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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.

Show MeSH
Phylogenetic tree of CYP450s. Phylogenetic tree of the P. vienamensis var. fuscidiscus CYP450s. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus CYP450s (bold letters) and other plant CYP450s involved in triterpenoid biosynthesis. Protein sequences were 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 MtCYP72A68v2 (BAL45204), 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).
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Fig7: Phylogenetic tree of CYP450s. Phylogenetic tree of the P. vienamensis var. fuscidiscus CYP450s. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus CYP450s (bold letters) and other plant CYP450s involved in triterpenoid biosynthesis. Protein sequences were 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 MtCYP72A68v2 (BAL45204), 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).

Mentions: For discovering the candidate CYP450s involved in ginsenosides biosynthesis in the transcriptomic data of P. vietnamensis var. fuscidicus, 251 unigenes which is annotated to be CYP450 (Additional file 15) were compared with CYP450s mentioned above. As shown in Figure 7, the orthologous genes of PgCYP716A52v2 (unigene 0046586), PgCYP716A53v2 (unigene0016477), and PgCYP716A47 (unigene0036796, unigene0036797, unigene0036795, and unigene0036798) were found. Besides, one unigene (unigene0027571) is also belong to CYP716A subfamily, indicate this unigene may has different functions from other CYP716A subfamily in P. ginseng. Furthermore, 4 unigenes (unigene0044841, unigene0044844, unigene0145860, and unigene0006105) are highly homologous to A. thaliana thalianol hydroxylase (AtCYP708A2). Unigene0038 is homologous to AsCYP51H10, unigene0042080 is homologous to GuCYP93E3 and AtCYP705A5, unigene0039295 and unigene0039296 are homologous to MtCYP72A67v2 and MtCYP72A8v2 (Figure 7). Two unigenes (unigene0039295 and unigene0039296) are the orthologous gene of 11-oxo-β-amyrin 30-oxidase and highly homologous to GuCYP72A154, MtCYP72A63, MtCYP72A61v2, and MtCYP72A68v2 [44,46]. Unigene00402080 is highly homologous to GuCYP93E3 and MtCYP93E2 [43,51], may encode enzyme catalyze C-24 hydroxylation of β-amyrin.Figure 7


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)

Phylogenetic tree of CYP450s. Phylogenetic tree of the P. vienamensis var. fuscidiscus CYP450s. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus CYP450s (bold letters) and other plant CYP450s involved in triterpenoid biosynthesis. Protein sequences were 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 MtCYP72A68v2 (BAL45204), 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).
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Related In: Results  -  Collection

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Fig7: Phylogenetic tree of CYP450s. Phylogenetic tree of the P. vienamensis var. fuscidiscus CYP450s. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus CYP450s (bold letters) and other plant CYP450s involved in triterpenoid biosynthesis. Protein sequences were 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 MtCYP72A68v2 (BAL45204), 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).
Mentions: For discovering the candidate CYP450s involved in ginsenosides biosynthesis in the transcriptomic data of P. vietnamensis var. fuscidicus, 251 unigenes which is annotated to be CYP450 (Additional file 15) were compared with CYP450s mentioned above. As shown in Figure 7, the orthologous genes of PgCYP716A52v2 (unigene 0046586), PgCYP716A53v2 (unigene0016477), and PgCYP716A47 (unigene0036796, unigene0036797, unigene0036795, and unigene0036798) were found. Besides, one unigene (unigene0027571) is also belong to CYP716A subfamily, indicate this unigene may has different functions from other CYP716A subfamily in P. ginseng. Furthermore, 4 unigenes (unigene0044841, unigene0044844, unigene0145860, and unigene0006105) are highly homologous to A. thaliana thalianol hydroxylase (AtCYP708A2). Unigene0038 is homologous to AsCYP51H10, unigene0042080 is homologous to GuCYP93E3 and AtCYP705A5, unigene0039295 and unigene0039296 are homologous to MtCYP72A67v2 and MtCYP72A8v2 (Figure 7). Two unigenes (unigene0039295 and unigene0039296) are the orthologous gene of 11-oxo-β-amyrin 30-oxidase and highly homologous to GuCYP72A154, MtCYP72A63, MtCYP72A61v2, and MtCYP72A68v2 [44,46]. Unigene00402080 is highly homologous to GuCYP93E3 and MtCYP93E2 [43,51], may encode enzyme catalyze C-24 hydroxylation of β-amyrin.Figure 7

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