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World Journal of Microbiology and Biotechnology

, Volume 28, Issue 1, pp 283–291 | Cite as

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

  • Liang Shi
  • Xing Fang
  • Mengjiao Li
  • Dashuai Mu
  • Ang Ren
  • Qi Tan
  • Mingwen Zhao
Original Paper

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.

Keywords

Ganoderma lucidum Agrobacterium tumefaciens-mediated transformation Promoter Fluorescence assay Histochemical staining assay 

Abbreviations

GPD

Glyceraldehyde-3-phosphate dehydrogenase

PEG

Polyethylene glycol

REMI

Restriction enzyme-mediated integration

ATMT

Agrobacterium tumefaciens-mediated transformation

EGFP

Enhanced green fluorescent protein

GUS

β-glucuronidase

AS

Acetosyringone

MES

2-(N-morpholino)ethanesulfonic acid

HPH

Hygromycin B phosphotransferase

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Liang Shi
    • 1
  • Xing Fang
    • 1
  • Mengjiao Li
    • 1
  • Dashuai Mu
    • 1
  • Ang Ren
    • 1
  • Qi Tan
    • 2
  • Mingwen Zhao
    • 1
  1. 1.Key Laboratory for Microbiological Engineering of the Agricultural Environment, Ministry of AgricultureCollege of Life Sciences, Nanjing Agricultural UniversityNanjingPeople’s Republic of China
  2. 2.National Engineering Research Center of Edible Fungi, Shanghai Key Laboratory of Agricultural Genetics and BreedingInstitute of Edible Fungi, Shanghai Academy of Agricultural ScienceShanghaiChina

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