Selection and Validation of Reference Genes in Clinacanthus nutans Under Abiotic Stresses, MeJA Treatment, and in Different Tissues

Clinacanthus nutans is a valuable traditional medicinal plant that contains enriched active compounds such as triterpenoids and flavonoids. Understanding the accuulation process of these secondary metabolites in C. nutans requires exploring gene expression regulation under abiotic stresses and hormonal stimuli. qRT-PCR is a powerful method for gene expression analysis, with the selection of suitable reference genes being paramount. However, reports on stably expressed reference genes in C. nutans and even across the entire family Acanthaceae are limited. In this study, we evaluated the expression stability of 12 candidate reference genes (CnUBQ, CnRPL, CnRPS, CnPTB1, CnTIP41, CnACT, CnUBC, CnGAPDH, Cn18S, CnCYP, CnEF1 alpha, and CnTUB) in C. nutans across different tissues and under abiotic stresses and MeJA treatment using three programs (geNorm, NormFinder, and BestKeeper). The integrated ranking results indicated that CnUBC, CnRPL, and CnCYP were the most stably expressed genes across different tissues. Under abiotic stress conditions, CnUBC, CnRPL, and CnEF1 alpha were the most stable, while under MeJA treatment, CnRPL, CnEF1 alpha, and CnGAPDH exhibited the highest stability. Additionally, CnRPL, CnUBC, and CnEF1 alpha were the most stable reference genes across all tested samples, whereas CnGAPDH was the least stable. CnRPL, consistently ranking among the top three most stable genes, may therefore serve as an ideal reference gene for qRT-PCR analysis in C. nutans. To further validate the selected reference genes, we assessed the expression of two key biosynthetic genes, CnPAL and CnHMGR. The results confirmed that using the most stable reference genes yielded expression patterns consistent with biological expectations, while using unstable reference genes led to significant deviations. These findings offer valuable insights for accurately quantifying target genes via qRT-PCR in C. nutans, facilitating investigations into the mechanisms underlying active compound accumulation.