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Fungal Genet Biol. 2014 Jan;62:1-10. doi: 10.1016/j.fgb.2013.10.012. Epub 2013 Nov 8.

Establishing a versatile Golden Gate cloning system for genetic engineering in fungi.

Author information

1
Institute for Microbiology, Center of Excellence on Plant Sciences (CEPLAS), Department of Biology, Heinrich-Heine University Düsseldorf, 40204 Düsseldorf, Germany.
2
Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, Georg-August-University, 37077 Göttingen, Germany.
3
Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada CICESE, 22860 Baja California, Mexico.
4
Institute for Microbiology, Center of Excellence on Plant Sciences (CEPLAS), Department of Biology, Heinrich-Heine University Düsseldorf, 40204 Düsseldorf, Germany. Electronic address: feldbrue@hhu.de.

Abstract

The corn pathogen Ustilago maydis is a well-studied fungal model organism. Along with a broad set of experimental tools, versatile strategies for the generation of gene replacement mutants by homologous recombination in U. maydis have been developed. Nevertheless, the production of corresponding linear DNA constructs still constitutes a time-limiting step. To overcome this bottleneck, various resistance cassette modules were adopted for use with the so-called Golden Gate cloning strategy. These modules allow not only simple gene deletions but also more sophisticated genetic manipulations like inserting sequences for C-terminal protein tagging. The type IIs restriction enzyme BsaI was selected for this novel approach as its recognition sites are comparatively rare in the U. maydis genome. To test the efficiency of the new strategy it was used to test the influence of varying flank lengths as well as the effect of non-homologous flank ends on homologous recombination. Importantly, to proof a broad applicability in other fungi the same strategy was used to generate mutants in the filamentous ascomycete Aspergillus nidulans. Hence, we present a highly efficient and economic cloning strategy that speeds up reverse genetic approaches in fungi.

KEYWORDS:

Filamentous fungi; Golden Gate cloning; Homologous recombination; Resistance-marker recycling

PMID:
24211735
DOI:
10.1016/j.fgb.2013.10.012
[Indexed for MEDLINE]

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