Volume 12 Supplement 5
Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers
- Hongmei Luo†1,
- Chao Sun†1,
- Yongzhen Sun1,
- Qiong Wu2,
- Ying Li1,
- Jingyuan Song1,
- Yunyun Niu1,
- Xianglin Cheng3,
- Hongxi Xu4,
- Chuyuan Li5,
- Juyan Liu5,
- André Steinmetz6 and
- Shilin Chen1Email author
© Luo et al. licensee BioMed Central Ltd 2011
Published: 23 December 2011
Panax notoginseng (Burk) F.H. Chen is important medicinal plant of the Araliacease family. Triterpene saponins are the bioactive constituents in P. notoginseng. However, available genomic information regarding this plant is limited. Moreover, details of triterpene saponin biosynthesis in the Panax species are largely unknown.
Using the 454 pyrosequencing technology, a one-quarter GS FLX titanium run resulted in 188,185 reads with an average length of 410 bases for P. notoginseng root. These reads were processed and assembled by 454 GS De Novo Assembler software into 30,852 unique sequences. A total of 70.2% of unique sequences were annotated by Basic Local Alignment Search Tool (BLAST) similarity searches against public sequence databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) assignment discovered 41 unique sequences representing 11 genes involved in triterpene saponin backbone biosynthesis in the 454-EST dataset. In particular, the transcript encoding dammarenediol synthase (DS), which is the first committed enzyme in the biosynthetic pathway of major triterpene saponins, is highly expressed in the root of four-year-old P. notoginseng. It is worth emphasizing that the candidate cytochrome P450 (Pn02132 and Pn00158) and UDP-glycosyltransferase (Pn00082) gene most likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis were discovered from 174 cytochrome P450s and 242 glycosyltransferases by phylogenetic analysis, respectively. Putative transcription factors were detected in 906 unique sequences, including Myb, homeobox, WRKY, basic helix-loop-helix (bHLH), and other family proteins. Additionally, a total of 2,772 simple sequence repeat (SSR) were identified from 2,361 unique sequences, of which, di-nucleotide motifs were the most abundant motif.
This study is the first to present a large-scale EST dataset for P. notoginseng root acquired by next-generation sequencing (NGS) technology. The candidate genes involved in triterpene saponin biosynthesis, including the putative CYP450s and UGTs, were obtained in this study. Additionally, the identification of SSRs provided plenty of genetic makers for molecular breeding and genetics applications in this species. These data will provide information on gene discovery, transcriptional regulation and marker-assisted selection for P. notoginseng. The dataset establishes an important foundation for the study with the purpose of ensuring adequate drug resources for this species.
Panax notoginseng (Burk) F.H. Chen is highly valued medicinal plant of the Araliaceae family . P. notoginseng is distributed in the southwestern region of China, Burma and Nepal . Presently, this species can only be found in cultivated forms . In China, P. notoginseng is cultiviated commercially in Wenshan County, Yunnan province . The roots of this plant, called notoginseng or sanchi, are commonly used as a hemostatic agent as well as a tonic to promote quality of life. In addition, the herb of sanqi possesses the bioactivities of antihypertensive, antithrombotic, anti-atherosclerotic and neuroprotective actions . The ingredients detected in P. notoginseng include triterpene saponins, non-protein amino acids, polyacetylenes, phytosterols, flavonoids, and polysaccharides, many of which have pharmacological activities and are useful in the treatment of some diseases . Among these compounds, triterpene saponins, a group of ginsenosides, are considered to be the principal bioactive components responsible for the pharmacological features [5–7]. Approximately 60 triterpene saponins have been isolated from P. notoginseng including ginsenosides, notoginsenosides, and gypenosides . The major ginsenosides are the dammarane glycosides, and the ginsenoside Rg1, Rb1, Rd, and notoginsenoside R1 are considered as the major constituents found in the P. notoginseng root . All the dammarane saponins have been classified as two groups: the protopanaxadiols group and the protopanaxatriols group . The oleanane-type saponin, Ro, which exists in Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), have not been found in P. notoginseng based on the evidence from phytochemical studies .
Despite its pharmacological importance, the transcriptomic and genomic data of P. notoginseng are very limited and only 95 ESTs are available in the National Center for Biotechnology Information (NCBI) database. The limited transcriptomic data hinder the study of triterpene saponin biosynthetic mechanisms in P. notoginseng. Expressed sequence tag (EST) analysis is a useful tool for the purposes of gene discovery especially in non-model plants for which no reference genome sequences are available . ESTs represent the expressed portion of a genome [17, 18] and can be used to characterize patterns of gene expression in special tissues . The discovery and prediction of genes involved in triterpene saponin and other secondary metabolite biosynthesis was performed based on EST analysis [15, 20, 21]. The triterpene carboxylic acid glucosyltransferase was characterized by mining ESTs from the developing seeds of Saponaria vaccaria . The licorice-amyrin 11-oxidase gene, which plays a key role in the biosynthesis of the triterpene sweetener glycyrrhizin, was identified from the ESTs generated from the stolons of Glycyrrhiza uralensis . In addition, ESTs are a rich source of gene-derived molecular markers (e.g. simple sequence repeat, SSR) which will be used for germplasm breeding or physical mapping . The next-generation sequencing (NGS) technologies improve sequencing depth and render large-scale EST projects more feasible [25–27].
Herein we present the results of the study designed to characterize the transcriptome of P. notoginseng root using NGS technology based on 454 GS FLX Titanium platform. Our ultimate goal is to discover the candidate genes that encode enzymes in the triterpene saponin biosynthetic pathway and provide an overview of transcriptome, as well as produce molecular markers of EST-SSRs for facilitation the marker-assisted breeding of this species.
Results and discussion
Transcriptome sequencing and sequence assembly
Summary of 454 sequencing and assembly for P. notoginseng
No. of sequences
No. of bases
Average HQ read length
410 ± 138 bp
Reads used in assembly
Reads assembled as contigs
Number of contigs
Average length of contigs
581 ± 404 bp
Range of contig lengths
95 - 10,423 bp
Contigs above 200 bp
Number of singletons
Average length of singletons
343 ± 166 bp
Range of singleton lengths
50 - 728 bp
Singletons above 200 bp
Number of unique sequencesa
Unique sequences above 200 bases
Unique sequence annotation and highly expressed transcript analyses
The annotation for P. notoginseng unique sequences was based on sequence similarity searches against public databases. These databases included SwissProt, KEGG, The Arabidopsis Information Resource (TAIR), NCBI non-redundant protein (Nr), and NCBI non-redundant nucleotide (Nt) database. The number and percentage of the annotated unique sequences were summarized in Additional file 1. In total, 21,672 (70.2%) P. notoginseng unique sequences were annotated and the remaining (29.8%) unique sequences had no match to any sequences in the public databases (Table 1). The annotation rate for P. notoginseng unique sequences is similar to that of P. quinquefolius root  and P. ginseng root  transcriptomes. The comparisons of the 30,852 P. notoginseng unique sequences with all the P. quinquefolius  and P. ginseng  unique sequences derived from 454 sequencing using BLAST search (E-value <= 1e-10) were performed. The result of the comparison between P. notoginseng and P. quinquefolius  unique sequences showed that 19,226 P. notoginseng unique sequences had sequence similarity to P. quinquefolius unique sequences, and the remained 11,626 unique sequences were the P. notoginseng special transcripts. Similarly, a total of 19,479 P. notoginseng unique sequences had sequence similarity to P. ginseng unique sequences , and the rest 11,373 unique sequences were the P. notoginseng special transcripts.
The 20 most abundant transcripts in P. notoginseng root
No. of reads
Reticuline oxidase -like protein
P. ginseng-specific abundant protein 3
P. ginseng-specific abundant protein 3
1,4-alpha-glucan- branching enzyme
Heat shock protein, putative
(S)-reticuline oxidase-like protein
P. ginseng dammarenediol-II synthase
Delta12-fatty acid acetylenase
P. ginseng squalene epoxidase
P. ginseng clone PG6L-4
Heat shock protein, putative
Heat shock protein 70-3
Sucrose synthase isoform 1
Tonoplast intrinsic protein
Heat shock protein
GO analysis and KEGG assignment
Genes involved in triterpene saponin biosynthesis in P. notoginseng 454-EST dataset
Acetyl-CoA acetyltransferase, AACT
Mevalonate kinase, MVK
IPP isomerase, IPPI
Squalene synthase, SS
Squalene epoxidase, SE
Dammarenediol-II synthase, DS
Simple sequence repeats (SSRs) are the most feasible genetic markers for plant breeding and genetic applications . A total of 2,772 putative SSR motifs were identified from 2,361 P. notoginseng unique sequences with 8.98% (2,772/30,852) frequency (Additional file 4). The frequency of SSRs identified among P. notoginseng 454-ESTs was similar to that of some dicotyledonous species . These motifs included di-, tri-, tetra-, penta- and hexa-nucleotides with the lengths ranging from 2 to 6 bp. Among the SSR-containing unique sequences, the majority (85.51%) had a single SSR motif in every sequence.
Summary of di- and tri-nucleotide repeats in P
No. of unique sequences (relative percentage)
Total of dinucleotide
Total of trinucleotide
Discovery of transcripts encoding putative transcription factors in P. notoginseng
Transcription factors, the sequence-specific DNA-binding proteins, play important roles in the regulation of gene expression in response to developmental programs and environmental changes in plants . Based on the searches of automated predictions using Inter-Pro, a total of 906 P. notoginseng unique sequences representing putative homologs belonging to different transcription factor (TF) families (Additional file 6), covering the ARF, AUX/IAA, B3, MYB, basic Helix-Loop-Helix (bHLH), bZIP, Homeobox, Homeodomain-like/related, pathogenesis-related/ERF, WRKY and Zinc finger family proteins (Additional file 7). Many protein members of the MYB, bZIP and WRKY transcription factors have been implicated in the regulation of stress responses . The most abundant TF family in P. notoginseng 454-EST dataset was the MYB family proteins characterized by DNA-binding domains. In Arabidopsis, the MYB family, comprising 163 genes, is also one of the largest transcription factor families . The Homeobox proteins were another set of highly expressed transcription factors in P. notoginseng. Homeobox genes regulate various developmental aspects in plants, such as the regulation of stem cell specification and organogenesis . The high expression level of MYB and Homeobox proteins in P. notoginseng may be linked to response to specific habitats and developmental regulation. Given that the functions of TFs vary in plants, the putative functions of these transcription factors potentially involved in environmental responses and/or developmental regulation in P. notoginseng will be characterized in a future study. It is noteworthy that the discovery of these candidate TFs in our 454-EST dataset may provide useful information for future research.
Candidate genes encoding enzymes involved in the biosynthesis of triterpene saponins
Discovery of the transcripts encoding the known enzymes invioved in triterpene saponin biosynthesis
The oleanane-type saponin (Ro) has not been detected in P. notoginseng based on the phytochemical studies . Surprisingly, two P. notoginseng singleton sequences (FW1NBNE02D50IR and FW1NBNE02D30JP) matched to β-AS of Panax ginseng. The presence of the β-ASs in P. notoginseng was further confirmed by RT-PCR and the PCR products were sequenced (Data not shown). The existence of the transcripts for β-ASs in P. notoginseng was seemingly conflicted with previous reports claiming that oleanane-type ginsenosides do not exist in P. notoginseng [2, 43]. Therefore, we presumed that either oleanane-type ginsenosides were present in P. notoginseng at levels too low to be detected phytochemically, or oleanane-type ginsenosides in fact did not exist in P. notoginseng, despite the presence of β-AS, might due to the lack of biosynthetic genes downstream of β-AS.
Discovery of the candidate CYP450s and UGTs might be involved in triterpene saponin biosynthesis by phylogenetic analysis
Characterization of specific CYP450s or UGTs involved in triterpene saponin biosynthesis in Panax genus will facilitate to elucidation of the triterpene saponin biosynthetic pathway. CYP450s are generally involved in the biosynthesis of terpenoids, sterols, lignins, hormones, fatty acids, pigments, and phytoalexins in plants . Some CYP450s are proposed to participate in the oxidation of the dammarane skeleton at C-12 and the other at C-6 toward the production of protopanaxadiol and protopanaxatriol, respectively [10, 45]. Previous studies have characterized CYP88D6 from Glycyrrhiza uralensis (CYP85 clan)  and CYP93E1 from Glycine max (CYP71 clan) , both of which were involved in triterpene saponin biosynthesis. Therefore, the CYP450s belonging to CYP85 and CYP71 clan might be involved in ginsenoside biosynthesis in Panax genus. Glycosylation, catalyzed by glycosyltransferases (GTs), transfers the activated saccharides to an aglycone substrate in the modification on ginsenoside biosynthesis. This enzymatic conjugation based on glycosylation can stabilize the product and alter its physiological activity . The dammarane- and oleanane-type aglycones have ginsenoside bioactivity after the glycosylation catalyzed by UGTs. UGT73K1 and UGT71G1 from Medicago truncatula  and UGT74M1 from Saponaria vaccaria  have been identified and characterized with functions in triterpene saponin biosynthesis. Recently, several CYP450s and UGTs were found as candidate genes involved in ginsenoside biosynthesis in P. quinquefolius and P. ginseng in our previous studies [15, 29]. Particularly, one CYP450 (contig00248) and four UGTs (contig01001, contig14976, contig15451, and contig16321) were selected as candidate genes most likely to be involved in ginsenoside biosynthesis based on their MeJA-inducible and tissue specific expression patterns in P. quinquefolius .
In this study, a total of 174 CYP450 (Additional file 8) and 242 GT (Additional file 9) unique sequences were found in the P. notoginseng cDNA library. As reported for P. ginseng and other plants, enzymes in the same biosynthetic pathway are usually co-expressed [14, 48, 49]. Hence, DS was expressed abundantly (with 1,018 reads) in the root of P. notoginseng, indicating that other genes in the triterpene saponin biosynthetic pathway might also expressed at higher levels in the P. notoginseng root. Based on this knowledge, CYP450- and UGT-unique sequences that contain more than 10 reads from the P. notoginseng root cDNA library were found as candidate enzymes involved in triterpene saponin biosynthesis. Thus, 25 CYP450 s and 16 UGT s were selected, among which 15 CYP450 s and 8 UGT s had full-length sequences after assembly and using RACE (r apid a mplification of c DNA e nd) method. The primers used for RACE were listed in Additional file 10. These cDNA sequences have been submitted to the NCBI database and given accession number GU997664-GU997678 for CYP450 s and GU997656-GU997663 for UGT s.
In this study, a large-scale 454-EST investigation of P. notoginseng root was performed based on 454 pyrosequencing. This 454-EST dataset from P. notoginseng root contribute significantly to provide a large number of transcripts for gene discovery in this medicinal plant. The description of the expressed genes and distribution of gene functions was illustrated according to GO analysis and KEGG assignment. A number of genes involved in triterpene saponin biosynthesis, including cytochrome P450s and glycosyltransferases, were discovered in our EST dataset. More importantly, a handful of candidate CYP450s and UGTs that are most likely to be involved in the biosynthesis of triterpene saponins were found based on phylogenetic analysis. Many transcription factors and EST-SSR markers were identified as well. These data will provide comprehensive information on gene discovery, transcriptome profiling, transcriptional regulation, and molecular markers for P. notoginseng. This study will contribute to further improvements on this medicinal plant through marker-assisted breeding or genetic engineering on this species as well as for other medicinal plants in the Araliaceae family.
The 4-year-old Panax notoginseng cultivated on farms was routinely harvested for medical purposes. The P. notoginseng was collected from the fields of Wenshan County, Yunnan Province, China. After cleaning, the root tissues were cut into small pieces and immediately frozen in liquid nitrogen, and stored at -80°C until further processing.
Total RNA was isolated using the Plant RNA Isolation Mini Kit (BioTeke, Beijing, China). The total RNA was treated with DNase I (TURBO DNase; Ambion, USA) at a concentration of 1.5 units/μg of total RNA. The RNA quality was tested using 1% ethidium bromide-stained (EtBr-stained) agarose gels and the concentration was assessed using a GeneQuant100 spectrophotometer (GE Healthcare, UK) prior to cDNA synthesis.
cDNA synthesis and 454 pyrosequencing
The first-strand cDNA was produced using 2.1 μg of total RNA extracted from the root of P. notoginseng according to the instructions provided with Clontech's SMART cDNA synthesis kit (Clontech, USA) with slight modifications as our previous study : in order to remove the long poly(A/T) tails in cDNA sequences, a modified synthetic poly (T) primer (5'-AAG CAG TGG TAT CAA CGC AGT GCA G T(20)VN-3') containing a Bsg I digestion site upstream of the poly (T) segment was used in combination with the Clontech SMART IV primer to synthesize the first-strand cDNA. The cDNA was amplified using PCR Advantage II polymerase (Clontech, USA) to synthesize the double-strand cDNA (ds cDNA) with the following thermal profile: 1 min at 95°C followed by 19 cycles of 95°C for 15 sec, 65°C for 30 sec, and 68°C for 6 min. And then, 5 μl of PCR product were electrophoresed in a 1% agarose gel to determine the amplification efficiency and quality. Approximately 13 μg of amplified ds cDNA was purified using the PureLink ™ PCR purification kit (Invitrogen, USA). Then the purified cDNA was treated with Bsg I (NEB, USA) overnight at 37°C and recovered by QIAquick PCR Purification Kit (Qiagen, USA). Finally, a total of 10 μg of ds cDNA was used for pyrosequencing with the GS FLX Titanium Kit.
454 EST assembly and annotation
The 454 raw read sequences were screened and trimmed for weak signals by GS FLX pyrosequencing software to yield high-quality (HQ) (> 99.5% accuracy of single-base reads) sequences. The resulting HQ reads were then submitted to the Short Read Archive at NCBI and assigned the accession number SRX017444. The primer and adapter sequences were trimmed from the HQ sequences, followed by removing the sequences shorter than 50 bp from the clean ESTs before assembly. Then, the data from the 454 read sequences were assembled into unique sequences (including contigs and singletons) using 454 GS De Novo Assembler software v2.0.01.14 (454 Life Sciences, Roche) with a quality score threshold set at 40.
The assembled unique sequences were first searched for sequence similarities against the NCBI non-redundant nucleotide (Nt) database using the BLASTN algorithm with E- value cut-off of 10-5 to find and remove ribosomal RNA sequences . And then, the remaining sequences were searched against the public databases including the Arabidopsis protein database at The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org) (version Tair9), SwissProt protein database (http://www.expasy.ch/sprot; released on 06/19/2009), and the NCBI non-redundant protein (Nr) database (http://www.ncbi.nlm.nih.gov; released on 06/23/2009) using the BLASTX algorithm with an E-value cut-off of 10-5. The functional categories of these unique sequences were further analyzed using the Gene Ontology (GO) database. The unique sequences were categorized according to GO terms based on AGI codes and TAIR GO slim provided by TAIR.
Pathway assignment with KEGG database
Pathway assignments were carried out according to the Kyoto Encyclopedia of Genes and Genome (KEGG) mapping (http://www.genome.ad.jp/kegg/kegg2.html) (version KEGG 50) . Enzyme commission (EC) numbers were assigned to unique sequences after BLASTX searches with an E- value cut-off of 10-5 upon against the KEGG database. The unique sequences were assigned to special biochemical pathways according to the corresponding EC distribution in the KEGG database.
Simple sequence repeat (SSR) detection
The total unique sequences were searched to determine the composition, frequency, and distribution of simple sequence repeats (SSRs) using an online SSR identification tool - SSRIT (Simple Sequence Repeat Identification Tool) (http://www.gramene.org/db/markers/ssrtool) . The search parameters for the maximum motif-length group were set to recognize hexamers and the minimum number of repeats was set to five.
Screening of CYP450 or UGT unique sequences encoding enzymes responsible for triterpene saponin biosynthesis
The DS transcript was much more abundant in P. notoginseng (1,018 reads), suggesting the other genes encoding enzymes in the same biosynthetic pathway were also expressed at much higher levels in the former species. For CYP450s and UGTs, each isozyme with more than ten reads in P. notoginseng is arbitrarily considered a candidate involved in the biosynthesis of triterpene saponins. The screening of CYP450s and UGTs was performed according to phylogenetic analysis.
Production of full-length cDNA sequences for CYP450s and UGTs using RACE technology
Primers listed in Additional file 10 were synthesized according to selected CYP450s and UGTs EST sequences. The 5' or 3' ends of cDNAs were amplified using a SMART RACE cDNA amplification kit (Clontech, USA) from total RNA of P. notoginseng root and cloned into T easy Vector (Promega, USA) for Sanger sequencing. Then the full-length cDNA of each gene was generated by assembly of the corresponding EST sequence and 5' and/or 3'end sequences.
Distances between each clone were calculated with the CLUSTAL W program . The indicated scale represents 0.1 amino acid substitutions per site. Amino acid sequences were aligned using the CLUSTAL W program and evolutionary distances were computed using the Poisson correction method, and a Neighbor-Joining (NJ) tree was constructed with MEGA4. Bootstrap values obtained after 1000 replications are given on the branches. Values less than 50% are not shown.
List of abbreviations
basic local alignment search tool
expressed sequence tag
Kyoto encyclopedia of genes and genomes
rapid amplification of cDNA end
simple sequence repeat
the Arabidopsis Information Resource
This work was supported by the National Natural Science Foundation of China (grant No. 81130069 and 30900113), and the preferred foundation of Ministry of human resources and social security of the People's Republic of China (2009-1011). The authors thank Dr. Chang Liu for his revision on this manuscript and Dr. Xi-Wen Li for his assistance with sample collection.
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