Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS

• Published in: Bioscience, biotechnology, and biochemistry Vol. 65; no. 7; pp. 1504 - 1510
• Main Authors: Maruyama, J. (Tokyo Univ. (Japan)), Nakajima, H, Kitamoto, K
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.07.2001; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Alcoholic Beverages - microbiology; Aspergillus nidulans; Aspergillus nidulans - genetics; Aspergillus nidulans - growth & development; Aspergillus nidulans - ultrastructure; ASPERGILLUS ORYZAE; Aspergillus oryzae - genetics; Aspergillus oryzae - growth & development; Aspergillus oryzae - ultrastructure; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Cell Nucleus - ultrastructure; conidia; EGFP; FACS; Fermentation; Flow Cytometry; Food Microbiology; Fundamental and applied biological sciences. Psychology; FUNGAL SPORES; Gene Expression; Green Fluorescent Proteins; H2B protein; Histones - genetics; Luminescent Proteins - genetics; Microbiology; Miscellaneous; Mission oriented research; Morphology, structure, chemical composition; Mycology; nuclei; NUCLEUS; PROTEINS; Recombinant Fusion Proteins - genetics; Transformation, Genetic; Fungi; Visualization; Aspergillus oryzae; Cell nucleus; Green fluorescent protein; Fluorescence; Fungi Imperfecti; Gene expression; Thallophyta; Conidia
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.65.1504

Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidulans histone H2B protein tagged by EGFP (H2B::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B::SEGFP, fluorescence was detected in nuctear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66% of conidia had two, 24% had one, and 10% had three or more in the A. oryzae transformant. The conidia expressing H2B::EGFP were put throtrgh FACS (fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were succesfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied nunber of nuclei existed in each conidium and some strains had a small number of uninucleate conidia.