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RNA-Seq Based De Novo Transcriptome Assembly and Gene Discovery of Cistanche deserticola Fleshy Stem.

Li Y, Wang X, Chen T, Yao F, Li C, Tang Q, Sun M, Sun G, Hu S, Yu J, Song S - PLoS ONE (2015)

Bottom Line: In all, we completed a global analysis of the C. deserticola fleshy stem transcriptome using RNA-seq technology.A collection of enzyme genes related to biosynthesis of lignin and phenylethanoid glysides were identified from the assembled and annotated transcripts, and the gene family of PAL was also predicted.The sequence data from this study will provide a valuable resource for conducting future phenylethanoid glysides biosynthesis researches and functional genomic studies in this important medicinal plant.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT

Backgrounds: Cistanche deserticola is a completely non-photosynthetic parasitic plant with great medicinal value and mainly distributed in desert of Northwest China. Its dried fleshy stem is a crucial tonic in traditional Chinese medicine with roles of mainly improving male sexual function and strengthening immunity, but few mechanistic studies have been conducted partly due to the lack of genomic and transcriptomic resources.

Results: In this study, we performed deep transcriptome sequencing in fleshy stem of C. deserticola, and about 80 million reads were generated using Illumina pair-end sequencing on HiSeq2000 platform. Using trinity assembler, we obtained 95,787 transcript sequences with transcript lengths ranging from 200 bp to 15,698 bp, having an average length of 950 bases and the N50 length of 1,519 bases. 63,957 transcripts were identified actively expressed with FPKM ≥ 0.5, in which 30,098 transcripts were annotated with gene descriptions or gene ontology terms by sequence similarity analyses against several public databases (Uniprot, NR and Nt at NCBI, and KEGG). Furthermore, we identified key enzyme genes involved in biosynthesis of lignin and phenylethanoid glycosides (PhGs) which are known to be the primary active ingredients. Four phenylalanine ammonia-lyase (PAL) genes, the first key enzyme in lignin and PhG biosynthesis, were identified based on sequences comparison and phylogenetic analysis. Two biosynthesis pathways of PhGs were also proposed for the first time.

Conclusions: In all, we completed a global analysis of the C. deserticola fleshy stem transcriptome using RNA-seq technology. A collection of enzyme genes related to biosynthesis of lignin and phenylethanoid glysides were identified from the assembled and annotated transcripts, and the gene family of PAL was also predicted. The sequence data from this study will provide a valuable resource for conducting future phenylethanoid glysides biosynthesis researches and functional genomic studies in this important medicinal plant.

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RT-qPCR analysis. Real-time PCR results for 10 selected genes related to lignin biosynthesis pathway.Plot in the top panel of the figure presents RNA-seq (line graph) and qPCR (bar graph) quantification results, and the bottom panel shows their high correlations (r>0.9). Corresponding gene names and unique gene identifier numbers in the assembly are bglX (comp10579_c0), 4CL (comp26895_c0), CAD_1 (comp30193_c0), CAD_2 (comp30398_c0), PER_1 (comp21305_c0), PER_2 (comp25087_c0), PER_3 (comp30272_c0), CYP98A3 (comp26902_c8), CSE (comp19323_c0), and cCOMT (comp28020_c1). (Notes: as comp30193_c0 and comp30398_c0 were both annotated as CAD gene, we added subscripts to differentiate them. The same thing was done to comp21305_c0, comp25087_c0 and comp30272_c0, which were all annotated as PER gene).
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pone.0125722.g004: RT-qPCR analysis. Real-time PCR results for 10 selected genes related to lignin biosynthesis pathway.Plot in the top panel of the figure presents RNA-seq (line graph) and qPCR (bar graph) quantification results, and the bottom panel shows their high correlations (r>0.9). Corresponding gene names and unique gene identifier numbers in the assembly are bglX (comp10579_c0), 4CL (comp26895_c0), CAD_1 (comp30193_c0), CAD_2 (comp30398_c0), PER_1 (comp21305_c0), PER_2 (comp25087_c0), PER_3 (comp30272_c0), CYP98A3 (comp26902_c8), CSE (comp19323_c0), and cCOMT (comp28020_c1). (Notes: as comp30193_c0 and comp30398_c0 were both annotated as CAD gene, we added subscripts to differentiate them. The same thing was done to comp21305_c0, comp25087_c0 and comp30272_c0, which were all annotated as PER gene).

Mentions: The four type lignins were biosynthesized by different pathways which were controlled by three key enzymes, cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), shikimate o-hydroxycinnamoyltransferase (HCT, EC 2.3.1.133), and ferulate-5-hydroxylase (F5H, EC 1.14.-.-). CCR was reported as a control point of lignins pathway [50, 51] which catalyzed X-CoA (X including p-coumaroyl, caffeoyl, feruloyl, 5-hydroxyl-feruloyl and sinapoyl) into Y-aldehyde (Y including p-coumar, caffeyl, coniferyl, 5-hydroxyl-coniferyl and sinap), while HCT catalyzed p-coumaroyl-CoA to p-coumaroyl shikimic acid/p-coumaroyl quinic acid. The two enzymes, just like a switch, regulated biosynthesis of P-hydroxyl-phenyl lignins or the other three type lignins. F5H was another branch switch that regulated syringyl lignin and 5-hydroxyl-guaiacyl lignin. Other important enzymes including caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.68), caffeoyl-CoA O-methyltransferase (CCoAOMT, EC 2.1.1.104) and cinnamyl-alcohol dehydrogenase (CAD, EC 1.1.1.195) were also detected expressed. Detailed expression information was listed in Table 6. These enzyme genes identified in this study will provide a valuable resource for functional genomic studies in this important medicinal plant. 10 genes related to lignins biosynthesis pathway in Table 6 were selected for RT-qPCR verification to confirm our RNA-seq results (Fig 4), and their high correlations (Pearson correlation coefficient: 0.90343) indicated high accuracy and reproducibility of our transcriptome analysis. S1 Dataset lists the primer sequences used in this analysis.


RNA-Seq Based De Novo Transcriptome Assembly and Gene Discovery of Cistanche deserticola Fleshy Stem.

Li Y, Wang X, Chen T, Yao F, Li C, Tang Q, Sun M, Sun G, Hu S, Yu J, Song S - PLoS ONE (2015)

RT-qPCR analysis. Real-time PCR results for 10 selected genes related to lignin biosynthesis pathway.Plot in the top panel of the figure presents RNA-seq (line graph) and qPCR (bar graph) quantification results, and the bottom panel shows their high correlations (r>0.9). Corresponding gene names and unique gene identifier numbers in the assembly are bglX (comp10579_c0), 4CL (comp26895_c0), CAD_1 (comp30193_c0), CAD_2 (comp30398_c0), PER_1 (comp21305_c0), PER_2 (comp25087_c0), PER_3 (comp30272_c0), CYP98A3 (comp26902_c8), CSE (comp19323_c0), and cCOMT (comp28020_c1). (Notes: as comp30193_c0 and comp30398_c0 were both annotated as CAD gene, we added subscripts to differentiate them. The same thing was done to comp21305_c0, comp25087_c0 and comp30272_c0, which were all annotated as PER gene).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4418726&req=5

pone.0125722.g004: RT-qPCR analysis. Real-time PCR results for 10 selected genes related to lignin biosynthesis pathway.Plot in the top panel of the figure presents RNA-seq (line graph) and qPCR (bar graph) quantification results, and the bottom panel shows their high correlations (r>0.9). Corresponding gene names and unique gene identifier numbers in the assembly are bglX (comp10579_c0), 4CL (comp26895_c0), CAD_1 (comp30193_c0), CAD_2 (comp30398_c0), PER_1 (comp21305_c0), PER_2 (comp25087_c0), PER_3 (comp30272_c0), CYP98A3 (comp26902_c8), CSE (comp19323_c0), and cCOMT (comp28020_c1). (Notes: as comp30193_c0 and comp30398_c0 were both annotated as CAD gene, we added subscripts to differentiate them. The same thing was done to comp21305_c0, comp25087_c0 and comp30272_c0, which were all annotated as PER gene).
Mentions: The four type lignins were biosynthesized by different pathways which were controlled by three key enzymes, cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), shikimate o-hydroxycinnamoyltransferase (HCT, EC 2.3.1.133), and ferulate-5-hydroxylase (F5H, EC 1.14.-.-). CCR was reported as a control point of lignins pathway [50, 51] which catalyzed X-CoA (X including p-coumaroyl, caffeoyl, feruloyl, 5-hydroxyl-feruloyl and sinapoyl) into Y-aldehyde (Y including p-coumar, caffeyl, coniferyl, 5-hydroxyl-coniferyl and sinap), while HCT catalyzed p-coumaroyl-CoA to p-coumaroyl shikimic acid/p-coumaroyl quinic acid. The two enzymes, just like a switch, regulated biosynthesis of P-hydroxyl-phenyl lignins or the other three type lignins. F5H was another branch switch that regulated syringyl lignin and 5-hydroxyl-guaiacyl lignin. Other important enzymes including caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.68), caffeoyl-CoA O-methyltransferase (CCoAOMT, EC 2.1.1.104) and cinnamyl-alcohol dehydrogenase (CAD, EC 1.1.1.195) were also detected expressed. Detailed expression information was listed in Table 6. These enzyme genes identified in this study will provide a valuable resource for functional genomic studies in this important medicinal plant. 10 genes related to lignins biosynthesis pathway in Table 6 were selected for RT-qPCR verification to confirm our RNA-seq results (Fig 4), and their high correlations (Pearson correlation coefficient: 0.90343) indicated high accuracy and reproducibility of our transcriptome analysis. S1 Dataset lists the primer sequences used in this analysis.

Bottom Line: In all, we completed a global analysis of the C. deserticola fleshy stem transcriptome using RNA-seq technology.A collection of enzyme genes related to biosynthesis of lignin and phenylethanoid glysides were identified from the assembled and annotated transcripts, and the gene family of PAL was also predicted.The sequence data from this study will provide a valuable resource for conducting future phenylethanoid glysides biosynthesis researches and functional genomic studies in this important medicinal plant.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT

Backgrounds: Cistanche deserticola is a completely non-photosynthetic parasitic plant with great medicinal value and mainly distributed in desert of Northwest China. Its dried fleshy stem is a crucial tonic in traditional Chinese medicine with roles of mainly improving male sexual function and strengthening immunity, but few mechanistic studies have been conducted partly due to the lack of genomic and transcriptomic resources.

Results: In this study, we performed deep transcriptome sequencing in fleshy stem of C. deserticola, and about 80 million reads were generated using Illumina pair-end sequencing on HiSeq2000 platform. Using trinity assembler, we obtained 95,787 transcript sequences with transcript lengths ranging from 200 bp to 15,698 bp, having an average length of 950 bases and the N50 length of 1,519 bases. 63,957 transcripts were identified actively expressed with FPKM ≥ 0.5, in which 30,098 transcripts were annotated with gene descriptions or gene ontology terms by sequence similarity analyses against several public databases (Uniprot, NR and Nt at NCBI, and KEGG). Furthermore, we identified key enzyme genes involved in biosynthesis of lignin and phenylethanoid glycosides (PhGs) which are known to be the primary active ingredients. Four phenylalanine ammonia-lyase (PAL) genes, the first key enzyme in lignin and PhG biosynthesis, were identified based on sequences comparison and phylogenetic analysis. Two biosynthesis pathways of PhGs were also proposed for the first time.

Conclusions: In all, we completed a global analysis of the C. deserticola fleshy stem transcriptome using RNA-seq technology. A collection of enzyme genes related to biosynthesis of lignin and phenylethanoid glysides were identified from the assembled and annotated transcripts, and the gene family of PAL was also predicted. The sequence data from this study will provide a valuable resource for conducting future phenylethanoid glysides biosynthesis researches and functional genomic studies in this important medicinal plant.

Show MeSH
Related in: MedlinePlus