Transformation of the Edible BasidiomyceteLentinus edodesby Restriction Enzyme-Mediated Integration of Plasmid DNA

Abstract

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.

The Tyrosinase-Encoding Gene ofLentinula edodes,Letyr, Is Abundantly Expressed in the Gills of the Fruit-Body during Post-Harvest Preservation

“…Genomic DNA was prepared by the method of Sato et al 12) from mycelia grown in 0.25 Â MYPG liquid culture for 2 weeks at 25 C with shaking.…”

Consolidated Bioprocessing Ethanol Production by Using a Mushroom

“…Genetic transformation was investigated using the pFvT vector and the protoplasts prepared as described above. The transformation procedures for Lentinus edodes (Sato et al, 1998) and Schizophyllum commune (Van Peer et al, 2009) were modified for the transformation of F. velutipes Fv-1. In the course of the transformation process, the effect of the structure of the plasmid DNA on transformation was evaluated using circular and linear pFvT plasmids.…”

“…Approximately 6-fold transformants were obtained when the plasmid DNA was linearized (Table 2). Because the REMI method is a popular transformation tool for fungi (Hirano et al, 2000;Maier & Schäfer, 1999;Riggle & Kumamoto, 1998;Sato et al, 1998), we evaluated the effect of REMI on the transformation for F. velutipes Fv-1. The F. velutipes Fv-1 strain was transformed by pFvT with a restriction enzyme, BglI, KpnI, or PstI.…”

Approaches to functional genomics in filamentous fungi

“…Conditions of transformation contribute significantly to the variable success rate of REMI. Although the enzyme type and concentration have an important impact on the number of transformants and the frequency of single-copy integrations, there are no clear rules for selecting either [56,64,66], and the npg concentration of RE that produces the maximum number of transformants is enzyme and host dependent [66][67][68]. REMI can give rise to a significant number of different integration events including single insertion with deletion of flanking RE sites, ectopic integration in the absence of an appropriate RE site, tandem insertion and large genome deletions or inversions [50,56,64,65,68,69].…”