Full-Length Transcriptome Sequences by A Combination of Sequencing Platforms Applied to Isoflavonoid and Triterpenoid Saponin Biosynthesis of Astragalus Mongholicus Bunge

Abstract Background: Astragalus mongholicus Bunge is an important medicinal plant and has been used in traditional Chinese medicine for a long history, which is rich in isoflavonoids and triterpenoid saponins. Although these active constituents in A. mongholicus have been discovered for a long time, the molecular genetic basis of the isoflavonoid and triterpenoid saponin biosynthesis pathways is virtually unknown due to the lack of a reference genome. The combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to analyze genes involved in the biosynthetic pathways of secondary metabolites in medicinal plants has been widely recognized.Results: In this study, NGS, SMRT sequencing, and targeted compounds were combined to investigate the association between isoflavonoids and triterpenoid saponins and gene expression in roots, stems and leaves of A. mongholicus. A total of four main isoflavonoids and four astragalosides (belong to triterpenoid saponins) were measured, and 44 differentially expressed genes (DEGs) of nine gene families, 44 DEGs of 16 gene families that encode for enzymes involved in isoflavonoid and triterpenoid saponin biosynthesis were identified, separately. Additionally, transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis were analyzed, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs and 11 bZIPs. The above transcripts exhibit different expression trends in different organs.Conclusions: Our study provides important genetic information for the essential genes of isoflavonoid and triterpenoid saponin biosynthesis in A. mongholicus, and provides a basis for developing its medicinal value.

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Full-length transcriptome sequences by a combination of sequencing platforms applied to isoflavonoid and triterpenoid saponin biosynthesis of Astragalus mongholicus Bunge

Plant Methods ◽

10.1186/s13007-021-00762-1 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Minzhen Yin ◽

Shanshan Chu ◽

Tingyu Shan ◽

Liangping Zha ◽

Huasheng Peng

Keyword(s):

Single Molecule ◽

Gene Families ◽

Specific Gene ◽

Triterpenoid Saponin ◽

Smrt Sequencing ◽

Triterpenoid Saponins ◽

Saponin Biosynthesis ◽

Medicinal Value ◽

Saponin Content ◽

Sequencing Platforms

Abstract Background Astragalus mongholicus Bunge is an important medicinal plant used in traditional Chinese medicine. It is rich in isoflavonoids and triterpenoid saponins. Although these active constituents of A. mongholicus have been discovered for a long time, the genetic basis of isoflavonoid and triterpenoid saponin biosynthesis in this plant is virtually unknown because of the lack of a reference genome. Here, we used a combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to identify genes involved in the biosynthetic pathway of secondary metabolites in A. mongholicus. Results In this study, NGS, SMRT sequencing, and targeted compound analysis were combined to investigate the association between isoflavonoid and triterpenoid saponin content, and specific gene expression in the root, stem, and leaves of A. mongholicus. Overall, 643,812 CCS reads were generated, yielding 121,107 non-redundant transcript isoforms with an N50 value of 2124 bp. Based on these highly accurate transcripts, 104,756 (86.50%) transcripts were successfully annotated by any of the seven databases (NR, NT, Swissprot, KEGG, KOG, Pfam and GO). Levels of four isoflavonoids and four astragalosides (triterpenoid saponins) were determined. Forty-four differentially expressed genes (DEGs) involved in isoflavonoid biosynthesis and 44 DEGs from 16 gene families that encode enzymes involved in triterpenoid saponin biosynthesis were identified. Transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs, and 11 bZIPs, were also identified. The above transcripts showed different expression trends in different plant organs. Conclusions This study provides important genetic information on the A. mongholicus genes that are essential for isoflavonoid and triterpenoid saponin biosynthesis, and provides a basis for developing the medicinal value of this plant.

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SMRT- and Illumina‑based RNA‑seq Analyses Unveil Triterpenoid Saponin Biosynthesis and Related Key Genes in Platycodon Grandiflorus (Jacq.) A. DC.

10.21203/rs.3.rs-139609/v1 ◽

2021 ◽

Author(s):

Hanwen Yu ◽

Mengli Liu ◽

Minzhen Yin ◽

Tingyu Shan ◽

Huasheng Peng ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Molecular Mechanisms ◽

Total Content ◽

Triterpenoid Saponin ◽

Triterpenoid Saponins ◽

Sequencing Technologies ◽

Saponin Biosynthesis ◽

Genes Encoding ◽

Key Genes

Abstract Background: Platycodon grandiflorus, a traditional Chinese medicine, contains considerable triterpene saponins with broad pharmacological activities. To date, information on the molecular mechanism of triterpenoid saponin biosynthesis in P. grandiflorus is limited. Here, single-molecule real-time (SMRT) and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and unveil triterpenoid saponin biosynthesis in P. grandiflorus.Results: We quantified four saponin monomers in P. grandiflorus, and found that the total content of the four saponins was the highest in the roots and the lowest in the stems and leaves. A total of 173,354 non-redundant transcripts generated from the PacBio platform were successfully annotated to seven functional databases, among which 1,765 transcripts were aligned to the "metabolism of terpenoids and polyketides" pathway in the KEGG database. Three full-length transcripts of β-amyrin synthase (β-AS), the key synthase of the β-amyrin, were identified. Furthermore, a total of 132,610 clean reads of BGISEQ sequences were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes (DEGs) involved were selected as candidates. Notably, 9 of the 96 DEGs showed the highest expression in the roots, which were considered key genes for synthesising triterpenoid saponins in P. grandiflorus. Furthermore, 3,469 genes encoding transcription factors (TFs) were identified and classified into 57 TF families, including MYB, bHLH, mTERF, and AP2-EREBP. The expression levels of genes were verified by quantitative real-time PCR.Conclusions: Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide new insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.

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A candidate gene identified in converting platycoside E to platycodin D from Platycodon grandiflorus by transcriptome and main metabolites analysis

Scientific Reports ◽

10.1038/s41598-021-89294-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xinglong Su ◽

Yingying Liu ◽

Lu Han ◽

Zhaojian Wang ◽

Mengyang Cao ◽

...

Keyword(s):

Candidate Gene ◽

Orthogonal Experiment ◽

Triterpenoid Saponin ◽

Gene Encoding ◽

Triterpenoid Saponins ◽

Platycodin D ◽

Polysaccharide Content ◽

Saponin Biosynthesis ◽

Polysaccharide Synthesis ◽

Experimental Use

AbstractPlatycodin D and platycoside E are two triterpenoid saponins in Platycodon grandiflorus, differing only by two glycosyl groups structurally. Studies have shown β-Glucosidase from bacteria can convert platycoside E to platycodin D, indicating the potential existence of similar enzymes in P. grandiflorus. An L9(34) orthogonal experiment was performed to establish a protocol for calli induction as follows: the optimal explant is stems with nodes and the optimum medium formula is MS + NAA 1.0 mg/L + 6-BA 0.5 mg/L to obtain callus for experimental use. The platycodin D, platycoside E and total polysaccharides content between callus and plant organs varied wildly. Platycodin D and total polysaccharide content of calli was found higher than that of leaves. While, platycoside E and total polysaccharide content of calli was found lower than that of leaves. Associating platycodin D and platycoside E content with the expression level of genes involved in triterpenoid saponin biosynthesis between calli and leaves, three contigs were screened as putative sequences of β-Glucosidase gene converting platycoside E to platycodin D. Besides, we inferred that some transcription factors can regulate the expression of key enzymes involved in triterpernoid saponins and polysaccharides biosynthesis pathway of P. grandiflorus. Totally, a candidate gene encoding enzyme involved in converting platycoside E to platycodin D, and putative genes involved in polysaccharide synthesis in P. grandiflorus had been identified. This study will help uncover the molecular mechanism of triterpenoid saponins biosynthesis in P. grandiflorus.

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SMRT Genome Assembly Corrects Reference Errors, Resolving the Genetic Basis of Virulence in Mycobacterium tuberculosis

10.1101/064840 ◽

2016 ◽

Author(s):

Afif Elghraoui ◽

Samuel J Modlin ◽

Faramarz Valafar

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Molecule ◽

Genetic Basis ◽

Reference Genome ◽

Reference Sequence ◽

Smrt Sequencing ◽

Virulence Attenuation ◽

Sequencing Platforms ◽

Genome Comparisons ◽

Reference Genomes

AbstractThe genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of its virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. We discuss how our results change the picture of virulence attenuation and the power of SMRT sequencing for producing high-quality reference genomes.

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Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

10.21203/rs.3.rs-24957/v1 ◽

2020 ◽

Author(s):

shaoshan zhang ◽

Qiong Liu ◽

Chengcheng Lyu ◽

Jinsong chen ◽

Renfeng xiao ◽

...

Keyword(s):

Single Molecule ◽

Phylogenetic Trees ◽

Leaf Tissue ◽

Stevia Rebaudiana ◽

Full Length ◽

Steviol Glycosides ◽

Smrt Sequencing ◽

Leaf Tissues ◽

Sequencing Platforms ◽

Insight Into

Abstract Background: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. Results: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a more complete and correct full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and one SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results.Conclusion: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the complete and correct full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

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Characterization of Gene Isoforms Related to Cellulose and Lignin Biosynthesis in Kenaf (Hibiscus cannabinus L.) Mutant

Plants ◽

10.3390/plants9050631 ◽

2020 ◽

Vol 9 (5) ◽

pp. 631

Author(s):

Jae Il Lyu ◽

Rahul Ramekar ◽

Dong-Gun Kim ◽

Jung Min Kim ◽

Min-Kyu Lee ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Sequence Similarity ◽

Molecular Genetic ◽

Lignin Biosynthesis ◽

Gene Families ◽

Hibiscus Cannabinus ◽

Growth Stages ◽

Sequencing Platform ◽

Apical Leaf

Kenaf is a source of fiber and a bioenergy crop that is considered to be a third world crop. Recently, a new kenaf cultivar, "Jangdae," was developed by gamma irradiation. It exhibited distinguishable characteristics such as higher biomass, higher seed yield, and earlier flowering than the wild type. We sequenced and analyzed the transcriptome of apical leaf and stem using Pacific Biosciences single-molecule long-read isoform sequencing platform. De novo assembly yielded 26,822 full-length transcripts with a total length of 59 Mbp. Sequence similarity against protein sequence allowed the functional annotation of 11,370 unigenes. Among them, 10,100 unigenes were assigned gene ontology terms, the majority of which were associated with the metabolic and cellular process. The Kyoto encyclopedia of genes and genomes (KEGG) analysis mapped 8875 of the annotated unigenes to 149 metabolic pathways. We also identified the majority of putative genes involved in cellulose and lignin-biosynthesis. We further evaluated the expression pattern in eight gene families involved in lignin-biosynthesis at different growth stages. In this study, appropriate biotechnological approaches using the information obtained for these putative genes will help to modify the desirable content traits in mutants. The transcriptome data can be used as a reference dataset and provide a resource for molecular genetic studies in kenaf.

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Comparative transcriptome analysis of root, stem, and leaf tissues of Entada phaseoloides reveals potential genes involved in triterpenoid saponin biosynthesis

BMC Genomics ◽

10.1186/s12864-020-07056-1 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Weifang Liao ◽

Zhinan Mei ◽

Lihong Miao ◽

Pulin Liu ◽

Ruijie Gao

Keyword(s):

Transcriptome Analysis ◽

Biosynthetic Pathway ◽

Triterpenoid Saponin ◽

Uridine Diphosphate ◽

Comparative Transcriptome ◽

Triterpenoid Saponins ◽

Comparative Transcriptome Analysis ◽

Saponin Biosynthesis ◽

Leaf Tissues ◽

Entada Phaseoloides

Abstract Background Entada phaseoloides (L.) Merr. is an important traditional medicinal plant. The stem of Entada phaseoloides is popularly used as traditional medicine because of its significance in dispelling wind and dampness and remarkable anti-inflammatory activities. Triterpenoid saponins are the major bioactive compounds of Entada phaseoloides. However, genomic or transcriptomic technologies have not been used to study the triterpenoid saponin biosynthetic pathway in this plant. Results We performed comparative transcriptome analysis of the root, stem, and leaf tissues of Entada phaseoloides with three independent biological replicates and obtained a total of 53.26 Gb clean data and 116,910 unigenes, with an average N50 length of 1218 bp. Putative functions could be annotated to 42,191 unigenes (36.1%) based on BLASTx searches against the Non-redundant, Uniprot, KEGG, Pfam, GO, KEGG and COG databases. Most of the unigenes related to triterpenoid saponin backbone biosynthesis were specifically upregulated in the stem. A total of 26 cytochrome P450 and 17 uridine diphosphate glycosyltransferase candidate genes related to triterpenoid saponin biosynthesis were identified. The differential expressions of selected genes were further verified by qPT-PCR. Conclusions The dataset reported here will facilitate the research about the functional genomics of triterpenoid saponin biosynthesis and genetic engineering of Entada phaseoloides.

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Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

BMC Genomics ◽

10.1186/s12864-020-07195-5 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Shaoshan Zhang ◽

Qiong Liu ◽

Chengcheng Lyu ◽

Jinsong Chen ◽

Renfeng Xiao ◽

...

Keyword(s):

Single Molecule ◽

Phylogenetic Trees ◽

Leaf Tissue ◽

Stevia Rebaudiana ◽

Full Length ◽

Steviol Glycosides ◽

Smrt Sequencing ◽

Leaf Tissues ◽

Sequencing Platforms ◽

Insight Into

Abstract Background Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. Results Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results. Conclusion Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

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Comparative transcriptome analysis of root, stem, and leaf tissues of Entada phaseoloides reveals potential genes involved in triterpenoid saponin biosynthesis

10.21203/rs.2.20018/v1 ◽

2020 ◽

Author(s):

Weifang Liao ◽

Lihong Miao ◽

Pulin Liu ◽

Ruijie Gao ◽

Zhinan Mei

Keyword(s):

Transcriptome Analysis ◽

Biosynthetic Pathway ◽

Triterpenoid Saponin ◽

Uridine Diphosphate ◽

Comparative Transcriptome ◽

Triterpenoid Saponins ◽

Comparative Transcriptome Analysis ◽

Saponin Biosynthesis ◽

Leaf Tissues ◽

Entada Phaseoloides

Abstract Background Entada phaseoloides (L.) Merr. is an important traditional medicinal plant. The stem of Entada phaseoloides is popularly used as traditional medicine because of its significance in dispelling wind and dampness and remarkable anti-inflammatory activities. Triterpenoid saponins are the major bioactive compounds of Entada phaseoloides. However, genomic or transcriptomic technologies have not been used to study the triterpenoid saponin biosynthetic pathway in this plant.Results We performed comparative transcriptome analysis of the root, stem, and leaf tissues of Entada phaseoloides with three independent biological replicates and obtained a total of 53.26 Gb clean data and 116,910 unigenes, with an average N50 length of 1218 bp. Putative functions could be annotated to 42,191 unigenes (36.1%) based on BLASTx searches against the Non-redundant, Uniprot, KEGG, Pfam, GO, KEGG and COG databases. Most of the unigenes related to triterpenoid saponin backbone biosynthesis were specifically upregulated in the stem. A total of 26 cytochrome P450 and 17 uridine diphosphate glycosyltransferase candidate genes related to triterpenoid saponin biosynthesis were identified. The differential expressions of selected genes were further verified by qPT-PCR.Conclusions The dataset reported here will facilitate the research about the functional genomics of triterpenoid saponin biosynthesis and genetic engineering of Entada phaseoloides .

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Full-Length Transcriptome Analyses of Genes Involved in Triterpenoid Saponin Biosynthesis of Psammosilene tunicoides Hairy Root Cultures With Exogenous Salicylic Acid

Frontiers in Genetics ◽

10.3389/fgene.2021.657060 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lingye Su ◽

Shufang Li ◽

Hanhan Qiu ◽

Hongfeng Wang ◽

Congcong Wang ◽

...

Keyword(s):

Salicylic Acid ◽

Real Time ◽

Hairy Root ◽

Hairy Roots ◽

Consensus Sequence ◽

Full Length ◽

Triterpenoid Saponin ◽

Rna Seq ◽

Triterpenoid Saponins ◽

Saponin Biosynthesis

Triterpenoid saponins constitute a diverse class of bioactive compounds in medicinal plants. Salicylic acid (SA) is an efficient elicitor for secondary metabolite production, but a transcriptome-wide regulatory network of SA-promoted triterpenoid saponin biosynthesis remains little understood. In the current study, we described the establishment of the hairy root culture system for Psammosilene tunicoides, a triterpenoid saponin-producing medicinal herb in China, using genetic transformation by Agrobacterium rhizogenes. Compared to controls, we found that total saponin content was dramatically increased (up to 2.49-fold) by the addition of 5 mg/L SA in hairy roots for 1 day. A combination of single-molecule real-time (SMRT) and next-generation sequencing (Illumina RNA-seq) was generated to analyze the full-length transcriptome data for P. tunicoides, as well as the transcript profiles in treated (8 and 24 h) and non-treated (0 h) groups with 5 mg/L SA in hairy roots. A total of 430,117 circular consensus sequence (CCS) reads, 16,375 unigenes and 4,678 long non-coding RNAs (lncRNAs) were obtained. The average length of unigenes (2,776 bp) was much higher in full-length transcriptome than that derived from single RNA-seq (1,457 bp). The differentially expressed genes (DEGs) were mainly enriched in the metabolic process. SA up-regulated the unigenes encoding SA-binding proteins and antioxidant enzymes in comparison with controls. Additionally, we identified 89 full-length transcripts encoding enzymes putatively involved in saponin biosynthesis. The candidate transcription factors (WRKY, NAC) and structural genes (AACT, DXS, SE, CYP72A) might be the key regulators in SA-elicited saponin accumulation. Their expression was further validated by quantitative real-time PCR (qRT-PCR). These findings preliminarily elucidate the regulatory mechanisms of SA on triterpenoid saponin biosynthesis in the transcriptomic level, laying a foundation for SA-elicited saponin augmentation in P. tunicoides.

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