RNA Extraction and qPCR Analysis

Ultrapure RNA Kit (cwbiotech, Beijing, China) was used to extract total RNA, then the first cDNA strand was synthesized using Evo M-MLV Mix Kit with gDNA Clean for qPCR (Accurate Biotechnology, Changsha, China). Expression of stress-responsive genes (NtDREB1A, NtERF5, NtKAT2, NtCOR15A, NtRAB18, NtERD11, NtNHX1, and NtSOS1) and ABA signaling genes from tobacco was detected. The qRT-PCR reactions were carried out in Roche LightCycler 480 Real-Time PCR instrument with SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Changsha, China). The tobacco ribosomal protein gene L25 (GenBank No. {"type":"entrez-nucleotide","attrs":{"text":"L18908","term_id":"310934","term_text":"L18908"}}L18908) was used as a control (Li Z. et al., 2021). All experimental data were obtained through three technical repetitions, and the relative expression level was calculated by 2^–ΔΔCT method (Livak and Schmittgen, 2001). The details of the primers in this study are provided in Supplementary Table 1.