Agrobacterium tumefaciens-Mediated Transformation of the King Tuber Medicinal Mushroom Lentinus tuber-regium (Agaricomycetes).

Lentinus tuber-regium is a sclerotium-forming basidiomycetous mushroom. It has increasingly aroused people's attention for its medicinal effects. In this study, we investigated the applicability of the Agrobacterium tume-faciens-mediated transformation (ATMT) method in L. tuber-regium. A. tumefaciens strain GV 3101 harboring the vector pPEH was used to transform the mycelium of L. tuber-regium strain ACCC50657. The genes for hygromycin B phosphotransferase (hph) and enhanced green fluorescent protein (egfp) under the control of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter of Pleurotus ostreatus were employed as the selection marker and reporter gene, respectively. The optimal cocultivation temperature and time for transformation were 3 days and 4 days at 25°C and 20°C, respectively. Southern blot analysis showed the variation in the copy number and position of hph, which indicated random integration of hph. Polymerase chain reaction and fluorescence microscopy indicated that the P. ostreatus gpd promoter can drive the fused hph-egfp gene expression in L. tuber-regium. This is the first report that the ATMT method was successfully applied to L. tuber-regium. This reliable and efficient transformation method could be a powerful tool for strain genetic improvement and gene function study in L. tuber-regium.