Quantification of FtZIP Transcripts via qRT-PCR

The expression levels of FtZIP transcripts were analyzed using quantitative real-time PCR (qRT-PCR) assay. Total RNA was extracted from the roots (7-d seedlings), stems (10-d seedlings), leaves (10-d seedlings), flowers, fruits and seeds using an RNAprep Pure Plant Kit (Tiangen, Beijing, China). Then, cDNA synthesis was performed in a 20 μl reaction mixture containing 1 μg of total RNA and a mixture of Hifair^® cDNA Synthesis Kit (Yeasen, Shanghai, China). The real-time PCR mixture contained 1 μl cDNA, 1 μl forward and reverse primers, and 10 μl 2 x SYBR Green (TaKaRa, Beijing, China). The qRT-PCR was performed using a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, Hercules, California, CA, USA). All reactions were performed in three triplicates with the following cycling conditions: 95°C for 3 min; 30 cycles each at 95°C for 10 s and 56°C for 30 s, and 72°C for 20 s. The 2^−ΔΔCt method was used for the analysis of qRT-PCR (Livak and Schmittgen, 2001 (link)). The housekeeping gene FtH3 (ID: HM628903) was used as an internal control (Li C et al., 2019 (link)). All primers are shown in Supplementary Table S1.