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 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.
Sato
T
T
Iwate Biotechnology Research Center, Japan. tosisato@sv02.ibrc.pref.iwate.jp
Yaegashi
K
K
Ishii
S
S
Hirano
T
T
Kajiwara
S
S
Shishido
K
K
Enei
H
H
D015139:Blotting, Southern; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D004271:DNA, Fungal / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D006921:Hygromycin B / Q000737:chemistry; D020074:Lentinula / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011523:Protoplasts / Q000737:chemistry; D015183:Restriction Mapping; D014170:Transformation, Genetic