Genome-Wide Identification of the CYP716 Gene Family in Platycodon grandiflorus (Jacq.) A. DC. and Its Role in the Regulation of Triterpenoid Saponin Biosynthesis.

Abstract

The main type of saponins occurring in the root of Platycodon grandiflorus (Jacq.) A. DC. are oleanolic acid glycosides. The CYP716 gene family plays a major role in catalyzing the conversion of β-amyrin into oleanolic acid. However, studies on the CYP716 genes in P. grandiflorus are limited, and its evolutionary history remains poorly understood. In this study, 22 PgCYP716 genes were identified, distributed among seven subfamilies. Cis-acting elements of the PgCYP716 promoters were mainly involved in plant hormone regulation and responses to abiotic stresses. PgCYP716A264, PgCYP716A391, PgCYP716A291, and PgCYP716BWv3 genes were upregulated in the root and during saponin accumulation, as shown by RNA-seq analysis, suggesting that these four genes play an important role in saponin synthesis. The results of subcellular localization indicated that these four genes encoded membrane proteins. Furthermore, the catalytic activity of these four genes was proved in the yeast, which catalyzed the conversion of β-amyrin into oleanolic acid. We found that the content of β-amyrin, platycodin D, platycoside E, platycodin D3, and total saponins increased significantly when either of the four genes was over expressed in the transgenic hair root. In addition, the expression of PgSS, PgGPPS2, PgHMGS, and PgSE was also upregulated while these four genes were overexpressed. These data support that these four PgCYP716 enzymes oxidize β-amyrin to produce oleanolic acid, ultimately promoting saponin accumulation by activating the expression of upstream pathway genes. Our results enhanced the understanding of the functional variation among the PgCYP716 gene family involved in triterpenoid biosynthesis and provided a theoretical foundation for improving saponin content and enriching the saponin biosynthetic pathway in P. grandiflorus.