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

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.

Fig 1. Overview of proteome analysis of G. pentaphyllum.

(A) G. pentaphyllum grown in the medicinal plant garden, (B) Roots, (C) Stems, (D) Leaves, (E) Summary of differentially expressed proteins; UP: Upregulated, DOWN: Downregulated. (F) GO enrichment analysis of differentially expressed proteins. The enriched GO annotation “biological process,” “cellular components,” and “molecular function” categories are shown. Further classification under each category is also listed. (A-D) Picture by our group. We own the copyright of these pictures, and we give permission to Plos Journal to publish these photos.

Fig 2. Quantitative proteome sequencing combined with transcriptome sequencing was used to identify genes or proteins in the G. pentaphyllum triterpene saponin pathway [14].

(A) Triterpene saponin synthesis pathway: Dark red font indicates that the candidate genes or proteins involved in triterpene saponin biosynthesis are identified by transcriptome sequencing and proteomics sequencing at the same time. Blue font indicates the candidate genes involved in triterpene saponin biosynthesis that can only be identified by transcriptome sequencing. LUS: Lupeol Synthase. LS: Lanosterol Synthase. DS: Dammarenediol Synthase. The other gene abbreviations are listed in S3 Table. (B-D) Correlation analysis of the overall proteome and transcriptome of G. pentaphyllum.: (B) Correlation analysis of the overall proteome and transcriptome of Stems vs Roots (Pearson:0.435, p<0.01); (C) Correlation analysis of the overall proteome and transcriptome of Leaves vs Roots (Pearson: 0.014, p = 0.380); (D) Correlation analysis of the overall proteome and transcriptome of Leaves vs Stems (Pearson: 0.015, p = 0.346); (E) Heat Map of Differential Expression of CYPs and UGTs Genes in G. pentaphyllum: Red represents highly expressed genes. Green represents low expressed genes. Black represents genes with intermediate expression.

Fig 3. Cloning and sequence analysis of G. pentaphyllum CYP and UGT genes.

(A) CYP phylogenetic tree analysis of G. pentaphyllum, Cucurbitaceae and other plants, the functional activities of which were identified [17]. *denotes the CYP protein of G. pentaphyllum. Gp: Gynostemma pentaphyllum. At: Arabidopsis thaliana. Pv: Phaseolus vulgaris. Ps: Pisum sativum. Lc: Lens culinaris. Gg: Glycyrrhiza glabra. Ca: Cicer arietinum. Ah: Arachis hypogaea. Gu: Glycyrrhiza uralensis. Mt: Medicago truncatula. Gm: Glycine max. Ml: Maesa lanceolate. Ks: Kalopanax septemlobus. Lj: Lotus japonicas. As: Avena strigose. Al: Arabidopsis lyrata subsp. Lyrata. Aa: Artemisia annua. Vv: Vitis vinifera. Sl: Solanum lycopersicum. Pg: Panax ginseng. Cq: Chenopodium quinoa. Bv: Barbarea vulgaris subsp. Arcuate. Ca: Centella asiatica. Ac: Aquilegia coerulea. Plg: Platycodon grandifloras. Cr: Catharanthus roseus. Bp: Betula platyphylla. Es: Eleutherococcus senticosus. Ob: Ocimum basilicum. Bf: Bupleurum falcatum. Cs: Cucumis sativus. Cp: Cucurbita pepo subsp. Pepo. Cm: Cucurbita moschata. Cum: Cucumis melo. Cma: Cucurbita maxima. Cme: Cucumis melo var. makuwa. (B) UGT phylogenetic tree analysis of G. pentaphyllum, Arabidopsis thaliana, Cucurbitaceae and other plants, the functions of which were identified [26]. *denotes the UGT protein of G. pentaphyllum. (C) Results of the MEME repeat of the corresponding CYP protein AGxDTT motif of G. pentaphyllum and Cucurbitaceae. (D) Results of the MEME repeat of the corresponding CYP protein ExxR motif of G. pentaphyllum and Cucurbitaceae. (E) Results of the MEME repeat of the corresponding CYP protein PER motif of G. pentaphyllum and Cucurbitaceae. (F) Results of the MEME repeat of the corresponding CYP protein FxxGxRxCxG motif of G. pentaphyllum and Cucurbitaceae. (G) Prediction of CYP protein tertiary structure of G. pentaphyllum: Red: CYP71B19; Green: CYP77A3; Yellow: CYP86A7; Magenta: CYP86A8; Cyan CYP89A2; Orange: CYP90A1; Light pink: CYP94A1. (H) Results of the MEME repeat of the corresponding UGT protein PSPG motif of G. pentaphyllum and Cucurbitaceae. (I) Prediction of the UGT protein tertiary structure of G. pentaphyllum: Red: UGT73B4; Green: UGT74F2; Blue: UGT76B1; Yellow: UGT91A1; Pink: UGT91C1.

Fig 4. Comparison of qPCR copy values of CYPs and UGTs of G. pentaphyllum roots, stems and leaves.

* means p<0.5; ** means P<0.01.