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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 UGTs. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus UGTs (bold letters) and other plant UGTs. Accession numbers in the NCBI GenBank database are as follows: Barbarea vulgaris BvUGT73C11 (AFN26667) and BvUGT73C10 (AFN26666); Arabidopsis thaliana AtUGT73C1 (NP_181213.1), AtUGT82A1 (NP_188864.1), AtUGT76B1 (NP_187742.1), AtUGT71B1 (NP_188812.1), AtUGT89B1 (NP_177529.2), AtUGT75B2 (NP_172044.1), AtUGT75C1 (NP_193146.1), AtUGT74C1 (NP_180738.1), AtUGT79B4 (Q9LJA6.1) and AtUGT79B1 (Q9LVW3.1); Solanum aculeatissimum SaGT4A (BAD89042); Medicago truncatula MtUGT73K1 (AAW56091), MtUGT73F3 (ACT34898) and MtUGT71G1 (AAW56092); Glycine max GmUGT73F4 (BAM29363); Panax notoginseng PnUGT1 (JX018210); Oryza sativa OsUGT709A4 (Q7XHR3); Saponaria vaccaria SvUGT74M1 (ABK76266); Linum usitatissimum LuUGT71A24 (AFJ52909), LuUGT82A2 (AFJ52979), LuUGT709D1 (AFJ53007), LuUGT75N1 (AFJ52962), LuUGT94G1 (AFJ53037.1), LuUGT79A3 (AFJ52973.1).
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Fig8: Phylogenetic tree of UGTs. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus UGTs (bold letters) and other plant UGTs. Accession numbers in the NCBI GenBank database are as follows: Barbarea vulgaris BvUGT73C11 (AFN26667) and BvUGT73C10 (AFN26666); Arabidopsis thaliana AtUGT73C1 (NP_181213.1), AtUGT82A1 (NP_188864.1), AtUGT76B1 (NP_187742.1), AtUGT71B1 (NP_188812.1), AtUGT89B1 (NP_177529.2), AtUGT75B2 (NP_172044.1), AtUGT75C1 (NP_193146.1), AtUGT74C1 (NP_180738.1), AtUGT79B4 (Q9LJA6.1) and AtUGT79B1 (Q9LVW3.1); Solanum aculeatissimum SaGT4A (BAD89042); Medicago truncatula MtUGT73K1 (AAW56091), MtUGT73F3 (ACT34898) and MtUGT71G1 (AAW56092); Glycine max GmUGT73F4 (BAM29363); Panax notoginseng PnUGT1 (JX018210); Oryza sativa OsUGT709A4 (Q7XHR3); Saponaria vaccaria SvUGT74M1 (ABK76266); Linum usitatissimum LuUGT71A24 (AFJ52909), LuUGT82A2 (AFJ52979), LuUGT709D1 (AFJ53007), LuUGT75N1 (AFJ52962), LuUGT94G1 (AFJ53037.1), LuUGT79A3 (AFJ52973.1).

Mentions: UGTs catalyze the glucosylation of C-3, C-12, C-20 hydroxyl, and C28-carboxyl for the biosynthesis of ginsenosides in P. vietnamensis var. fuscidicus. Even though UGTs catalyze the last committed step of ginsenoside biosynthesis; no UGT was functionally characterized from Panax species, only one putative UGT gene (PnUGT1) was cloned from P. notoginseng [52], which had relative close relationship to the triterpene UDP-glucosyltransferase of M. truncatula UGT71G1 [53]. In cDNA library of P. vietnamensis var. fuscidicus, 282 unigenes were found to encode UGTs (Additional file 16). The phylogenetic relationship between UGTs from P. vietnamensis var. fuscidicus and characterized UGTs from other plants was depicted in Figure 8. Except the orthologous genes of PnUGT1 (unigene0045236), unigene0071620 is highly homologous to Barbarea vulgaris UGT73C11 and UGT73C10, which catalyze sapogenin 3-O-glucosylation [54], suggested that unigene0071620 has the same function in P. vietnamensis var. fuscidicus. Besides, unigene005064, unigene0031030, and unigene0031036 have close relationship to Solanum aculeatissimum steroidal saponin UDP-glucosyltransferase SaGT4A [55], M. truncatula UGT73F3 [56], MtUGT73K1, MtUGT71G1 [53], and soybean UGT73F4 [57], indicated that those unigenes are also involved in ginsenoside biosynthesis. Furthermore, 2 unigenes (unigene0063740 and unigene0063744) have close relationship to Saponaria vaccaria UGT74M1, which is a triterpene carboxylic acid glucosyltransferase [58], suggested that these unigenes may catalyze the glucosylation of C28-carboxyl for the biosynthesis of ginsenoside Ro.Figure 8


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 UGTs. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus UGTs (bold letters) and other plant UGTs. Accession numbers in the NCBI GenBank database are as follows: Barbarea vulgaris BvUGT73C11 (AFN26667) and BvUGT73C10 (AFN26666); Arabidopsis thaliana AtUGT73C1 (NP_181213.1), AtUGT82A1 (NP_188864.1), AtUGT76B1 (NP_187742.1), AtUGT71B1 (NP_188812.1), AtUGT89B1 (NP_177529.2), AtUGT75B2 (NP_172044.1), AtUGT75C1 (NP_193146.1), AtUGT74C1 (NP_180738.1), AtUGT79B4 (Q9LJA6.1) and AtUGT79B1 (Q9LVW3.1); Solanum aculeatissimum SaGT4A (BAD89042); Medicago truncatula MtUGT73K1 (AAW56091), MtUGT73F3 (ACT34898) and MtUGT71G1 (AAW56092); Glycine max GmUGT73F4 (BAM29363); Panax notoginseng PnUGT1 (JX018210); Oryza sativa OsUGT709A4 (Q7XHR3); Saponaria vaccaria SvUGT74M1 (ABK76266); Linum usitatissimum LuUGT71A24 (AFJ52909), LuUGT82A2 (AFJ52979), LuUGT709D1 (AFJ53007), LuUGT75N1 (AFJ52962), LuUGT94G1 (AFJ53037.1), LuUGT79A3 (AFJ52973.1).
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Related In: Results  -  Collection

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Fig8: Phylogenetic tree of UGTs. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus UGTs (bold letters) and other plant UGTs. Accession numbers in the NCBI GenBank database are as follows: Barbarea vulgaris BvUGT73C11 (AFN26667) and BvUGT73C10 (AFN26666); Arabidopsis thaliana AtUGT73C1 (NP_181213.1), AtUGT82A1 (NP_188864.1), AtUGT76B1 (NP_187742.1), AtUGT71B1 (NP_188812.1), AtUGT89B1 (NP_177529.2), AtUGT75B2 (NP_172044.1), AtUGT75C1 (NP_193146.1), AtUGT74C1 (NP_180738.1), AtUGT79B4 (Q9LJA6.1) and AtUGT79B1 (Q9LVW3.1); Solanum aculeatissimum SaGT4A (BAD89042); Medicago truncatula MtUGT73K1 (AAW56091), MtUGT73F3 (ACT34898) and MtUGT71G1 (AAW56092); Glycine max GmUGT73F4 (BAM29363); Panax notoginseng PnUGT1 (JX018210); Oryza sativa OsUGT709A4 (Q7XHR3); Saponaria vaccaria SvUGT74M1 (ABK76266); Linum usitatissimum LuUGT71A24 (AFJ52909), LuUGT82A2 (AFJ52979), LuUGT709D1 (AFJ53007), LuUGT75N1 (AFJ52962), LuUGT94G1 (AFJ53037.1), LuUGT79A3 (AFJ52973.1).
Mentions: UGTs catalyze the glucosylation of C-3, C-12, C-20 hydroxyl, and C28-carboxyl for the biosynthesis of ginsenosides in P. vietnamensis var. fuscidicus. Even though UGTs catalyze the last committed step of ginsenoside biosynthesis; no UGT was functionally characterized from Panax species, only one putative UGT gene (PnUGT1) was cloned from P. notoginseng [52], which had relative close relationship to the triterpene UDP-glucosyltransferase of M. truncatula UGT71G1 [53]. In cDNA library of P. vietnamensis var. fuscidicus, 282 unigenes were found to encode UGTs (Additional file 16). The phylogenetic relationship between UGTs from P. vietnamensis var. fuscidicus and characterized UGTs from other plants was depicted in Figure 8. Except the orthologous genes of PnUGT1 (unigene0045236), unigene0071620 is highly homologous to Barbarea vulgaris UGT73C11 and UGT73C10, which catalyze sapogenin 3-O-glucosylation [54], suggested that unigene0071620 has the same function in P. vietnamensis var. fuscidicus. Besides, unigene005064, unigene0031030, and unigene0031036 have close relationship to Solanum aculeatissimum steroidal saponin UDP-glucosyltransferase SaGT4A [55], M. truncatula UGT73F3 [56], MtUGT73K1, MtUGT71G1 [53], and soybean UGT73F4 [57], indicated that those unigenes are also involved in ginsenoside biosynthesis. Furthermore, 2 unigenes (unigene0063740 and unigene0063744) have close relationship to Saponaria vaccaria UGT74M1, which is a triterpene carboxylic acid glucosyltransferase [58], suggested that these unigenes may catalyze the glucosylation of C28-carboxyl for the biosynthesis of ginsenoside Ro.Figure 8

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