Hybrid sequencing of the Gynostemma pentaphyllum transcriptome provides new insights into gypenoside biosynthesis

Abstract

Background: Gypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis.

Results: Following the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis.

Conclusions: We developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.

Keywords: Biosynthesis; Gynostemma pentaphyllum; Gypenosides; Iso-Seq; Transcriptome.

Fig. 1

The putative gypenoside biosynthetic pathway

Fig. 2

Flowchart of the experimental design and analyses for hybrid sequencing

Fig. 3

Sequence length distribution, annotation and functional classification of unigenes. a Sequence length distribution. b Venn diagram of the KEGG, SwissProt, Pfam and GO results for the G. pentaphyllum transcriptome. c Functional classification by KEGG. The abscissa indicates the number of genes annotated to the pathway, and the ordinate indicates the subcategories. The pathway is divided into four categories in this analysis, including Cellular Processes, Environmental Information Processing, Genetic Information Processing and Metabolism

Fig. 4

Phylogenetic analyses of the GpOSCs from G. pentaphyllum and characterized OSCs from other plants. Amino acid sequences were aligned using the program ClustalW, and evolutionary distances were computed using the Poisson correction method with MEGA6. Lanosterol synthase from Homo sapiens (HsLS) is used as the outgroup. Values less than 50% are not shown. The GenBank accession numbers of the sequences are EsCAS (AFC67276), PnCAS (ABY60426), CaCAS (AAS01524), PgCAS (BAA33460), RcCAS (Q2XPU6), PsCAS (BAA23533), CpCAS (Q6BE25), AtLS (BAE95408), LjLS (BAE95410), PgPNZ1 (BAA33462), HsLS (AAB36220), BpLUS (BAB83087), LjLUS (BAE53430), ToLUS (BAA86932), GgLUS (BAD08587), GuLUS (BAL41371), OeLUS (BAA86930), PqDS (AGI15962), PgDS (AEO27862), PsDS (ANB82450), PgDSII (BAF33291), AeBAS (ADK12003), EsBAS (APZ88354), BcBAS (ADM89633), PgBAS1 (O82140), GsBAS (ACO24697.1), and PqBAS (AGG09939)

Fig. 5

Expression analysis of the selected GpOSCs, GpCYP450s, GpUGTs, GpWRKYs and GpbHLHs in different tissues and MeJA-treated leaves by real-time PCR. a Heatmap of expression levels based on their real-time PCR analysis in three tissues, including roots, stems, and leaves. b Expression analysis of the upstream genes in MeJA-treated leaves by real-time PCR. Here, five time points, 0 h, 6 h, 12 h, 18 h and 24 h, and 0 h were used as a reference. c Expression analysis of the GpOSCs, GpCYP450 and GpUGTs in MeJA-treated leaves by real-time PCR. d Expression analysis of the GpWRKYs and GpbHLH in MeJA-treated leaves by real-time PCR

Fig. 6

Phylogenetic analyses of the GpCYP450s belonged to 85 clans from G. pentaphyllum and characterized CYP450s from P. ginseng. The GenBank accession numbers of the sequences are PgCYP716A47 (AEY75212.1), PgCYP716A53v2 (AFO63031.1), and PgCYP716A52v2 (AFO63032.1)

Fig. 7

Phylogenetic analyses of 68 GpUGTs from G. pentaphyllum and characterized UGTs from P. ginseng. The GenBank accession numbers of the sequences are PgUGT74AE2 (AGR44631.1), PgUGT94Q2 (AGR44632.1), UGTPg1 (AIE12479.1), UGTPg100 (AKQ76388.1), and UGTPg101(AKQ76389.1). Those who did not indicate the family belonged to the novel family

Fig. 8

Distribution of transcription factors