Fig1

Fig 1. Construction of an OsMDHAR-expressing yeast vector and the stress response of OsMDHAR-expressing yeast to hydrogen peroxide. (A) Schematic diagram of the p426GPD::OsMDHAR construct. The OsMDHAR gene (approximately 1.5 kbp) was subcloned to generate the p426GPD::OsMDHAR construct with OsMDHAR under the control of the constitutive GPD promoter. Semi-quantitative RT-PCR (B), immunoblotting (C), and an enzymatic assay (D) were performed to examine whether OsMDHAR is expressed in this yeast strain. PDA1 and tubulin (Tub) were used as housekeeping controls for RT-PCR and western blotting, respectively. The molecular size of the PCR product and molecular weight of the detected band were approximately 494 bp and 47 kDa, respectively. Stress tolerance to hydrogen peroxide was evaluated by cell survival, growth kinetics, and spotting assays. (E) To monitor cell viability, yeast cells precultured in YPD medium were inoculated into fresh YPD medium and exposed to different concentrations of H2O2 for 16 h at 28°C. Then, the optical density at 600 nm (OD600) was measured. Circles, cells transformed with p426GPD-OsMDHAR (TC cells); squares, wild-type (WT) cells transformed with an empty vector. (F) For the growth kinetics assay, precultured yeast cells were inoculated into YPD medium containing 5 mM H2O2, and the OD600 was measured at 2-h intervals for 36 h. A streaking assay was also performed, in which mid-log phase yeast cells (OD600 ≈ 2.0) were streaked onto YPD agar plates supplemented with 5 mM H2O2. WT (squares) and TC (circles) cells in the absence of 5 mM H2O2; WT (upward triangles) and TC (diamonds) cells in the presence of 5 mM H2O2. (G) Mid-log phase yeast cells were exposed to 20 mM H2O2 for 1 h with shaking, and serially diluted with YPD medium. A 5-µL aliquot of each dilution was spotted onto YPD agar plates.