| Summary: | <i>Panax ginseng</i> as a traditional medicinal plant with a long history of medicinal use. Ginsenoside Ro is the only oleanane-type ginsenoside in ginseng, and has various pharmacological activities, including anti-inflammatory, detoxification, and antithrombotic activities. UDP-dependent glycosyltransferase (UGT) plays a key role in the synthesis of ginsenoside, and the excavation of UGT genes involved in the biosynthesis of ginsenoside Ro has great significance in enriching ginsenoside genetic resources and further revealing the synthesis mechanism of ginsenoside. In this work, ginsenoside-Ro-synthesis-related genes were mined using the <i>P. ginseng</i> reference-free transcriptome database. Fourteen hub transcripts were identified by differential expression analysis and weighted gene co-expression network analysis. Phylogenetic and synteny block analyses of <i>PgUGAT252645</i>, a <i>UGT</i> transcript among the hub transcripts, showed that <i>PgUGAT252645</i> belonged to the <i>UGT73</i> subfamily and was relatively conserved in ginseng plants. Functional analysis showed that <i>PgUGAT252645</i> encodes a glucuronosyltransferase that catalyzes the glucuronide modification of the C3 position of oleanolic acid using uridine diphosphate glucuronide as the substrate. Furthermore, the mutation at 622 bp of its open reading frame resulted in amino acid substitutions that may significantly affect the catalytic activity of the enzyme, and, as a consequence, affect the biosynthesis of ginsenoside Ro. Results of the in vitro enzyme activity assay of the heterologous expression product in <i>E. coli</i> of <i>PgUGAT252645</i> verified the above analyses. The function of <i>PgUGAT252645</i> was further verified by the result that its overexpression in ginseng adventitious roots significantly increased the content of ginsenoside Ro. The present work identified a new <i>UGT</i> gene involved in the biosynthesis of ginsenoside Ro, which not only enriches the functional genes in the ginsenoside synthesis pathway, but also provides the technical basis and theoretical basis for the in-depth excavation of ginsenoside-synthesis-related genes.
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