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De novo sequencing and comparative analysis of holy and sweet basil transcriptomes.

Rastogi S, Meena S, Bhattacharya A, Ghosh S, Shukla RK, Sangwan NS, Lal RK, Gupta MM, Lavania UC, Gupta V, Nagegowda DA, Shasany AK - BMC Genomics (2014)

Bottom Line: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively.Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism.Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Divison, CSIR-Central Institute of Medicinal and Aromatic Plants, P,O, CIMAP, 226015 Lucknow, U,P, India. da.nagegowda@cimap.res.in.

ABSTRACT

Background: Ocimum L. of family Lamiaceae is a well known genus for its ethnobotanical, medicinal and aromatic properties, which are attributed to innumerable phenylpropanoid and terpenoid compounds produced by the plant. To enrich genomic resources for understanding various pathways, de novo transcriptome sequencing of two important species, O. sanctum and O. basilicum, was carried out by Illumina paired-end sequencing.

Results: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively. Out of the total transcripts, 59648 (86.30%) and 105470 (81.10%) from O. sanctum and O. basilicum, and respectively were annotated by uniprot blastx against Arabidopsis, rice and lamiaceae. KEGG analysis identified 501 and 952 transcripts from O. sanctum and O. basilicum, respectively, related to secondary metabolism with higher percentage of transcripts for biosynthesis of terpenoids in O. sanctum and phenylpropanoids in O. basilicum. Higher digital gene expression in O. basilicum was validated through qPCR and correlated to higher essential oil content and chromosome number (O. sanctum, 2n = 16; and O. basilicum, 2n = 48). Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism. Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes.

Conclusion: This is the first report of a comparative transcriptome analysis of Ocimum species and can be utilized to characterize genes related to secondary metabolism, their regulation, and breeding special chemotypes with unique essential oil composition in Ocimum.

Show MeSH
Mevalonate (MVA) and Non- mevalonate (MEP) biosynthetic pathways inOcimumsps. Enzymes found in this study are colored in blue. Graphs represent the average log2fold change observed in the digital gene expression analysis. Abbreviations: AACT, acetoacetyl-CoA thiolase; ADS, amorpha-4,11-diene synthase; ALDH1, aldehyde dehydrogenase 1; BFS, β-farnesene synthase; CPR, cytochrome P450 reductase; CPS, β-caryophyllene synthase; CYP71AV1, amorphadiene-12-hydroxylase; DBR2, artemisinic aldehyde reductase; ECS, epi-cedrol synthase; FDS, farnesyl diphosphate synthase; GAS, germacrene A synthase; HMGR, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; HMGS, 3-hydroxy-3-methyl-glutaryl coenzyme A synthase; IDI, isopentenyl diphosphate isomerase; MVK, mevalonate kinase; PMD, diphosphomevalonate decarboxylase; PMK, phosphomevalonate kinase; SMO, squalene monooxygenase; SQS, squalene synthase; CMK, 4-cytidine 5′-diphospho-2-C-methyl-Derythritol kinase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; GGDS, geranylgeranyl diphosphate synthase; GDS, geranyl diphosphate synthase; HDR, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; HDS, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; IDI, isopentenyl diphosphate isomerase; MCT, 2-C-methyl-D-erythritol-4-(cytidyl-5-diphosphate) transferase; MCS, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (adapted from Olfosson et al. [67]).
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Fig5: Mevalonate (MVA) and Non- mevalonate (MEP) biosynthetic pathways inOcimumsps. Enzymes found in this study are colored in blue. Graphs represent the average log2fold change observed in the digital gene expression analysis. Abbreviations: AACT, acetoacetyl-CoA thiolase; ADS, amorpha-4,11-diene synthase; ALDH1, aldehyde dehydrogenase 1; BFS, β-farnesene synthase; CPR, cytochrome P450 reductase; CPS, β-caryophyllene synthase; CYP71AV1, amorphadiene-12-hydroxylase; DBR2, artemisinic aldehyde reductase; ECS, epi-cedrol synthase; FDS, farnesyl diphosphate synthase; GAS, germacrene A synthase; HMGR, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; HMGS, 3-hydroxy-3-methyl-glutaryl coenzyme A synthase; IDI, isopentenyl diphosphate isomerase; MVK, mevalonate kinase; PMD, diphosphomevalonate decarboxylase; PMK, phosphomevalonate kinase; SMO, squalene monooxygenase; SQS, squalene synthase; CMK, 4-cytidine 5′-diphospho-2-C-methyl-Derythritol kinase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; GGDS, geranylgeranyl diphosphate synthase; GDS, geranyl diphosphate synthase; HDR, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; HDS, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; IDI, isopentenyl diphosphate isomerase; MCT, 2-C-methyl-D-erythritol-4-(cytidyl-5-diphosphate) transferase; MCS, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (adapted from Olfosson et al. [67]).

Mentions: O. sanctum and O. basilicum analyzed in this investigation accumulate different types of phenylpropanoids/terpenoids in the essential oil. O. sanctum contains mainly eugenol (83.56%), β-elemene (7.47%) and β-caryophyllene (6.93%)[26] whereas O. basilicum accumulates methylchavicol (62.54%) and linalool (24.61%)[19]. Precursor molecules for phenylpropanoid biosynthesis are derived from the shikimate pathway (Figure 4) while terpenoid biosynthesis utilizes isoprenoid precursors from cytosolic MVA (mevalonate) as well as plastidial MEP pathways (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate/non-mevalonate pathways) (Figure 5)[7]. Uniprot annotations against lamiaceae family were used to identify genes encoding enzymes involved in different steps of phenylpropanoid and terpenoid backbone biosynthesis. Both O. sanctum and O. basilicum annotations comprised of all most all the genes involved in the biosynthesis of essential oil specific phenylproanoids and terpenoids indicating the completeness of transcriptome data (Tables 3,4 and5). Higher number of transcripts for 4CL (4-coumarate: coenzyme A ligase), ADH (alcohol dehydrogenase), TAT (tyrosine aminotransferase) from phenylpropanoid biosynthetic pathway and DXS (1-deoxy-D-xylulose 5-phosphate synthase), GPPS (geranyl diphosphate synthase), and TPS (terpene synthase) were detected for terpenoid biosynthetic pathway. The multiplicity of terpenoids produced by a single plant is achieved both by the expression of multiple TPS genes and by the ability of some TPSs to catalyze the production of multiple products[27]. Evidently, annotation of transcriptome data from both Ocimum species against Arabidopsis and lamiaceae family in uniprot revealed several candidates of probable terpene synthases involved in biosynthesis of terpenoids like- menthofuran, geraniol, limonene, linalool, kaurene, cadinene, selinene, germacrene-D and zingiberene (Figure 6).Figure 4


De novo sequencing and comparative analysis of holy and sweet basil transcriptomes.

Rastogi S, Meena S, Bhattacharya A, Ghosh S, Shukla RK, Sangwan NS, Lal RK, Gupta MM, Lavania UC, Gupta V, Nagegowda DA, Shasany AK - BMC Genomics (2014)

Mevalonate (MVA) and Non- mevalonate (MEP) biosynthetic pathways inOcimumsps. Enzymes found in this study are colored in blue. Graphs represent the average log2fold change observed in the digital gene expression analysis. Abbreviations: AACT, acetoacetyl-CoA thiolase; ADS, amorpha-4,11-diene synthase; ALDH1, aldehyde dehydrogenase 1; BFS, β-farnesene synthase; CPR, cytochrome P450 reductase; CPS, β-caryophyllene synthase; CYP71AV1, amorphadiene-12-hydroxylase; DBR2, artemisinic aldehyde reductase; ECS, epi-cedrol synthase; FDS, farnesyl diphosphate synthase; GAS, germacrene A synthase; HMGR, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; HMGS, 3-hydroxy-3-methyl-glutaryl coenzyme A synthase; IDI, isopentenyl diphosphate isomerase; MVK, mevalonate kinase; PMD, diphosphomevalonate decarboxylase; PMK, phosphomevalonate kinase; SMO, squalene monooxygenase; SQS, squalene synthase; CMK, 4-cytidine 5′-diphospho-2-C-methyl-Derythritol kinase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; GGDS, geranylgeranyl diphosphate synthase; GDS, geranyl diphosphate synthase; HDR, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; HDS, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; IDI, isopentenyl diphosphate isomerase; MCT, 2-C-methyl-D-erythritol-4-(cytidyl-5-diphosphate) transferase; MCS, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (adapted from Olfosson et al. [67]).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4125705&req=5

Fig5: Mevalonate (MVA) and Non- mevalonate (MEP) biosynthetic pathways inOcimumsps. Enzymes found in this study are colored in blue. Graphs represent the average log2fold change observed in the digital gene expression analysis. Abbreviations: AACT, acetoacetyl-CoA thiolase; ADS, amorpha-4,11-diene synthase; ALDH1, aldehyde dehydrogenase 1; BFS, β-farnesene synthase; CPR, cytochrome P450 reductase; CPS, β-caryophyllene synthase; CYP71AV1, amorphadiene-12-hydroxylase; DBR2, artemisinic aldehyde reductase; ECS, epi-cedrol synthase; FDS, farnesyl diphosphate synthase; GAS, germacrene A synthase; HMGR, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; HMGS, 3-hydroxy-3-methyl-glutaryl coenzyme A synthase; IDI, isopentenyl diphosphate isomerase; MVK, mevalonate kinase; PMD, diphosphomevalonate decarboxylase; PMK, phosphomevalonate kinase; SMO, squalene monooxygenase; SQS, squalene synthase; CMK, 4-cytidine 5′-diphospho-2-C-methyl-Derythritol kinase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; GGDS, geranylgeranyl diphosphate synthase; GDS, geranyl diphosphate synthase; HDR, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; HDS, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; IDI, isopentenyl diphosphate isomerase; MCT, 2-C-methyl-D-erythritol-4-(cytidyl-5-diphosphate) transferase; MCS, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (adapted from Olfosson et al. [67]).
Mentions: O. sanctum and O. basilicum analyzed in this investigation accumulate different types of phenylpropanoids/terpenoids in the essential oil. O. sanctum contains mainly eugenol (83.56%), β-elemene (7.47%) and β-caryophyllene (6.93%)[26] whereas O. basilicum accumulates methylchavicol (62.54%) and linalool (24.61%)[19]. Precursor molecules for phenylpropanoid biosynthesis are derived from the shikimate pathway (Figure 4) while terpenoid biosynthesis utilizes isoprenoid precursors from cytosolic MVA (mevalonate) as well as plastidial MEP pathways (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate/non-mevalonate pathways) (Figure 5)[7]. Uniprot annotations against lamiaceae family were used to identify genes encoding enzymes involved in different steps of phenylpropanoid and terpenoid backbone biosynthesis. Both O. sanctum and O. basilicum annotations comprised of all most all the genes involved in the biosynthesis of essential oil specific phenylproanoids and terpenoids indicating the completeness of transcriptome data (Tables 3,4 and5). Higher number of transcripts for 4CL (4-coumarate: coenzyme A ligase), ADH (alcohol dehydrogenase), TAT (tyrosine aminotransferase) from phenylpropanoid biosynthetic pathway and DXS (1-deoxy-D-xylulose 5-phosphate synthase), GPPS (geranyl diphosphate synthase), and TPS (terpene synthase) were detected for terpenoid biosynthetic pathway. The multiplicity of terpenoids produced by a single plant is achieved both by the expression of multiple TPS genes and by the ability of some TPSs to catalyze the production of multiple products[27]. Evidently, annotation of transcriptome data from both Ocimum species against Arabidopsis and lamiaceae family in uniprot revealed several candidates of probable terpene synthases involved in biosynthesis of terpenoids like- menthofuran, geraniol, limonene, linalool, kaurene, cadinene, selinene, germacrene-D and zingiberene (Figure 6).Figure 4

Bottom Line: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively.Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism.Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Divison, CSIR-Central Institute of Medicinal and Aromatic Plants, P,O, CIMAP, 226015 Lucknow, U,P, India. da.nagegowda@cimap.res.in.

ABSTRACT

Background: Ocimum L. of family Lamiaceae is a well known genus for its ethnobotanical, medicinal and aromatic properties, which are attributed to innumerable phenylpropanoid and terpenoid compounds produced by the plant. To enrich genomic resources for understanding various pathways, de novo transcriptome sequencing of two important species, O. sanctum and O. basilicum, was carried out by Illumina paired-end sequencing.

Results: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively. Out of the total transcripts, 59648 (86.30%) and 105470 (81.10%) from O. sanctum and O. basilicum, and respectively were annotated by uniprot blastx against Arabidopsis, rice and lamiaceae. KEGG analysis identified 501 and 952 transcripts from O. sanctum and O. basilicum, respectively, related to secondary metabolism with higher percentage of transcripts for biosynthesis of terpenoids in O. sanctum and phenylpropanoids in O. basilicum. Higher digital gene expression in O. basilicum was validated through qPCR and correlated to higher essential oil content and chromosome number (O. sanctum, 2n = 16; and O. basilicum, 2n = 48). Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism. Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes.

Conclusion: This is the first report of a comparative transcriptome analysis of Ocimum species and can be utilized to characterize genes related to secondary metabolism, their regulation, and breeding special chemotypes with unique essential oil composition in Ocimum.

Show MeSH