Development of a modified positive selection medium that allows to isolate Aspergillus oryzae strains cured of the integrated niaD-based plasmid.
The nitrate reductase gene (niaD) is the most frequently utilized as a selectable marker for homologous integration at the niaD locus of Aspergillus oryzae. In this study we developed a method for curing of the niaD-based plasmid integrated on the A. oryzae genome. Positive selection using a modified chlorate medium containing leucine as a nitrogen source enabled efficient isolation of the strains deficient in nitrate assimilation from the niaD(+) transformant. PCR analysis of the strains confirmed that the homologously integrated plasmid carrying the h2b-egfp fusion gene was cured by intrachromosomal recombination which was accompanied by the loss of the EGFP-fluorescence.
Ishi
Kazutomo
K
Department of Biotechnology, The University of Tokyo, Japan.
Watanabe
Taisuke
T
Juvvadi
Praveen Rao
PR
Maruyama
Jun-Ichi
J
Kitamoto
Katsuhiko
K
D001233:Aspergillus nidulans / Q000235:genetics; D001236:Aspergillus oryzae / Q000235:genetics* / Q000254:growth & development*; D002704:Chlorates / Q000494:pharmacology; D003470:Culture Media; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D018698:Glutamic Acid / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D050901:Nitrate Reductase / Q000235:genetics* / Q000502:physiology; D010957:Plasmids / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism
D002704:Chlorates; D003470:Culture Media; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D050901:Nitrate Reductase; C022842:chloric acid