Gene excavation and expression analysis of CYP and UGT related to the post modifying stage of gypenoside biosynthesis in Gynostemma pentaphyllum (Thunb.) Makino by comprehensive analysis of RNA and proteome sequencing.

Yangmei Zhang,Xidong Yuan,Yuanheng Huang,Huiyu Liu,Yaosheng Wu,Jian Sun,Huiming Xu,Ruiqiang Zhao,Qicong Chen,Shilin Chen

doi:10.1371/journal.pone.0260027

Yangmei Zhang, Xidong Yuan + Show 8 more

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https://doi.org/10.1371/journal.pone.0260027

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Abstract

Previous studies have revealed that gypenosides produced from Gynostemma pentaphyllum (Thunb.) Makino are mainly dammarane-type triterpenoid saponins with diverse structures and important biological activities, but the mechanism of diversity for gypenoside biosynthesis is still unclear. In this study, a combination of isobaric tags for relative and absolute quantification (iTRAQ) proteome analysis and RNA sequencing transcriptome analysis was performed to identify the proteins and genes related to gypenoside biosynthesis. A total of 3925 proteins were identified by proteomic sequencing, of which 2537 were quantified. Seventeen cytochrome P450 (CYP) and 11 uridine 5’-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) candidate genes involved in the side chain synthesis and modification of gypenosides were found. Seven putative CYPs (CYP71B19, CYP77A3, CYP86A7, CYP86A8, CYP89A2, CYP90A1, CYP94A1) and five putative UGTs (UGT73B4, UGT76B1, UGT74F2, UGT91C1 and UGT91A1) were selected as candidate structural modifiers of triterpenoid saponins, which were cloned for gene expression analysis. Comprehensive analysis of RNA sequencing and proteome sequencing showed that some CYPs and UGTs were found at both the transcription and translation levels. In this study, an expression analysis of 7 CYPs and 5 UGTs that contributed to gypenoside biosynthesis and distribution in G. pentaphyllum was performed, providing consistent results that will inspire more future research on vital genes/proteins involved in gypenoside biosynthesis.

Highlights

Gynostemma pentaphyllum (Thunb.) Makino, a slender creeping or climbing plant of the genus Gynostemma of the Cucurbitaceae family, possesses multiple medicinal capabilities and is associated with liver protection, antitumor activity, anti-inflammatory activity, and blood sugar and lipid balance [1–7]
Understanding the expression patterns of key proteins involved in the biosynthesis of gypenosides in G. pentaphyllum and their distribution characteristics in different tissues will reveal the mechanism accounting for the diversity of gypenosides
G. pentaphyllum is well known for its abundant and diversiform triterpene saponins compared with other medicinal herbs

Summary

Introduction

Gynostemma pentaphyllum (Thunb.) Makino, a slender creeping or climbing plant of the genus Gynostemma of the Cucurbitaceae family, possesses multiple medicinal capabilities and is associated with liver protection, antitumor activity, anti-inflammatory activity, and blood sugar and lipid balance [1–7]. As the main active ingredient of G. pentaphyllum, gypenosides display a typical dammarane-style structure of tetracyclic triterpenes. Triterpene saponins are secondary metabolites of isoprenoid compounds. Compared with Panax ginseng, G. pentaphyllum generates almost five times higher production of triterpene saponins and grows faster. The expression pattern of key proteins involved in the biosynthesis of gypenosides is still unclear. We used differential proteomics to determine the protein sets involved in the synthesis of triterpene saponins and analyzed the expression pattern of important proteins in different tissues of G. pentaphyllum (roots, stems, and leaves). Understanding the expression patterns of key proteins involved in the biosynthesis of gypenosides in G. pentaphyllum and their distribution characteristics in different tissues will reveal the mechanism accounting for the diversity of gypenosides

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