Polyethylene glycol (PEG)-mediated transformation of the fused egfp-hph gene into Pleurotus ostreatus

Abstract

A transformation system for the basidiomycete Pleurotus ostreatus was established using PEG-mediated transformation. The homologous glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter and terminator regions of P. ostreatus were amplified and cloned onto the restriction site of the pUC19 vector. Enhanced green fluorescent protein-encoding gene (egfp) and a selection marker, the hygromycin phosphotransferase gene (hph), were then cloned and an expression vector, pUEGFP-hph, was constructed. Protoplasts of P. ostreatus were prepared and used as the recipients in transformation procedures. An improved transformation method was established in the present study and about 100 to 200 resistant colonies per microgram DNA per 107 viable protoplasts were obtained. After subculturing, putative transformants were screened by PCR and verified by Southern blot and RT-PCR analyses showing the correct insertion into the genomic DNA and transcription of the transformed DNA. Moreover, the quantitative PCR and green fluorescence observation were carried out, thus demonstrating expression of the egfp gene driven by the homologous gpd promoter. This was the first report of transformation of P. ostreatus using homologous gpd promoter by PEG-mediated methods. These data will be helpful in future investigations using PEG-mediated transformation for functional characterisation of genes in the edible fungi basidiomycete P. ostreatus. Key words : Pleurotus ostreatus, PEG-mediated transformation, EGFP.