Quantifying ScCIPK Expression Levels

Using the 7500 qRT-PCR system (Applied Biosystems, South San Francisco, CA, USA), the relative expression levels of ScCIPKs under different exogenous stresses were assessed. The qRT-PCR primers of ScCIPKs were designed using Beacon Designer 8.12 software. The Cullin (CUL) [91 (link)] and Clathrin adaptor complex (CAC) [91 (link)] genes were used to normalize relative transcript levels. The qRT-PCR reaction system was prepared using the SYBR Green Master Mix (TaKaRa), following the manufacturer’s instructions. Each qRT-PCR was repeated thrice, and the reaction conditions were listed as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The qRT-PCR data was analyzed using the 2^-ΔΔCtmethod [92 (link)]. Statistical analysis was conducted using Data Processing System v9.50 software (China). Significance (p < 0.05) was calculated using one-way ANOVA, followed by Duncan’s new multiple range test. All of the primers used in qRT-PCR are listed in Supplementary Table S5.