Pepper ABC Transporter Gene Expression

Total RNA was isolated from pepper fruits (6, 16 and 25 dpa) by using the Plant RNA mini spin kit (Macherey-Nagel). First-strand cDNA was synthesized with 1 μg total RNA per sample by using the Super Script First-Strand Synthesis system (Invitrogen). To identify in the three Capsicum genomes the orthologs of the markers previously reported by [5 (link)] for the ABC transporter family, the CDS sequences for the CA06g14430 and CA11g09150 genes were downloaded from the Sol Genomics database (https://solgenomics.net/) [49 (link)] and a BLASTN search was performed (identity ≥ 98% and coverage ≥ 70%) across the three pepper genomes. Gene-specific primers for the selected Capsicum ABC transporter orthologs were designed by using Primer3Plus (http://www.primer3plus.com/). The qRT-PCR analysis involved a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a total volume of 20 μL containing 1 μL cDNA template, 2 μL forward and reverse primers (10 μM), 10 μL SYBR Green PCR Master (ROX) (Roche, Shanghai) and 7 μL sterile distilled water. For each sample, three replicates were run to compute the average Ct values. The data were analyzed by the 2−ΔΔCt method [50 (link)]. Relative gene expression was normalized against that of the endogenous control β-tubulin [51 (link)].