Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations
Identifieur interne : 000A02 ( Main/Exploration ); précédent : 000A01; suivant : 000A03Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations
Auteurs : Satiander Rana ; Wajid Waheed Bhat ; Niha Dhar ; Shahzad A. Pandith ; Sumeer Razdan ; Ram Vishwakarma ; Surrinder K. LattooSource :
- BMC Biotechnology [ 1472-6750 ] ; 2014.
Abstract
Pharmacological investigations position withanolides as important bioactive molecules demanding their enhanced production. Therefore, one of the pivotal aims has been to gain knowledge about complete biosynthesis of withanolides in terms of enzymatic and regulatory genes of the pathway. However, the pathway remains elusive at the molecular level. P450s monooxygenases play a crucial role in secondary metabolism and predominantly help in functionalizing molecule core structures including withanolides.
In an endeavor towards identification and characterization of different P450s, we here describe molecular cloning, characterization and expression analysis of two A-type P450s,
Two A-types P450
The online version of this article (doi:10.1186/s12896-014-0089-5) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/s12896-014-0089-5
PubMed: 25416924
PubMed Central: 4247701
Affiliations:
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Molecular characterization of two A-type P450s, <italic>WsCYP98A</italic> and <italic>WsCYP76A</italic> from <italic>Withania somnifera</italic> (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations</title><author><name sortKey="Rana, Satiander" sort="Rana, Satiander" uniqKey="Rana S" first="Satiander" last="Rana">Satiander Rana</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Bhat, Wajid Waheed" sort="Bhat, Wajid Waheed" uniqKey="Bhat W" first="Wajid Waheed" last="Bhat">Wajid Waheed Bhat</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Dhar, Niha" sort="Dhar, Niha" uniqKey="Dhar N" first="Niha" last="Dhar">Niha Dhar</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Pandith, Shahzad A" sort="Pandith, Shahzad A" uniqKey="Pandith S" first="Shahzad A" last="Pandith">Shahzad A. Pandith</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Razdan, Sumeer" sort="Razdan, Sumeer" uniqKey="Razdan S" first="Sumeer" last="Razdan">Sumeer Razdan</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Vishwakarma, Ram" sort="Vishwakarma, Ram" uniqKey="Vishwakarma R" first="Ram" last="Vishwakarma">Ram Vishwakarma</name><affiliation><nlm:aff id="Aff2">Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Lattoo, Surrinder K" sort="Lattoo, Surrinder K" uniqKey="Lattoo S" first="Surrinder K" last="Lattoo">Surrinder K. Lattoo</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25416924</idno><idno type="pmc">4247701</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4247701</idno><idno type="RBID">PMC:4247701</idno><idno type="doi">10.1186/s12896-014-0089-5</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000556</idno><idno type="wicri:Area/Pmc/Curation">000555</idno><idno type="wicri:Area/Pmc/Checkpoint">000759</idno><idno type="wicri:Area/Ncbi/Merge">001906</idno><idno type="wicri:Area/Ncbi/Curation">001906</idno><idno type="wicri:Area/Ncbi/Checkpoint">001906</idno><idno type="wicri:Area/Main/Merge">000A03</idno><idno type="wicri:Area/Main/Curation">000A02</idno><idno type="wicri:Area/Main/Exploration">000A02</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Molecular characterization of two A-type P450s, <italic>WsCYP98A</italic> and <italic>WsCYP76A</italic> from <italic>Withania somnifera</italic> (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations</title><author><name sortKey="Rana, Satiander" sort="Rana, Satiander" uniqKey="Rana S" first="Satiander" last="Rana">Satiander Rana</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Bhat, Wajid Waheed" sort="Bhat, Wajid Waheed" uniqKey="Bhat W" first="Wajid Waheed" last="Bhat">Wajid Waheed Bhat</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Dhar, Niha" sort="Dhar, Niha" uniqKey="Dhar N" first="Niha" last="Dhar">Niha Dhar</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Pandith, Shahzad A" sort="Pandith, Shahzad A" uniqKey="Pandith S" first="Shahzad A" last="Pandith">Shahzad A. Pandith</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Razdan, Sumeer" sort="Razdan, Sumeer" uniqKey="Razdan S" first="Sumeer" last="Razdan">Sumeer Razdan</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Vishwakarma, Ram" sort="Vishwakarma, Ram" uniqKey="Vishwakarma R" first="Ram" last="Vishwakarma">Ram Vishwakarma</name><affiliation><nlm:aff id="Aff2">Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author><author><name sortKey="Lattoo, Surrinder K" sort="Lattoo, Surrinder K" uniqKey="Lattoo S" first="Surrinder K" last="Lattoo">Surrinder K. Lattoo</name><affiliation><nlm:aff id="Aff1">Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001 India</nlm:aff><wicri:noCountry code="subfield">Tawi-180001 India</wicri:noCountry></affiliation></author></analytic><series><title level="j">BMC Biotechnology</title><idno type="eISSN">1472-6750</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Pharmacological investigations position withanolides as important bioactive molecules demanding their enhanced production. Therefore, one of the pivotal aims has been to gain knowledge about complete biosynthesis of withanolides in terms of enzymatic and regulatory genes of the pathway. However, the pathway remains elusive at the molecular level. P450s monooxygenases play a crucial role in secondary metabolism and predominantly help in functionalizing molecule core structures including withanolides.</p></sec><sec><title>Results</title><p>In an endeavor towards identification and characterization of different P450s, we here describe molecular cloning, characterization and expression analysis of two A-type P450s, <italic>WsCYP98A</italic> and <italic>WsCYP76A</italic> from <italic>Withania somnifera</italic>. Full length cDNAs of <italic>WsCYP98A</italic> and <italic>WsCYP76A</italic> have open reading frames of 1536 and 1545 bp encoding 511 (58.0 kDa) and 515 (58.7 kDa) amino acid residues, respectively. Entire coding sequences of <italic>WsCYP98A</italic> and <italic>WsCYP76A</italic> cDNAs were expressed in <italic>Escherichia coli</italic> BL21 (DE3) using pGEX4T-2 expression vector. Quantitative real-time PCR analysis indicated that both genes express widely in leaves, stalks, roots, flowers and berries with higher expression levels of <italic>WsCYP98A</italic> in stalks while <italic>WsCYP76A</italic> transcript levels were more obvious in roots. Further, transcript profiling after methyl jasmonate, salicylic acid, and gibberellic acid elicitations displayed differential transcriptional regulation of <italic>WsCYP98A</italic> and <italic>WsCYP76A</italic>. Copious transcript levels of both P450s correlated positively with the higher production of withanolides.</p></sec><sec><title>Conclusions</title><p>Two A-types P450 <italic>WsCYP98A</italic> and <italic>WsCYP76A</italic> were isolated, sequenced and heterologously expressed in <italic>E. coli</italic>. Both P450s are spatially regulated at transcript level showing differential tissue specificity. Exogenous elicitors acted as both positive and negative regulators of mRNA transcripts. Methyl jasmonate and salicylic acid resulted in copious expression of <italic>WsCYP98A</italic> and <italic>WsCYP76A</italic>. Enhanced mRNA levels also corroborated well with the increased accumulation of withanolides in response to elicitations. The empirical findings suggest that elicitors possibly incite defence or stress responses of the plant by triggering higher accumulation of withanolides.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1186/s12896-014-0089-5) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Cheng, Q" uniqKey="Cheng Q">Q Cheng</name></author><author><name sortKey="Guengerich, Fp" uniqKey="Guengerich F">FP Guengerich</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nelson, Dr" uniqKey="Nelson D">DR Nelson</name></author><author><name sortKey="Nebert, Dw" uniqKey="Nebert D">DW Nebert</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nelson, D" uniqKey="Nelson D">D Nelson</name></author><author><name sortKey="Ming, R" uniqKey="Ming R">R 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Rana</name></author><author><name sortKey="Dhar, N" uniqKey="Dhar N">N Dhar</name></author><author><name sortKey="Lattoo, Sk" uniqKey="Lattoo S">SK Lattoo</name></author><author><name sortKey="Dhar, Rs" uniqKey="Dhar R">RS Dhar</name></author><author><name sortKey="Vishwakarma, Ra" uniqKey="Vishwakarma R">RA Vishwakarma</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list></list><tree><noCountry><name sortKey="Bhat, Wajid Waheed" sort="Bhat, Wajid Waheed" uniqKey="Bhat W" first="Wajid Waheed" last="Bhat">Wajid Waheed Bhat</name><name sortKey="Dhar, Niha" sort="Dhar, Niha" uniqKey="Dhar N" first="Niha" last="Dhar">Niha Dhar</name><name sortKey="Lattoo, Surrinder K" sort="Lattoo, Surrinder K" uniqKey="Lattoo S" first="Surrinder K" last="Lattoo">Surrinder K. Lattoo</name><name sortKey="Pandith, Shahzad A" sort="Pandith, Shahzad A" uniqKey="Pandith S" first="Shahzad A" last="Pandith">Shahzad A. Pandith</name><name sortKey="Rana, Satiander" sort="Rana, Satiander" uniqKey="Rana S" first="Satiander" last="Rana">Satiander Rana</name><name sortKey="Razdan, Sumeer" sort="Razdan, Sumeer" uniqKey="Razdan S" first="Sumeer" last="Razdan">Sumeer Razdan</name><name sortKey="Vishwakarma, Ram" sort="Vishwakarma, Ram" uniqKey="Vishwakarma R" first="Ram" last="Vishwakarma">Ram Vishwakarma</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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