Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum

Abstract

In this study, we report the development of a simple and efficient system for genetic transformation of the medicinal fungus Ganoderma lucidum. Various parameters were optimized to obtain successful Agrobacterium tumefaciens-mediated transformation. Co-cultivation of bacteria and protoplast at a ratio of 1,000:1 at 25°C in medium containing 0.2 mM acetosyringone was found to be the optimum condition for high efficiency transformation. Four plasmids, each carrying a different promoter driving the expression of an antibiotic resistance marker, were tested. The construct carrying the Ganoderma lucidum glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter showed good transformation efficiency, whereas constructs with the GPD promoter from ascomycetes were ineffective. Our analysis showed that over 70% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. We were able to detect the expression of EGFP and GUS reporter genes in the Ganoderma lucidum transformants by fluorescence imaging and histochemical staining assays respectively. Our results demonstrate a new transgenic approach that will facilitate Ganoderma lucidum research.

UDP-glucose pyrophosphorylase influences polysaccharide synthesis, cell wall components, and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose

“…The mycelia were then ground to powder with liquid nitrogen. Total DNA was extracted from the fungus by a method adapted from the CTAB method, as described previously (Shi et al, 2012a).…”

“…The construction of a fungal overexpression vector, pGl-gpd, has been described in our previous study (Shi et al, 2012a). This vector was used for the construction of ugp overexpression strains of G. lucidum.…”

“…This vector was used for the construction of ugp overexpression strains of G. lucidum. G. lucidum strains were transformed by Agrobacterium tumefaciens-mediated transformation (ATMT), as described by Shi et al (2012a). The ugp gene was amplified with the primers listed in Table S1.…”

Functional analysis of the role of glutathione peroxidase (GPx) in the ROS signaling pathway, hyphal branching and the regulation of ganoderic acid biosynthesis in Ganoderma lucidum

“…The lack of basic research on G. lucidum limits the further development and utilization of this organism. Luckily, the development of methodological platforms for genome sequencing, genetic transformation and transcriptional gene silencing has made it possible to conduct additional research on G. lucidum (Chen et al, 2012;Mu et al, 2012;Shi et al, 2012). Because of these studies, G. lucidum is now considered an adequate model system for the study of complicated growth patterns and the regulation of secondary metabolic pathways in higher basidiomycetes.…”

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

“…Basidiomycete mushrooms generally exhibit higher resistance to genetic transformation compared to ascomycetous fungi (37). Despite the great requirement for G. lucidum, only a few reports exist regarding its genetic transformation using heterologous resistance genes as selection marker (16,34,38). In addition, the application of these methods to express other genes except marker genes has never been reported.…”