The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae.
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.
National Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam.
Khuyen Thi
KT
Nguyen
National Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam.
Quynh Ngoc
QN
Ho
National Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam.
Thu Ha
TH
Pham
National Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam.|Faculty of Biology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam.
Tuan-Nghia
TN
Phan
National Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam. tuantran@vnu.edu.vn.|Faculty of Biology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam. tuantran@vnu.edu.vn.
Van-Tuan
VT
Tran
D016960:Agrobacterium tumefaciens / Q000502:physiology*; D001236:Aspergillus oryzae / Q000235:genetics*; D005800:Genes, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D014170:Transformation, Genetic
D011994:Recombinant Proteins
Agrobacterium-mediated transformation; Aspergillus oryzae; Binary vector; Fluorescent reporter genes; pyrG auxotrophic marker