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. 2016 May 19:7:673.
doi: 10.3389/fpls.2016.00673. eCollection 2016.

Candidate Genes Involved in the Biosynthesis of Triterpenoid Saponins in Platycodon grandiflorum Identified by Transcriptome Analysis

Chun-Hua Ma  1 , Zheng-Jie Gao  1 , Jia-Jin Zhang  1 , Wei Zhang  2 , Jian-Hui Shao  3 , Mei-Rong Hai  1 , Jun-Wen Chen  1 , Sheng-Chao Yang  1 , Guang-Hui Zhang  1
Affiliations

Candidate Genes Involved in the Biosynthesis of Triterpenoid Saponins in Platycodon grandiflorum Identified by Transcriptome Analysis

Chun-Hua Ma et al. Front Plant Sci. .

Abstract

Background: Platycodon grandiflorum is the only species in the genus Platycodon of the family Campanulaceae, which has been traditionally used as a medicinal plant for its lung-heat-clearing, antitussive, and expectorant properties in China, Japanese, and Korean. Oleanane-type triterpenoid saponins were the main chemical components of P. grandiflorum and platycodin D was the abundant and main bioactive component, but little is known about their biosynthesis in plants. Hence, P. grandiflorum is an ideal medicinal plant for studying the biosynthesis of Oleanane-type saponins. In addition, the genomic information of this important herbal plant is unavailable.

Principal findings: A total of 58,580,566 clean reads were obtained, which were assembled into 34,053 unigenes, with an average length of 936 bp and N50 of 1,661 bp by analyzing the transcriptome data of P. grandiflorum. Among these 34,053 unigenes, 22,409 unigenes (65.80%) were annotated based on the information available from public databases, including Nr, NCBI, Swiss-Prot, KOG, and KEGG. Furthermore, 21 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely involved in triterpenoid saponins biosynthesis pathway were discovered from the transcriptome sequencing of P. grandiflorum. In addition, 10,626 SSRs were identified based on the transcriptome data, which would provide abundant candidates of molecular markers for genetic diversity and genetic map for this medicinal plant.

Conclusion: The genomic data obtained from P. grandiflorum, especially the identification of putative genes involved in triterpenoid saponins biosynthesis pathway, will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level.

Keywords: Platycodon grandiflorum; biosynthesis; platycodin D; transcriptome; triterpenoid saponins.

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Figures

FIGURE 1
FIGURE 1
Putative pathway for triterpenoid saponin biosynthesis in Platycodon grandiflorum. Enzymes found in this study are boxed. AACT, acetyl-CoA acetyltransferase; HMGS, HMG-CoA synthase; IPPI, IPP isomerase; HMGR, HMG-CoA reductase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; GPPS, geranylgeranyl pyrophosphate synthase; FPPS, farnesyl diphosphate synthase; SS, squalene synthase; SE, squalene epoxidase; β-AS, β-amyrin synthase; β-A28O, β-amyrin 28-oxidase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GPP, geranyl pyrophosphate; IPP, isopentenyl diphosphate; GT, glycosyltransferase; CYPs, cytochrome P450.
FIGURE 2
FIGURE 2
Overview of the P. grandiflorum transcriptome assembly.
FIGURE 3
FIGURE 3
Gene ontology classification of assembled unigenes.
FIGURE 4
FIGURE 4
Eukaryotic Orthologous Groups (KOGs) function classification of P. grandiflorum.
FIGURE 5
FIGURE 5
Pathway assignment based on KEGG. (A) Classification based on metabolism categories. (B) Classification based on biosynthesis of other secondary metabolites.
FIGURE 6
FIGURE 6
Phylogenetic tree of the P. grandiflorum CYP450s. Phylogenetic tree is constructed based on the deduced amino acid sequences for the P. grandiflorum CYP450s (bold letters) and other plant CYP450s involved in triterpenoid biosynthesis. Protein sequences are retrieved from NCBI GenBank using the following accession numbers: Vitis vinifera VvCYP716A15 (BAJ84106.1) and VvCYP716A17 (BAJ84107.1); Medicago truncatula MtCYP716A12 (ABC59076.1), MtCYP93E2 (ABC59085), MtCYP72A63 (H1A981.1), MtCYP72A65v2 (BAL45202), MtCYP72A67v2 (BAL45203) and MtCYP72A67v2 (BAL45203), and MtCYP72A61v2 (BAL45199); Panax ginseng PgCYP716A52v2 (AFO63032.1), PgCYP716A53v2 (I7CT85.1) and PgCYP716A47 (H2DH16.2); Arabidopsis thaliana AtCYP708A2 (NP_001078732.1) and AtCYP705A5 (EFH40098); Glycyrrhiza uralensis GuCYP88D6 (B5BSX1.1), GuCYP93E3 (BAG68930) and GuCYP72A154 (H1A988.1); Avena strigosa AsCYP51H10 (ABG88965.1); Glycine max GmCYP93E1 (NP_001236154.1); BfCYP716Y1.
FIGURE 7
FIGURE 7
Phylogenetic tree constructed based on the deduced amino acid sequences for the P. grandiflorum UGTs (bold letters) and other plant UGTs. Accession numbers in the NCBI GenBank database are as follows: Barbarea vulgaris BvUGT73C11 (AFN26667) and BvUGT73C10 (AFN26666); Arabidopsis thaliana AtUGT73C1 (NP_181213.1), AtUGT82A1 (NP_188864.1), AtUGT76B1 (NP_187742.1), AtUGT71B1 (NP_188812.1), AtUGT89B1 (NP_177529.2), AtUGT75B2 (NP_172044.1), AtUGT75C1 (NP_193146.1), AtUGT74C1 (NP_180738.1), AtUGT79B4 (Q9LJA6.1) and AtUGT79B1 (Q9LVW3.1); Solanum aculeatissimum SaGT4A (BAD89042); M. truncatula MtUGT73K1 (AAW56091), MtUGT73F3 (ACT34898) and MtUGT71G1 (AAW56092); G max GmUGT73F4 (BAM29363); Panax notoginseng PnUGT1 (JX018210); Oryza sativa OsUGT709A4 (Q7XHR3); Saponaria vaccaria SvUGT74M1 (ABK76266); Linum usitatissimum LuUGT71A24 (AFJ52909), LuUGT82A2 (AFJ52979), LuUGT709D1 (AFJ53007), LuUGT75N1 (AFJ52962), LuUGT94G1 (AFJ53037.1), LuUGT79A3 (AFJ52973.1).
FIGURE 8
FIGURE 8
Validation of candidate unigenes involved in triterpene saponin biosynthesis in P. grandiflorum by qPCR. Bars represent the mean (± SD) of four experiments. Statistical analysis is performed with one way ANOVA with Tukey’s test to compare the difference in the mean expression level of a given gene among different tissues. P ≤ 0.05 was considered statistically significant.
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