Development of a modified positive selection medium that allows to isolate Aspergillus oryzae strains cured of the integrated niaD-based plasmid.
نویسندگان
چکیده
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.
منابع مشابه
Recombinational inactivation of the gene encoding nitrate reductase in Aspergillus parasiticus.
Functional disruption of the gene encoding nitrate reductase (niaD) in Aspergillus parasiticus was conducted by two strategies, one-step gene replacement and the integrative disruption. Plasmid pPN-1, in which an internal DNA fragment of the niaD gene was replaced by a functional gene encoding orotidine monophosphate decarboxylase (pyrG), was constructed. Plasmid pPN-1 was introduced in linear ...
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ورودعنوان ژورنال:
- Bioscience, biotechnology, and biochemistry
دوره 69 12 شماره
صفحات -
تاریخ انتشار 2005