Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum
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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.
KeywordsGanoderma lucidum Agrobacterium tumefaciens-mediated transformation Promoter Fluorescence assay Histochemical staining assay
Restriction enzyme-mediated integration
Agrobacterium tumefaciens-mediated transformation
Enhanced green fluorescent protein
Hygromycin B phosphotransferase
This work was financially supported by the National Natural Science Foundation of China (Project No. 30970042, 30871767), the Fundamental Research Funds for the Central Universities (Project No. KYZ201121), and Shanghai Committee of Science and Technology, China (Project No. 08JC1418100).
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