Subcellular Localization of Rice Small G Protein OsRab5b in BY-2 Cells - 林木分子遗传育种 (MGBFT)

To investigate the subcellular localization of rice OsRab5b and the Gly2 role in the protein subcellular localization. OsRab5b-GFP and OsRab5b（Gly2Ala）-GFP plasmids were transformed into BY-2 cells. The laser confocal microscope was employed to observe the transgenic cells treated with drugs, a marker protein VSRAt-1 or an endocytosis tracer dye FM4-64. Results showed Punctuate and diffuse signals were observed in the OsRab5b-GFP transgenic cells. Wortmannin at a concentration of 16.5 μmol/L led to the change of the OsRab5b-GFP marked organelles into small vacuoles. The signals of OsRab5b-GFP were aggregated after the treatment of Brefeldin A（BFA）at a concentration of 100 μg/mL, but not at 10 μg/mL. Most of the OsRab5b-GFP positive signals colocalized with the VSRAt-1 positive organelles in the cells with and without wortmannin or BFA treatment. Additionally, most of the OsRab5b-GFP marked organelles colocalized with the endosomal marker FM4-64 after 60-min incubation. The diffuse signals of the mutation protein OsRab5b（Gly2Ala）-GFP were observed in the nuclei and cytoplasm of the transgenic cells, which were not affected by the wortmannin or BFA treatment. OsRab5b is localized to the prevacuolar compartment of BY-2 cells and the Gly2 site is essential for the correct subcellular localization of OsRab5b.