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Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties.

Rama Reddy NR, Mehta RH, Soni PH, Makasana J, Gajbhiye NA, Ponnuchamy M, Kumar J - PLoS ONE (2015)

Bottom Line: A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf.Several differentially expressed genes found functionally associated with sennoside biosynthesis.We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.

View Article: PubMed Central - PubMed

Affiliation: ICAR-Directorate of Medicinal and Aromatic Plants Research (DMAPR), Anand, Gujarat, India.

ABSTRACT
Senna (Cassia angustifolia Vahl.) is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090)', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.

No MeSH data available.


FPKM values based gene expression of annotated cytochrome P450s (CYPs) in leaf transctiptome of Cassia angustifolia.
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pone.0129422.g009: FPKM values based gene expression of annotated cytochrome P450s (CYPs) in leaf transctiptome of Cassia angustifolia.

Mentions: Differential gene expression profile between the young and mature leaf transcripts was created using multiple experiment viewer (MEV v4.8.1) to identify genes with differential expression level in the young leaf compared to mature leaf (as control), initially we used the FPKM method (fragments per kilobase of transcript per million fragments mapped) to calculate the expression level of the CDS. Differentially expressed gene identified in control and experimental conditions were analyzed by hierarchical clustering. A heat map was constructed using the log-transformed and normalized value of genes based on Pearson uncentered correlation distance as well as based on complete linkage method (Fig 8). Based on the common hit accession of functionally annotated CDS in young and mature leaf CDS, a total of 10,763 CDS expressing in both young and mature leaf libraries of which a total of 333 (3.09%) CDS were down-regulated in young leaf compared to mature leaf whereas 2,343 (21.7%) CDS were up-regulated in young leaf compared to mature leaf with the log 2 fold change value of greater than zero (S3 Table). Gene ontology (GO) enrichment analysis was performed with the 2,343 CDS up-regulated in young leaf compared to mature leaf (S4 Table). The GO terms ‘metabolic process’ (GO:0008152) was mostly significantly enriched, followed by ‘translation’ (GO:0006412), ‘oxidation-reduction process’ (GO:0055114), ‘protein phosphorylation’ (GO:0006468) and ‘proteolysis’ (GO:0006508). The BLASTX search was performed with the 2,343 CDS up-regulated in young leaf compared to mature leaf in DGE data, we obtained number of them to be functionally involved in the anthraquinone biosynthetic pathway, such as three CDS (CDS_2968, CDS_5934 and CDS_9955) encoding ACAT and one CDS each encoding HMGS (CDS_14246), HMGR (CDS_4947), MVD (CDS_19447), DXPS (CDS_3283), ISPD (CDS_11678), IPP (CDS_6069), DAHPS (CDS_4830), DHQS (CDS_4942), menF (CDS_18013) and menB (CDS_32013) were up-regulated in young leaf compared to mature leaf however with varying log2 fold change (Table 5). BLAST search also identified 42 CDS encoding for CYPs found differentially expressing (Fig 9; S5 Table) of which 19 CYPs were up-regulated in young leaf compared to mature leaf suggests these to be possible candidates associated with sennoside biosynthesis.


Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties.

Rama Reddy NR, Mehta RH, Soni PH, Makasana J, Gajbhiye NA, Ponnuchamy M, Kumar J - PLoS ONE (2015)

FPKM values based gene expression of annotated cytochrome P450s (CYPs) in leaf transctiptome of Cassia angustifolia.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476680&req=5

pone.0129422.g009: FPKM values based gene expression of annotated cytochrome P450s (CYPs) in leaf transctiptome of Cassia angustifolia.
Mentions: Differential gene expression profile between the young and mature leaf transcripts was created using multiple experiment viewer (MEV v4.8.1) to identify genes with differential expression level in the young leaf compared to mature leaf (as control), initially we used the FPKM method (fragments per kilobase of transcript per million fragments mapped) to calculate the expression level of the CDS. Differentially expressed gene identified in control and experimental conditions were analyzed by hierarchical clustering. A heat map was constructed using the log-transformed and normalized value of genes based on Pearson uncentered correlation distance as well as based on complete linkage method (Fig 8). Based on the common hit accession of functionally annotated CDS in young and mature leaf CDS, a total of 10,763 CDS expressing in both young and mature leaf libraries of which a total of 333 (3.09%) CDS were down-regulated in young leaf compared to mature leaf whereas 2,343 (21.7%) CDS were up-regulated in young leaf compared to mature leaf with the log 2 fold change value of greater than zero (S3 Table). Gene ontology (GO) enrichment analysis was performed with the 2,343 CDS up-regulated in young leaf compared to mature leaf (S4 Table). The GO terms ‘metabolic process’ (GO:0008152) was mostly significantly enriched, followed by ‘translation’ (GO:0006412), ‘oxidation-reduction process’ (GO:0055114), ‘protein phosphorylation’ (GO:0006468) and ‘proteolysis’ (GO:0006508). The BLASTX search was performed with the 2,343 CDS up-regulated in young leaf compared to mature leaf in DGE data, we obtained number of them to be functionally involved in the anthraquinone biosynthetic pathway, such as three CDS (CDS_2968, CDS_5934 and CDS_9955) encoding ACAT and one CDS each encoding HMGS (CDS_14246), HMGR (CDS_4947), MVD (CDS_19447), DXPS (CDS_3283), ISPD (CDS_11678), IPP (CDS_6069), DAHPS (CDS_4830), DHQS (CDS_4942), menF (CDS_18013) and menB (CDS_32013) were up-regulated in young leaf compared to mature leaf however with varying log2 fold change (Table 5). BLAST search also identified 42 CDS encoding for CYPs found differentially expressing (Fig 9; S5 Table) of which 19 CYPs were up-regulated in young leaf compared to mature leaf suggests these to be possible candidates associated with sennoside biosynthesis.

Bottom Line: A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf.Several differentially expressed genes found functionally associated with sennoside biosynthesis.We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.

View Article: PubMed Central - PubMed

Affiliation: ICAR-Directorate of Medicinal and Aromatic Plants Research (DMAPR), Anand, Gujarat, India.

ABSTRACT
Senna (Cassia angustifolia Vahl.) is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090)', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.

No MeSH data available.