Real-Time qPCR Analysis of Plant Genes

Total RNA was extracted from the frozen tissue using Tiangen’s RNA prep pure plant kit (Cat.DP432, Beijing, China) according to the manufacturer’s instructions. Next, 2 μg RNA was used for the first strand of cDNA synthesis using reverse transcriptase (Thermo Scientific, #EP0441). Real-time PCR amplification was carried out with the Bio-Rad CFX96 system using SYBR Green I (Takara, DRR081A, Dalian, China). PCR conditions were 3 min at 95°C followed by 40 cycles of the following: 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. The primer pairs used were FtCuAO 5'-ACCTCAGGTGAAGCAGTCAA-3' and 5'-GGGATTTCGCACCCTCATTC-3'; FtRPB1 5'-CTCACGACAACCACCATTCC-3' and 5'-CCTCCTTGTGTGGAGTGTCT-3'; and FtDHE1 5'-CAGAGGAGCTTGCTTGGTTG-3' and 5'-CGCAAATGGCAGACACTGAT-3'. The FtH3 gene was amplified as a reference gene, since its expression is unaffected by abiotic treatment (Li et al., 2019 (link)), using primers 5'-GAAATTCGCAAGTACCAGAAGAG-3' and 5'-CCAACAAGGTATGCCTCAGC-3'.