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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.

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Data validation using HPLC and Real Time PCR analysis. (A) Estimation of triterpenoid content in the leaves of O. sanctum and O. basilicum.(B) Validation of the expression pattern of selected pathway genes was carried out using total RNA isolated from O. sanctum and O. basilicum leaf tissues through quantitative Real time PCR. Error bars represent standard deviation between three replicates. (C) Digital gene expression of PAL, CCR, CS3′H, EGS, CVOMT, HPPR, BAS, PMK.
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Fig7: Data validation using HPLC and Real Time PCR analysis. (A) Estimation of triterpenoid content in the leaves of O. sanctum and O. basilicum.(B) Validation of the expression pattern of selected pathway genes was carried out using total RNA isolated from O. sanctum and O. basilicum leaf tissues through quantitative Real time PCR. Error bars represent standard deviation between three replicates. (C) Digital gene expression of PAL, CCR, CS3′H, EGS, CVOMT, HPPR, BAS, PMK.

Mentions: Recently, presence of pentacyclic triterpenoids like ursolic, oleanolic and betulinic acids has been reported in Ocimum spp.[28]. This non-aromatic class of compounds have pharmacological importance such as anti-HIV, antibacterial, antiviral, anticancer and anti-inflammatory activities[29]. Like other sesquiterpenoids these triterpenoids also share their origin to farnesyl diphosphate (FDP). FDP is converted to squalene and then to oxidosqualene respectively by squalene synthase (SQS) and squalene epoxidase (SQE) enzymes. Subsequently, oxidosqualene in presence of multifunctional oxidosqualene cyclases (OSCs) viz.α-amyrin synthase (aAS), β-amyrin synthase (bAS) or lupeol synthase (LUP) which are then converted to α-amyrin, β-amyrin or lupeol, respectively. OSCs catalyzing the formation of α-amyrin, also produce β-amyrin, finally synthesizing diverse triterpenoids with the help of CypP450s members. Hence, the bAS expression cannot be directly correlated with the triterpene accumulation. Similar reports of triterpenoids biosynthesis from these OSCs are available from Catharanthus roseus and O. basilicum[30, 31]. In this investigation a total of 12 transcripts from O. basilicum and 8 transcripts from O. sanctum were homologous to β-amyrin synthase as per the Arabidopsis annotation. HPLC analysis from the dried leaves of both the Ocimum species detected oleanolic and ursolic acids however betulinic acid remained undetected. O. sanctum was observed to be having higher content of oleanolic and ursolic acids as compared to O. basilicum (Figure 7A).Figure 7


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)

Data validation using HPLC and Real Time PCR analysis. (A) Estimation of triterpenoid content in the leaves of O. sanctum and O. basilicum.(B) Validation of the expression pattern of selected pathway genes was carried out using total RNA isolated from O. sanctum and O. basilicum leaf tissues through quantitative Real time PCR. Error bars represent standard deviation between three replicates. (C) Digital gene expression of PAL, CCR, CS3′H, EGS, CVOMT, HPPR, BAS, PMK.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Data validation using HPLC and Real Time PCR analysis. (A) Estimation of triterpenoid content in the leaves of O. sanctum and O. basilicum.(B) Validation of the expression pattern of selected pathway genes was carried out using total RNA isolated from O. sanctum and O. basilicum leaf tissues through quantitative Real time PCR. Error bars represent standard deviation between three replicates. (C) Digital gene expression of PAL, CCR, CS3′H, EGS, CVOMT, HPPR, BAS, PMK.
Mentions: Recently, presence of pentacyclic triterpenoids like ursolic, oleanolic and betulinic acids has been reported in Ocimum spp.[28]. This non-aromatic class of compounds have pharmacological importance such as anti-HIV, antibacterial, antiviral, anticancer and anti-inflammatory activities[29]. Like other sesquiterpenoids these triterpenoids also share their origin to farnesyl diphosphate (FDP). FDP is converted to squalene and then to oxidosqualene respectively by squalene synthase (SQS) and squalene epoxidase (SQE) enzymes. Subsequently, oxidosqualene in presence of multifunctional oxidosqualene cyclases (OSCs) viz.α-amyrin synthase (aAS), β-amyrin synthase (bAS) or lupeol synthase (LUP) which are then converted to α-amyrin, β-amyrin or lupeol, respectively. OSCs catalyzing the formation of α-amyrin, also produce β-amyrin, finally synthesizing diverse triterpenoids with the help of CypP450s members. Hence, the bAS expression cannot be directly correlated with the triterpene accumulation. Similar reports of triterpenoids biosynthesis from these OSCs are available from Catharanthus roseus and O. basilicum[30, 31]. In this investigation a total of 12 transcripts from O. basilicum and 8 transcripts from O. sanctum were homologous to β-amyrin synthase as per the Arabidopsis annotation. HPLC analysis from the dried leaves of both the Ocimum species detected oleanolic and ursolic acids however betulinic acid remained undetected. O. sanctum was observed to be having higher content of oleanolic and ursolic acids as compared to O. basilicum (Figure 7A).Figure 7

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