Screening and validation of optimal real-time PCR reference genes for Abelmoschus Manihot

Abelmoschus Manihot is an important medicinal and edible plant known for its functional secondary metabolites. However, little is known about the key genes involved in production of secondary metabolites in A. manihot. This is largely due to the lack of effective gene expression detection systems for A. manihot, making the screening of real-time PCR reference genes a prerequisite. In this study, 11 candidate reference genes were screened and cloned from A. manihot, and their expression stability was evaluated in different tissues under different flowering stages using four algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The expression stability of eIF and PP2A1 was the highest, while that of tubulin alpha (TUA) was the lowest. The combined use of the two most stable reference genes, eIF and PP2A1, met the experimental requirements for normalizing gene expression in A. manihot. Furthermore, the gene expression of transcription factors bHLH147 and bHLH148 was further validated by data normalization. This study identified potential reference genes in different A. manihot tissues, paving the way for functional gene analysis and dissecting metabolite regulation mechanisms in A. manihot.