Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation

Abstract

Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our experiments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 microg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experimental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0x10(8) cells/mL of blastospores, 200 microL in transformation volume, 6 microg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/microg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR detection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis.