| Pleurotus ostreatus is an important cultivated species of edible mushroom. The development of a genetic transformation system for this fungus will play an essential role in further transgenic manipulation of P. ostreatus, facilitating efforts to improve the food value of this species as well as other edible mushroom fungi.; Genetic transformation was accomplished by electroporation of protoplasts based on positive selection for resistance to hygromycin B (HmB) using a plasmid vector pAN7-1. The vector pAN7-1 contains the Escherichia coli hygromycin B phosphotransferase gene (hph), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Pgpd), and the tryptophan synthetase gene terminator signal (ttrpC) from Aspergillus nidulans. Expression of the hph gene confers resistance to HmB. Optimal conditions for protoplast formation and transformation of P. ostreatus from dikaryotic mycelia were determined. Selection for HmB resistance of this fungus, indicative of transformation, resulted in 3-48 transformants per {dollar}mu{dollar}g of pAN7-1 per 10{dollar}sp7{dollar} protoplasts. Southern blot analysis of certain transformants revealed unexpected replicative plasmids that contain sequences both from pAN7-1 and from the fungal chromosome. These replicative plasmids were modified for size, methylation and restriction enzyme pattern when compared to the initial transforming vector. Such plasmids, generated through in vivo recombination in P. ostreatus, have been rescued in E. coli strain DH5{dollar}alpha{dollar}. Two of the plasmids, pPO1 and pPO2, then were introduced and recovered from the methylation-minus SURE strain of E. coli. Both plasmids were thus demethylated, based on evidences from isoschizomer analysis. Demethylated plasmids are designated pPO1{dollar}alpha{dollar} and pPO2{dollar}alpha{dollar}. Use of pPO1{dollar}alpha{dollar} to transform P. ostreatus indicated extrachromosomal maintenance of this plasmid. A recombined fungal chromosome-derived sequence of 1148 bp from pPO1{dollar}alpha{dollar} has been characterized by DNA sequencing. This sequence lacks an open-reading-frame and possesses several general features characteristic of a replicon. |
