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Liposome-mediated mycelial transformation of filamentous fungi

Ran Chai1, Guang Zhang, Qiang Sun

  • 1College of Life Sciences, Henan Agricultural University, 95 Wenhua Road, Zhengzhou 450002, China; Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, 95 Wenhua Road, Zhengzhou 450002, China.

Fungal Biology
|September 10, 2013
PubMed

Insights

This study introduces a novel liposome-mediated DNA transformation method for filamentous fungi, bypassing the need for protoplast preparation. This effective and simple technique achieves transformation efficiencies comparable to traditional methods.

Area of Science:

  • Mycology
  • Molecular Biology
  • Biotechnology

Background:

  • Liposome-mediated transformation is widely used for cell wall-lacking organisms.
  • Its application in organisms with cell walls, like fungi, is limited due to technical challenges.
  • Traditional methods for fungal transformation often require protoplast preparation, which can be time-consuming and reduce viability.

Purpose of the Study:

  • To develop a direct liposome-mediated DNA transformation protocol for filamentous fungi.
  • To introduce DNA into mycelium without prior protoplast isolation.
  • To establish an efficient, fast, and simple transformation method for fungal species.

Main Methods:

  • Developed a procedure involving grinding fungal mycelia (Rhizopus nigricans and Pleurotus ostreatus) in mannitol to create a suspension of mycelial fragments.
  • Mixed the suspension with DNA (plasmid pEGFP-C1 for R. nigricans, 7.2 kb linear DNA for P. ostreatus) and Lipofectamine 2000.
  • Incubated on ice, plated on potato dextrose agar (PDA), and cultivated at 28 °C for screening transformants.

Main Results:

  • Successfully transformed R. nigricans and P. ostreatus using the direct liposome-mediated method.
  • Confirmed integration of both plasmid and linear DNA into the host chromosomes.
  • Achieved transformation efficiencies comparable to established methods like electroporation-mediated protoplast transformation (EMPT) and PEG/CaCl2-mediated protoplast transformation (PMT).

Conclusions:

  • The developed liposome-mediated procedure is an effective, rapid, and straightforward method for transforming filamentous fungi.
  • This technique eliminates the need for protoplast preparation, simplifying the transformation process.
  • The method holds promise for genetic manipulation of various filamentous fungal species.
Keywords:
Fungal myceliumLiposome-mediated transformationPleurotus ostreatusRhizopus nigricans

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