title: Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria.
abstract: We report here a development of the MultiSite Gateway(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 carrier-proteins 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.
authors.affiliation: Department of Biotechnology, The University of Tokyo, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Mabashi
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikuma
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Maruyama
authors.forename: Manabu
authors.initials: M
authors.lastname: Arioka
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Kitamoto
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D002950:Citrate (si)-Synthase / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002950:Citrate (si)-Synthase