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 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 mitochondrialtargeting 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.

The distribution of starch-degrading enzymes in dormant rice grains was investigated with specific antibodies against α-glucosidases, α-amylases, and β-amylases. The α-glucosidases were predominantly localized in the inner endosperm. The α-amylases were mainly localized in the outer layers, and the β-amylases were distributed in whole grains. We propose a model to demonstrate how these enzymes degrade starch during rice cooking.

No abstract

No abstract

The pullulanase and α-glucosidase in brown rice is predominately found in the endosperm of rice grains of Koshihikari and Nipponbare cultivars. However, the exact localization of these enzymes in the endosperm layers is not clear. To investigate enzyme localization in non-glutinous brown rice, immuno-fluorescence detection of these enzymes was carried out using frozen sections of rice grains (Koshihikari, Nipponbare and Milkyqueen cultivars). While pullulanase-like immuno-reactivity was dominantly detected in amyloplasts, α-glucosidase-like immuno-reactivity was mainly detected along endosperm cell walls among the three non-glutinous cultivars. Immuno-reactivity of both enzymes overlapped in the aleurone layer and embryo among the three cultivars. During rice cooking, pullulanase-like immuno-reactivity partly overlapped with α-glucosidase-like immuno-reactivity along endosperm cell walls among the three cultivars. These results indicate that each enzyme has a unique localization, and these unique localizations are common among the three non-glutinous cultivars.