[Reference genes screening and expression analysis of genes involved in terpenoid biosynthesis in Curcuma...

Investigating optimal reference genes and expression patterns of key enzyme genes in terpenoid biosynthesis in Curcuma kwangsiensis across different developmental stages and tissues, this study selected 13 candidate reference genes(including ACTIN1, ACTIN2, ACTIN3, EF1, EF2, EF3, EF4, GAPDH1, GAPDH2, β-TUB, UBC, CYP1, and CYP2) and evaluated their stability by using ΔCT, GeNorm, NormFinder, BestKeeper, and RefFinder, with tuberous roots, rhizomes, leaves, and petioles at different developmental stages taken as experimental materials. The reliability of the reference genes was validated by detecting the expression of CkHMGR1. EF2 gene was identified as the most suitable reference gene in tissues of C. kwangsiensis across different developmental stages. Transcriptomic and metabolomic data were used to screen key enzyme genes involved in terpenoid biosynthesis, and their expression patterns in different tissues of C. kwangsiensis across different developmental stages were analyzed with EF2 gene as the reference gene. The results revealed that the expression patterns of 17 key enzyme genes(including CkHMGR1, CkHMGR2, CkDXS1, CkDXS2, CkDXR1, CkCMK1, CkHDS1, CkHDS2, CkHDS3, CkHDS4, CkHDR1, CkHDR2, CkHDR3, CkTPS1, CkTPS2, CkTPS3, and CkTPS4) exhibited significant differences across various tissues and developmental stages. Most genes showed high expression during the middle to late developmental stages of tuberous root and rhizome. Notably, CkTPS1 and CkTPS4 exhibited high expression during the late developmental stages of tuberous root and rhizome. This study lays a foundation for functional validation of key enzyme genes and elucidation of the terpenoid biosynthesis pathway in C. kwangsiensis.