Transformation of the edible basidiomycete Lentinus edodes by restriction enzyme-mediated integration of plasmid DNA

We have used the restriction enzyme-mediated DNA integration (REMI) method to establish a transformation system in Lentinus edodes using the recombina nt plasmid pLC1-hph, which contains the L. edodes transcriptional signals a nd an Escherichia coli hygromycin B phosphotransferase gene. Protoplasts of L. edodes were treated by the PEG transformation mixture containing 50 uni ts of SalI, which cleaves pLC1-hph at a single site, yielding about 15 tran sformants per 2.5 mu g of DNA, The conventional PEG transformation without SalI, however, yielded only 1.5 transformants per 25 mu g of DNA. The optim al amount of SalI for increased transformation was 50 units. In the case of transformation with SphI, which cleaves the plasmid at one site, the optim al amount of the enzyme was 2.5 units. Southern blot analysis of the SphI-d erived transformants suggested that 50% of the plasmid integrations were RE MI events.