[Expression and stability analysis of 9 candidate reference genes in three tissues of Bletilla striata during...

Bletilla striata, a perennial herbaceous plant of the genus Bletilla in the Orchidaceae family, possesses significant pharmacological activities, including promotion of wound healing, astringent hemostasis, anti-inflammatory effects, antioxidation, and immunomodulation. These pharmacological activities are primarily attributed to its core active constituent, militarine. However, the metabolic patterns and quality formation mechanisms of militarine remain poorly understood. Quantitative reverse transcription PCR(qRT-PCR) is a pivotal tool in molecular biology and functional gene research, and appropriate and stable reference genes serve as the basis for investigating the quality formation mechanisms in B. striata. In this study, nine candidate reference genes, namely ATP-binding cassette protein 1(ABC1), actin(ACT), α-tubulin 3(α-TUB3), eukaryotic translation elongation factor 1α(EF1α), eukaryotic translation elongation factor 2(EF2), eukaryotic translation initiation factor 4A(EIF4A), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), protein phosphatase 2A(PP2A), and ribosomal protein L7(RPL7), were selected. qRT-PCR was performed to assess the expression levels of these genes in various tissues at seven distinct developmental stages of B. striata. Gene expression stability was analyzed using the ΔCt method, BestKeeper, NormFinder, and geNorm, and comprehensive evaluation was conducted using RefFinder. The results showed that GAPDH exhibited the most stable expression across the three tissues at the seven growth stages, followed by EIF4A, while RPL7 demonstrated the lowest stability. The stability of the selected reference genes was further validated by assessing the expression of key enzyme genes involved in the biosynthesis of militarine. The results showed that when GAPDH and EIF4A were used as the reference genes, the expression trends of 2-isobutylmalate synthase(BsIBMS) and UDP-glycosyltransferase(BsUGT) genes were consistent with the relative expression levels obtained from transcriptomic data, indicating that GAPDH and EIF4A are reliable and accurate reference genes. These findings offer a reference for future studies on the quality formation mechanisms of major bioactive constituents in B. striata.