RT-PCR analysis of ATG genes and western blot analysis of ATG8 in Cucumis sativus during stress conditions

Description

The expression of different splice variance of ATG genes in Cucumis sativus under stresses conditions including nitrogen starvation, oxidative stress, and pathogen infection were examined using realtime-PCR. The nitrogen starvation experiment was performed using five-day-old seedlings of C. sativus grown on liquid 1/2 MS medium with and without nitrogen for 2, 4 and 6 days (Oka et al., 2012). Oxidative stress was conducted using two-week-old seedlings grown on soil. These plants were sprayed once with 50 µM Methyl viologen (MV) prepared in 0.05% (v/v) Tween 20 (Song et al., 2005), and leaves samples were collected at 3, 6, 9, 12 and 15 days after the treatment. For pathogen responses, seven-day-old seedlings of C. sativus grown on soil were inoculated with sporangia of the fungi causing downy mildew disease (Pseudoperonospora cubensis). Samples were collected at 6, 12, 24, 72 and 168 h after inoculation. For each experiment, three replicates were collected for each time point before frozen in liquid nitrogen and stored at - 80 °C The real-time PCR analysis was performed in duplicate assays in 96-well plates; each 10 µl reaction consisted of 2x QPCR Green Master Mix (Biotechrabbit, Germany), 5 µmol forward and reverse primers and 1 ng cDNA. The primers were designed to span the junction that differ between transcript variances. The data of Ct values obtained from two different experimental RNA samples were directly normalized to a housekeeping gene, CsActin. The fold-change in expression of the gene of interest between each timepoint was then calculated as 2^ (-ΔΔCt). Statistical differences in the level expression compared to the expression at time point 0 or that of ATG7 were analyzed with student’s T test with two-tailed distribution. Protein extraction and immunoblot analysis Samples were ground and resuspended in extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) IGEPAL CA-360, 1 mM phenylmethylsulfonyl fluoride) on ice. Crude proteins were separated on18% SDS-PAGE before stained using Coomassie blue or transferred onto a PVDF membrane (GE Healthcare). Immunodetection of ATG8 was performed using anti-CrATG8 (Pérez-Pérez et al., 2010), with a working concentration of the primary antibody at 1:1000 in PBS containing 2.5% BSA. The Coomassie Brilliant Blue staining of the gel was used to estimate the amount of protein loaded.

Steps to reproduce

The nitrogen starvation experiment was performed using five-day-old seedlings of C. sativus grown on liquid 1/2 MS medium with and without nitrogen for 2, 4 and 6 days (Oka et al., 2012). Oxidative stress was conducted using two-week-old seedlings grown on soil. These plants were sprayed once with 50 µM Methyl viologen (MV) prepared in 0.05% (v/v) Tween 20 (Song et al., 2005), and leaves samples were collected at 3, 6, 9, 12 and 15 days after the treatment. For pathogen responses, seven-day-old seedlings of C. sativus grown on soil were inoculated with sporangia of the fungi causing downy mildew disease (Pseudoperonospora cubensis). Samples were collected at 6, 12, 24, 72 and 168 h after inoculation. For each experiment, three replicates were collected for each time point before frozen in liquid nitrogen and stored at - 80 °C The real-time PCR analysis was performed in duplicate assays in 96-well plates; each 10 µl reaction consisted of 2x QPCR Green Master Mix (Biotechrabbit, Germany), 5 µmol forward and reverse primers and 1 ng cDNA. The primers were designed to span the junction that differ between transcript variances. The data of Ct values obtained from two different experimental RNA samples were directly normalized to a housekeeping gene, CsActin. The fold-change in expression of the gene of interest between each timepoint was then calculated as 2^ (-ΔΔCt). Statistical differences in the level expression compared to the expression at time point 0 or that of ATG7 were analyzed with student’s T test with two-tailed distribution.

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