Comprehensive Candidate Gene for Ginsenoside Rg1 Biosynthesis: Identification, Systematic Analysis, and Verification

Ginsenoside Rg1 has a broad spectrum of pharmacological activities, and its concentration is a key indicator of ginseng quality. Because ginsenoside levels are quantitative traits controlled by multiple genes and environmental factors, identifying genes involved in Rg1 biosynthesis is essential to improve its production in ginseng. Transcriptome analysis of 344 4-year-old ginseng roots identified 33 Candidate Genes implicated in Rg1 biosynthesis. These candidates were detected through differential expression analysis, SNP/InDel mutation screening, correlation analysis with Rg1 content variation, and co-expression network analysis. By integrating GO functional annotation, gene expression profiles, and Rg1 content correlations, a putative biosynthetic pathway for Rg1 was inferred and the roles of these genes were preliminarily clarified. Further study of MeJA regulation confirmed that PgRg1-021 is highly associated with ginsenoside Rg1 biosynthesis. Functional verification was performed via overexpression of PgRg1-021 and RNAi in ginseng hairy roots. RT-qPCR analysis showed that PgRg1-021 negatively regulated ginsenoside Rg1 biosynthesis: Rg1 saponin levels decreased after overexpression and increased after RNAi. This study was the first to verify PgRg1-021's role in Rg1 biosynthesis and provided essential gene resources for pathway analysis. It established a foundation for targeted breeding and serves as a reference for identifying other genes associated with complex traits.