Screening and Stability Validation of RT-qPCR Reference Genes in Portulaca oleracea L. in Diverse Tissues and Under Abiotic Stress Conditions

Purslane (Portulaca oleracea L.) is an important plant species that has been increasingly used in functional gene studies and molecular analyses. However, reference genes that exhibit stable expression across multiple tissues and stress conditions have not been systematically validated in purslane, which limits the accuracy of reverse transcription quantitative PCR (RT-qPCR) based gene expression analyses. In this study, ten candidate reference genes from six gene families (Actin, PP2A, CYP, eIF4A, Ubiquitin, and eIF5A) were selected based on transcriptome data. A combination of bioinformatic analyses and experimental validation was employed to comprehensively characterize these candidates, including their physicochemical properties, chromosomal localization, phylogenetic relationships, gene structures, and promoter cis-acting elements. Furthermore, the expression stability of the candidate genes was systematically evaluated across different tissues (seed, root, stem, leaf, and flower) and under multiple stress treatments, including salinity, temperature stress, drought, and hormone treatments. Based on conventional PCR amplification specificity, melting curve analysis, Ct value distribution, and amplification efficiency, ACT-2 and eIF5A-1 were identified as the most stably expressed reference genes under diverse experimental conditions. This study provides reliable reference gene candidates for accurate normalization of gene expression in purslane and establishes a systematic framework for reference gene selection in non-model plant species.