Construction of Aspergillus oryzae strains with organelles labeled with fluorescence and observation of organelle morphology

Abstract:

The organelles in the multi-nucleated filamentous fungus Aspergillus oryzae present polymorphism. To observe the organelle morphology in A. oryzae and provide references for the localization prediction of unknown proteins and the disclosure of biological reaction pathways in A. oryzae, we fused different subcellular localization signals with green fluorescent protein (GFP) to obtain different subcellular localization vectors, which were then transferred into A. oryzae by Agrobacterium tumefaciens-mediated transformation. The A. oryzae reporter strains with fluorescence-labeled nuclei, mitochondria, endoplasmic reticulum, vacuole, lipid droplets, peroxisome, and Golgi apparatus were successfully constructed. Furthermore, staining with small-molecule specific dyes was carried out to validate the co-localization of fluorescence-labeled mitochondria, nuclei, and lipid droplets in the reporter strains, which further confirmed that the reporter strains were successfully constructed. The distribution and morphology of fluorescence-labeled organelles were observed at different growth stages and under different culture conditions. The constructed reporter strains provide basic tools for studying the organelle morphology, localization of unknown target proteins, and subcellular localization in A. oryzae.