RNA Extraction and qRT-PCR Analysis

The total RNA from different tissues and whole seedlings under different treatments were extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China). The RNA concentration and purity were determined using the NaNo drop 1,000 spectrophotometer and agarose gel electrophoresis (Xie et al., 2018 (link)). The FastQuant first strand cDNA synthesis kit (TIANGEN, Beijing, China) was used for the synthesis of cDNA following the manufacturer’s protocol. The SuperReal PreMix Plus kit (TIANGEN, Beijing, China) and a Roche LightCycler instrument were used for qRT-PCR. The reaction system of qRT-PCR was as follows: 10 μL 2×SuperReal PreMix Plus, 0.6 μL 10 μM forward primers, 0.6 μL 10 μM reverse primers, 2 μL cDNA and 6.8 μL RNase-free ddH₂O. The qRT-PCR procedure was as follow: 95 °C for 15 min and 40 cycles of 95 °C for 10 s and 60 °C for 20 s. CsActin was used as an internal reference gene (Zhou et al., 2017 (link)). The primers of the cucumber TPS genes and CsActin for qRT-PCR were designed and synthesized using Sangon Biotech online software (Table 1). Three technical replicates were performed for each reaction.