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Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

Zhang GH, Ma CH, Zhang JJ, Chen JW, Tang QY, He MH, Xu XZ, Jiang NH, Yang SC - BMC Genomics (2015)

Bottom Line: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China.We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions.The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

View Article: PubMed Central - PubMed

Affiliation: Yunnan Research Center on Good Agricultural Practice for Dominant Chinese Medicinal Materials, Yunnan Agricultural University, Kunming, 650201, Yunnan, People's Republic of China. zgh73107310@163.com.

ABSTRACT

Background: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking.

Results: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD).

Conclusions: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

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Putative pathway for triterpene saponin biosynthesis. Putative pathway for triterpene saponin biosynthesis in P. vietnamensis var. fuscidicus. Two proposed pathways (A and B) for the biosynthesis of ocotillol-type saponins, mainlymajonoside R2 in the horizontally grown rhizome (C) of P. vietnamensis var. fuscidicus(D). Enzymes found in this study are boxed. Abbreviations: AACT, acetyl-CoA acetyltransferase; β-AS, β-amyrin synthase; DMAPP, dimethylallyl diphosphate; DS, dammarenediol-II synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; Glc, glucose; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl pyrophosphate synthase; GT, glycosyltransferase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IPP, isopentenyl diphosphate; IPPI, IPP isomerase; MVD, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; P450, cytochrome P450; PMK, phosphomevalonate kinase; SE, squalene epoxidase; SS, squalene synthase.
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Fig1: Putative pathway for triterpene saponin biosynthesis. Putative pathway for triterpene saponin biosynthesis in P. vietnamensis var. fuscidicus. Two proposed pathways (A and B) for the biosynthesis of ocotillol-type saponins, mainlymajonoside R2 in the horizontally grown rhizome (C) of P. vietnamensis var. fuscidicus(D). Enzymes found in this study are boxed. Abbreviations: AACT, acetyl-CoA acetyltransferase; β-AS, β-amyrin synthase; DMAPP, dimethylallyl diphosphate; DS, dammarenediol-II synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; Glc, glucose; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl pyrophosphate synthase; GT, glycosyltransferase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IPP, isopentenyl diphosphate; IPPI, IPP isomerase; MVD, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; P450, cytochrome P450; PMK, phosphomevalonate kinase; SE, squalene epoxidase; SS, squalene synthase.

Mentions: 2,3-oxidosqualene (OS), a precursor of terpenoids is synthesized via the mevalonic acid (MVA) pathway [7]. After the cyclization of 2,3-oxidosqalene by oxidosqualene cyclase (OSC), the triterpene skeletons are modified by hydroxylation and glycosidation that leads to the production of various ginsenosides, that are further catalyzed by cytochrome P450 monoxygenases (CYP450s) and uridine diphosphate (UDP)-dependent glycosyl-transferases (UGTs) [8] (Figure 1). The biosynthesis of protopanaxadiol, protopanaxatriol and oleanolic acid has been studied well, many genes involved in this pathway have been cloned and identified [9-17]. Recently, many putative triterpene saponin-biosynthetic genes in Panax species were detected using de novo sequencing and transcriptome analysis, especially in P. ginseng, P. quinquefolius, and P. notoginseng [18-22]. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis [1]. P. vietnamensis is the only species found in the narrow habitat in central Vietnam with high content of ocotillol-type saponins, which is also in the list of endanger species.Figure 1


Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

Zhang GH, Ma CH, Zhang JJ, Chen JW, Tang QY, He MH, Xu XZ, Jiang NH, Yang SC - BMC Genomics (2015)

Putative pathway for triterpene saponin biosynthesis. Putative pathway for triterpene saponin biosynthesis in P. vietnamensis var. fuscidicus. Two proposed pathways (A and B) for the biosynthesis of ocotillol-type saponins, mainlymajonoside R2 in the horizontally grown rhizome (C) of P. vietnamensis var. fuscidicus(D). Enzymes found in this study are boxed. Abbreviations: AACT, acetyl-CoA acetyltransferase; β-AS, β-amyrin synthase; DMAPP, dimethylallyl diphosphate; DS, dammarenediol-II synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; Glc, glucose; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl pyrophosphate synthase; GT, glycosyltransferase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IPP, isopentenyl diphosphate; IPPI, IPP isomerase; MVD, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; P450, cytochrome P450; PMK, phosphomevalonate kinase; SE, squalene epoxidase; SS, squalene synthase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4355973&req=5

Fig1: Putative pathway for triterpene saponin biosynthesis. Putative pathway for triterpene saponin biosynthesis in P. vietnamensis var. fuscidicus. Two proposed pathways (A and B) for the biosynthesis of ocotillol-type saponins, mainlymajonoside R2 in the horizontally grown rhizome (C) of P. vietnamensis var. fuscidicus(D). Enzymes found in this study are boxed. Abbreviations: AACT, acetyl-CoA acetyltransferase; β-AS, β-amyrin synthase; DMAPP, dimethylallyl diphosphate; DS, dammarenediol-II synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; Glc, glucose; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl pyrophosphate synthase; GT, glycosyltransferase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IPP, isopentenyl diphosphate; IPPI, IPP isomerase; MVD, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; P450, cytochrome P450; PMK, phosphomevalonate kinase; SE, squalene epoxidase; SS, squalene synthase.
Mentions: 2,3-oxidosqualene (OS), a precursor of terpenoids is synthesized via the mevalonic acid (MVA) pathway [7]. After the cyclization of 2,3-oxidosqalene by oxidosqualene cyclase (OSC), the triterpene skeletons are modified by hydroxylation and glycosidation that leads to the production of various ginsenosides, that are further catalyzed by cytochrome P450 monoxygenases (CYP450s) and uridine diphosphate (UDP)-dependent glycosyl-transferases (UGTs) [8] (Figure 1). The biosynthesis of protopanaxadiol, protopanaxatriol and oleanolic acid has been studied well, many genes involved in this pathway have been cloned and identified [9-17]. Recently, many putative triterpene saponin-biosynthetic genes in Panax species were detected using de novo sequencing and transcriptome analysis, especially in P. ginseng, P. quinquefolius, and P. notoginseng [18-22]. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis [1]. P. vietnamensis is the only species found in the narrow habitat in central Vietnam with high content of ocotillol-type saponins, which is also in the list of endanger species.Figure 1

Bottom Line: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China.We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions.The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

View Article: PubMed Central - PubMed

Affiliation: Yunnan Research Center on Good Agricultural Practice for Dominant Chinese Medicinal Materials, Yunnan Agricultural University, Kunming, 650201, Yunnan, People's Republic of China. zgh73107310@163.com.

ABSTRACT

Background: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking.

Results: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD).

Conclusions: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

Show MeSH
Related in: MedlinePlus