Evaluating ScADH3 Expression under Stress

The relative expression levels of ScADH3 under different exogenous stresses were detected using qRT-PCR. Beacon Designer 8.12 software was employed to design the qRT-PCR primers of ScADH3. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was selected as the reference gene [78 , 79 (link)]. The qRT-PCR reaction system (SYBR Green Master Mix: 10 μL, 10 μM forward and reverse primers: 0.8 μL, 20 × diluted cDNA template: 1.0 μL, and sterile distilled water: 7.4 μL) was constructed with reference to the manual of SYBR Green Master Mix (TaKaRa). The reaction procedure was as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The 2^-ΔΔCt method was used to normalize the relative expression level of qRT-PCR data [80 (link)]. Each of the samples had three biological replicates. Three technical replicates were performed. All primers used in qRT-PCR are listed in Supplementary Table S8. Data Processing System v9.50 software (China) was used to conduct the statistical analysis. Data were expressed as the mean ± standard error (SE), Significance (p < 0.05) was calculated using one-way ANOVA, followed by Duncan’s new multiple range test.