Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus

• Published in: FEMS microbiology letters Vol. 181; no. 2; pp. 225 - 228
• Main Authors: Kim, B.G, Magae, Y, Yoo, Y.B, Kwon, S.T
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-12-1999; Blackwell; Oxford University Press
• Subjects: Acid resistance; Aspartate Carbamoyltransferase - genetics; Auxotrophs; Basidiospores; Biological and medical sciences; Biotechnology; Blotting, Southern; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - genetics; crossing; Deoxyribonucleic acid; DNA; DNA, Fungal - genetics; Drug Resistance, Microbial; Fruit bodies; fruiting bodies; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungi; genes; Genetic engineering; Genetic technics; Genetic transformation; Genetic Vectors - genetics; Genomes; Methods. Procedures. Technologies; Microbiology; Miscellaneous; Monokaryons; Monophosphate decarboxylase; Multienzyme Complexes - genetics; Mutation - radiation effects; Mycological methods and techniques used in mycology; Mycology; Orotic acid; Orotic Acid - analogs & derivatives; Orotic Acid - pharmacology; Orotidine; orotidine-5'-phosphate decarboxylase; plasmids; Plasmids - genetics; Pleurotus - drug effects; Pleurotus - genetics; Pleurotus - metabolism; Pleurotus - radiation effects; Pleurotus ostreatus; Protoplasts; Prototrophy; Saccharomyces cerevisiae Proteins; Southern blotting; Synthetic digonucleotides and genes. Sequencing; Transformation; Transformation, Genetic; Trichoderma - genetics; Trichoderma reesei; Ultraviolet Rays; Uracil; Uracil - metabolism; Uracil auxotrophic marker; Transformation; Monophosphate decarboxylase; Uracil auxotrophic marker; Orotidine; Fungi; Auxotrophy; Pleurotus ostreatus; Genetic transformation; Biological marker; Basidiomycetes; Isolation; Method; Uracil; Edible fungi; Thallophyta
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.1999.tb08848.x

Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA). Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei. Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts. Using 2 X 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microgram of DNA. Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy. These results indicate the integrated DNA was stably inherited.