Integrated transcriptomics and WGCNA reveal candidate hub genes associated with terpenoid biosynthesis in Rehmannia...

Terpenoids are the key bioactive constituents of Rehmannia glutinosa, a valuable medicinal plant. The rising economic and pharmaceutical values of R. glutinosa have necessitated elucidating the metabolic pathways governing terpenoid metabolism. Herein, we integrated transcriptome sequencing (RNA-seq) and weighted gene co-expression network analysis (WGCNA) to identify co-expression modules and hub genes closely linked to terpenoid biosynthesis in tuberous roots of cultivar 'Wen85-5' across eight developmental stages. A total of 20996 differentially expressed genes (DEGs) were identified, with GO/KEGG annotation analysis confirming enrichment in various metabolic and cellular processes. Further, we screened 16 terpenoid biosynthesis-related DEGs, mapping to the MVA (6 genes) and MEP (10 genes) pathways. WGCNA clustered 19957 DEGs into 16 modules, of which 9 modules (containing 28 hub genes) were potentially participated in the regulation of terpenoid biosynthesis. Functional annotation of these 9 modules revealed enrichment in secondary metabolic processes, as well as the biosynthetic pathways of terpenoids and polyketides, secondary metabolites, sesquiterpenes, and triterpenes. Among the 28 hub genes, 11 and 17 were mapped to the MVA and MEP pathways, respectively. Co-expression network analysis revealed intricate interactions between hub genes and between hub genes and key transcription factors. Notably, 11 of these hub genes exhibited conserved co-expression patterns across multiple modules and served as candidate genes potentially associated with terpenoid biosynthesis. The expression profiles of these 11 hub genes, inferred from FPKM values, were further validated by RT-qPCR, demonstrating consistent expression trends. This study provides the first systematic characterization of terpenoid biosynthetic network in R. glutinosa, offering critical insights and a valuable genetic resource for metabolic engineering to enhance terpenoid production.