qRT-PCR
The mRNA expression levels of 5 genes were analyzed by quantitative
real-time PCR (qRT-PCR) using SYBR Green PCR Master Mix (2X) (Biofact,
South Korea) according to the manufacturer’s protocols. All analysis was
carried out on StepOneTM 48-well Real-Time PCR System
(Applied Biosystems). The qRT-PCR was performed with preliminary hold at
95 °C for 15 min, 45 cycles of 95 °C for 15 s, annealing temperature
depending on the primer Tm for 25 s and 72 °C for 40 s (Table 1). The
experiment was performed with three biological replicates for each
sample and two technical replicates. Primer efficiencies (E) for all
primers and Ct values were determined using LinRegPCR software version
2012.3 (Ruijter et al., 2009) and the Pfaffl method was used to
calculate the relative gene expression (Pfaffl, 2001). The cucumberactin gene (XM_004147305.2) was used as the internal reference
for the normalization of expression level (Wan et al. 2010; Castellano
et al. 2016).