Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance.
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.
Hirano
T
T
Iwate Biotechnology Research Center, Kitakami, Japan.
Sato
T
T
Yaegashi
K
K
Enei
H
H
D001483:Base Sequence; D015139:Blotting, Southern; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006921:Hygromycin B / Q000494:pharmacology*; D008969:Molecular Sequence Data; D020072:Polyporales / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D006921:Hygromycin B; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D004262:DNA Restriction Enzymes