Cucumber Heat Stress Gene Expression

Two contrasting genotypes, WBC-13 and DGPC-59 (Supplementary Figure 1) were used for gene expression analysis under two different stress conditions (35°C/30°C and 40°C/35°C) along with control without any stress (30°C/25°C). Total RNA from cucumber leaves under different stress conditions was extracted using Trizol reagent. RNA was quantified by spectrophotometric analysis and the quality was evaluated through agarose gel electrophoresis. First-strand complementary DNA (cDNA) synthesis was carried out using the user instruction (Promega, USA). Relative expression of 18 important genes associated with heat tolerance were conducted using two contrasting genotypes under two different stress conditions (Table S1). Quantitative real-time PCR was carried out using Light Cycler (Roche) with Light Cycler Fast Start DNA Master SYBR Green kit (Roche). Amplification of stress-related genes was carried out according to the manufacturer’s protocol. Reaction mixture of 20 μl contains 1.5 μl cDNA, 0.3 μl of primer (forward and reverse), 12.5 μl SYBR Premix, and 5.4 μl dH₂O. Expression analysis of all genes were tested in triplicate with appropriate primers along with Actin used as an internal control. The gene expression data were calculated comparative to Actin, and Ct values of the used target genes were normalized using the Ct values of Actin. The levels of mRNA were also normalized with Actin and its value was expressed relative to that of the control, which was given an arbitrary value 1 (Liu et al., 2012 (link)). The relative differential gene expression was measured according to the equation 2−ΔΔCt (Livak and Schmittgen, 2001 (link)). The final data of RT-PCR were calculated from three experimental replicates.