kim D, Tamai Y, Azuma T, Harada A, Ando A, Sakuma Y, Miura K. Analysis of the electrophoretic karyotype of Flammulina velutipes. J Wood Sci 2000;46:466-9.   DOI   ScienceOn
11 van de Rhee MD, Graça RM, Huizing HJ, Mooibroek H. Transformation of the cultivated mushroom, Agaricus bisporus, to hygromycin B resistance. Mol Gen Genet 1996;250:252-8.
12 Sun L, Cai H, Xu W, Hu Y, Gao Y, Lin Z. Efficient transformation of the medicinal mushroom Ganoderma lucidum. Plant Mol Biol Rep 2001;19:383a-j.   DOI
13 Kim BG, Joh JH, Yoo YB, Magae Y. Transformation of the edible basidiomycete, Pleurotus ostreatus to phleomycin resistance. Mycobiology 2003;31:42-5.   과학기술학회마을   DOI   ScienceOn
14 Ogawa K, Yamazaki T, Hasebe T, Kajiwara S, Watanabe A, Asada Y, Shishido K. Molecular breeding of the bisidiomycete Coprinus cinereus strains with high lignin-decolorization and -degradation activities using novel heterologous protein expression vectors. Appl Microbiol Biotechnol 1998;49:285-9.   DOI