Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum.
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.
Key Laboratory for Microbiological Engineering of the Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, Jiangsu, People's Republic of China.
Liang
L
Shi
Xing
X
Fang
Mengjiao
M
Li
Dashuai
D
Mu
Ang
A
Ren
Qi
Q
Tan
Mingwen
M
Zhao
D016960:Agrobacterium tumefaciens / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008516:Medicine, Chinese Traditional; D008938:Mitosis / Q000235:genetics; D019936:Probiotics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D020940:Reishi / Q000166:cytology / Q000235:genetics* / Q000502:physiology; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D011994:Recombinant Proteins; C036483:T-DNA; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005966:Glucuronidase