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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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Typical chromatograms of triterpenoid saponins in roots. Typical chromatograms of triterpenoid saponins in P. vietnamensis var. fuscidiscus roots. (A) HPLC-ELSD chromatograms of majonoside R2 in P. vietnamensis var. fuscidiscus roots; (B) HPLC-ELSD chromatograms of authentic majonoside R2. (C) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside Rd in P. vietnamensis var. fuscidiscus roots. (D) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside standards.
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Fig10: Typical chromatograms of triterpenoid saponins in roots. Typical chromatograms of triterpenoid saponins in P. vietnamensis var. fuscidiscus roots. (A) HPLC-ELSD chromatograms of majonoside R2 in P. vietnamensis var. fuscidiscus roots; (B) HPLC-ELSD chromatograms of authentic majonoside R2. (C) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside Rd in P. vietnamensis var. fuscidiscus roots. (D) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside standards.

Mentions: The content of main component is the most widely used indicator to measure the quality of herb, so quantitative analysis of main component has important practical significance. According to previous research [2], majonoside R2, ginsenoside Rg1, Rb1, Rd and notoginsenoside R1 are considered as the five main components of P. vietnamensis var. fuscidicus. Herein, the content of five major triterpene saponins in roots of P. vietnamensis var. fuscidicus was determined. High performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was employed for quantitative analysis of majonoside R2, due to its low UV absorptivity. As shown in Figure 10A and B, the peak of majonoside R2 was identified by direct comparing the retention times of the peaks with those of the standard majonoside R2 eluted under the same conditions. The content of majonoside R2 in in roots of P. vietnamensis var. fuscidicus is about 68 mg/g, indicated that majonoside R2 were rich in the roots of this species. Quantitative analysis of other four triterpene saponins in the roots of this herb were performed using high performance liquid chromatography (HPLC). As shown in Figure 10C and D, the investigated saponins were well separated within 55 min. The content of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside Rd in the roots of this herb were approximately 52.7, 17.9, 17.8 and 3.2 mg/g, respectively. The above results were approximately in accordance with previous studies [2], indicated that our quantitative results are reliable. We believe that these data will be useful for pharmacological evaluation and quality control of this new variety.Figure 10


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)

Typical chromatograms of triterpenoid saponins in roots. Typical chromatograms of triterpenoid saponins in P. vietnamensis var. fuscidiscus roots. (A) HPLC-ELSD chromatograms of majonoside R2 in P. vietnamensis var. fuscidiscus roots; (B) HPLC-ELSD chromatograms of authentic majonoside R2. (C) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside Rd in P. vietnamensis var. fuscidiscus roots. (D) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside standards.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig10: Typical chromatograms of triterpenoid saponins in roots. Typical chromatograms of triterpenoid saponins in P. vietnamensis var. fuscidiscus roots. (A) HPLC-ELSD chromatograms of majonoside R2 in P. vietnamensis var. fuscidiscus roots; (B) HPLC-ELSD chromatograms of authentic majonoside R2. (C) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside Rd in P. vietnamensis var. fuscidiscus roots. (D) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside standards.
Mentions: The content of main component is the most widely used indicator to measure the quality of herb, so quantitative analysis of main component has important practical significance. According to previous research [2], majonoside R2, ginsenoside Rg1, Rb1, Rd and notoginsenoside R1 are considered as the five main components of P. vietnamensis var. fuscidicus. Herein, the content of five major triterpene saponins in roots of P. vietnamensis var. fuscidicus was determined. High performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was employed for quantitative analysis of majonoside R2, due to its low UV absorptivity. As shown in Figure 10A and B, the peak of majonoside R2 was identified by direct comparing the retention times of the peaks with those of the standard majonoside R2 eluted under the same conditions. The content of majonoside R2 in in roots of P. vietnamensis var. fuscidicus is about 68 mg/g, indicated that majonoside R2 were rich in the roots of this species. Quantitative analysis of other four triterpene saponins in the roots of this herb were performed using high performance liquid chromatography (HPLC). As shown in Figure 10C and D, the investigated saponins were well separated within 55 min. The content of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside Rd in the roots of this herb were approximately 52.7, 17.9, 17.8 and 3.2 mg/g, respectively. The above results were approximately in accordance with previous studies [2], indicated that our quantitative results are reliable. We believe that these data will be useful for pharmacological evaluation and quality control of this new variety.Figure 10

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