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The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae

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Abstract

Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

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Acknowledgements

The authors thank Dr. Jun-ichi Maruyama (Department of Biotechnology, The University of Tokyo, Japan) for kindly providing the A. oryzae AUT1-PlD strain. This work was funded by the Ministry of Science and Technology of Vietnam through the research grant KLEPT.14.01 with the regulation of Vietnam National University (VNU), Hanoi.

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Authors and Affiliations

  1. National Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

    Khuyen Thi Nguyen, Quynh Ngoc Ho, Thu Ha Pham, Tuan-Nghia Phan & Van-Tuan Tran

  2. Faculty of Biology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

    Tuan-Nghia Phan & Van-Tuan Tran

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  1. Khuyen Thi Nguyen
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  2. Quynh Ngoc Ho
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  4. Tuan-Nghia Phan
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Correspondence to Van-Tuan Tran.

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Nguyen, K.T., Ho, Q.N., Pham, T.H. et al. The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae . World J Microbiol Biotechnol 32, 204 (2016). https://doi.org/10.1007/s11274-016-2168-3

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