Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria

• Published in: Bioscience, biotechnology, and biochemistry Vol. 70; no. 8; pp. 1882 - 1889
• Main Authors: Mabashi, Y.(Tokyo Univ. (Japan)), Kikuma, T, Maruyama, J, Arioka, M, Kitamoto, K
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01-08-2006; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: ASPERGILLUS ORYZAE; Aspergillus oryzae - enzymology; Aspergillus oryzae - genetics; Aspergillus oryzae - growth & development; Biological and medical sciences; Citrate (si)-Synthase - genetics; Cloning, Molecular; EGFP; EXPRESION GENICA; EXPRESSION DES GENES; expression plasmid; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; GENETIC ENGINEERING; Genetic Vectors - genetics; GENIE GENETIQUE; Green Fluorescent Proteins - genetics; INGENIERIA GENETICA; MITOCHONDRIA; Mitochondria - enzymology; Mitochondria - genetics; MITOCHONDRIE; MITOCONDRIA; PLASMIDE; PLASMIDIOS; PLASMIDS; Plasmids - genetics; protein expression; PROTEINAS; PROTEINE; PROTEINS; Recombinant Fusion Proteins - genetics; Visualization; Gene expression; EGFP; Protein; Fungi; Plasmid; expression plasmid; Mitochondria; Aspergillus oryzae; Fungi Imperfecti; Application; protein expression; Thallophyta
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.60052

We report here a development of the MultiSite Gatewaysup(TM)-based versatile plasmid construction system applicable for the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrierproteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein. Using this system, plasmids for expression of the EGFP fused with the mitochondrial-targeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1-EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae.