Development of a transformation system for Aspergillus sojae based on the Agrobacterium tumefaciens-mediated approach

BMC Microbiology

Marcelo Fernandez-Lahore²  Amira M Rizk²  Judith Zimmermann¹  Rodrigo Mora-Lugo² 

[1] Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, Bremen, 28359, Germany;Jacobs University Bremen gGmbH, Campus Ring 1, Bremen, 28759, Germany

关键词: Western blot;    Transfer DNA (T-DNA);    Filamentous fungus;    Enhanced green fluorescent protein (EGFP);    Ble gene;   

Others  :  1170529
DOI  :  10.1186/s12866-014-0247-x

 received in 2014-06-04, accepted in 2014-09-15,  发布年份 2014

【 摘 要 】

Background

Aspergillus sojae has been an important filamentous fungus in Biotechnology due to its use in diverse fermentative processes for the production of various food products. Furthermore, this fungus is a common expression system for the production of enzymes and other metabolites. The availability of molecular genetic tools to explore its biology is thus of big interest. In this study, an Agrobacterium tumefaciens-mediated transformation (ATMT) system for A. sojae was developed and its applicability evaluated.

Results

The donor plasmid named pRM-eGFP was constructed for ATMT of A. sojae. This plasmid contains the ble and egfp genes in its transfer DNA element (T-DNA) to confer phleomycin resistance and express the enhanced green fluorescent protein (EGFP) in A. sojae, respectively. Agrobacterium tumefaciens (LBA4404) harboring the donor plasmid and A. sojae (ATCC 20235) were co-cultured under diverse conditions to achieve ATMT. The maximum number of transformed fungi was obtained after three days of co-culturing at 28°C, and selection with 50 ?g/ml phleomycin. Polymerase chain reaction (PCR), fluorescence microscopy and Western Blot analysis for EGFP expression confirmed successful genomic integration of the T-DNA element in A. sojae. The T-DNA was mitotically stable in approximately 40% of the fungal transformants after four generations of sub-culturing under phleomycin pressure.

Conclusion

We successfully established a new ATMT protocol for A. sojae. This transformation system should enable further protein expression studies on this filamentous fungus.

【 授权许可】

   
2014 Mora-Lugo et al.; licensee BioMed Central Ltd.

【 图 表 】

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