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

To construct a vector for high-level expression of heterologous genes in Lentinus edodes, the L. edodes GPD promoter, which is expressed constitutively and strongly, was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selective marker. Using the resulting pLG-hph construct, L. edodes was efficiently transformed to hygromycin resistance by restriction enzyme-mediated integration (REMI). The restriction enzyme concentrations yielding the maximal numbers of transformants by the REMI method were 10 U per transformation in the case of BglII and 25-50 U in the case of HindIII. Southern analysis of the transformants indicated that some 50% of plasmid integrations were REMI-mediated events. These results indicate that pLG is a useful vector for transformation of L. edodes. Deletion analysis of the GPD promoter region suggested that the segment between positions -442 bp and -270 bp relative to the transcription start point may be essential for function.

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