Functional Characterization of Sesquiterpene Synthase from Polygonum minus
Identifieur interne : 001282 ( Pmc/Curation ); précédent : 001281; suivant : 001283Functional Characterization of Sesquiterpene Synthase from Polygonum minus
Auteurs : Su-Fang Ee [Malaisie] ; Zeti-Azura Mohamed-Hussein [Malaisie] ; Roohaida Othman [Malaisie] ; Noor Azmi Shaharuddin [Malaisie] ; Ismanizan Ismail [Malaisie] ; Zamri Zainal [Malaisie]Source :
- The Scientific World Journal [ 1537-744X ] ; 2014.
Abstract
Url:
DOI: 10.1155/2014/840592
PubMed: 24678279
PubMed Central: 3942395
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Functional Characterization of Sesquiterpene Synthase from <italic>Polygonum minus</italic></title><author><name sortKey="Ee, Su Fang" sort="Ee, Su Fang" uniqKey="Ee S" first="Su-Fang" last="Ee">Su-Fang Ee</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Mohamed Hussein, Zeti Azura" sort="Mohamed Hussein, Zeti Azura" uniqKey="Mohamed Hussein Z" first="Zeti-Azura" last="Mohamed-Hussein">Zeti-Azura Mohamed-Hussein</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Othman, Roohaida" sort="Othman, Roohaida" uniqKey="Othman R" first="Roohaida" last="Othman">Roohaida Othman</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Shaharuddin, Noor Azmi" sort="Shaharuddin, Noor Azmi" uniqKey="Shaharuddin N" first="Noor Azmi" last="Shaharuddin">Noor Azmi Shaharuddin</name><affiliation wicri:level="1"><nlm:aff id="I3">Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Ismail, Ismanizan" sort="Ismail, Ismanizan" uniqKey="Ismail I" first="Ismanizan" last="Ismail">Ismanizan Ismail</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Zainal, Zamri" sort="Zainal, Zamri" uniqKey="Zainal Z" first="Zamri" last="Zainal">Zamri Zainal</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24678279</idno><idno type="pmc">3942395</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942395</idno><idno type="RBID">PMC:3942395</idno><idno type="doi">10.1155/2014/840592</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">001284</idno><idno type="wicri:Area/Pmc/Curation">001282</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Functional Characterization of Sesquiterpene Synthase from <italic>Polygonum minus</italic></title><author><name sortKey="Ee, Su Fang" sort="Ee, Su Fang" uniqKey="Ee S" first="Su-Fang" last="Ee">Su-Fang Ee</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Mohamed Hussein, Zeti Azura" sort="Mohamed Hussein, Zeti Azura" uniqKey="Mohamed Hussein Z" first="Zeti-Azura" last="Mohamed-Hussein">Zeti-Azura Mohamed-Hussein</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Othman, Roohaida" sort="Othman, Roohaida" uniqKey="Othman R" first="Roohaida" last="Othman">Roohaida Othman</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Shaharuddin, Noor Azmi" sort="Shaharuddin, Noor Azmi" uniqKey="Shaharuddin N" first="Noor Azmi" last="Shaharuddin">Noor Azmi Shaharuddin</name><affiliation wicri:level="1"><nlm:aff id="I3">Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Ismail, Ismanizan" sort="Ismail, Ismanizan" uniqKey="Ismail I" first="Ismanizan" last="Ismail">Ismanizan Ismail</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author><author><name sortKey="Zainal, Zamri" sort="Zainal, Zamri" uniqKey="Zainal Z" first="Zamri" last="Zainal">Zamri Zainal</name><affiliation wicri:level="1"><nlm:aff id="I1">School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor</wicri:regionArea></affiliation></author></analytic><series><title level="j">The Scientific World Journal</title><idno type="eISSN">1537-744X</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Polygonum minus</italic> is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C<sub>15</sub>H<sub>24</sub>. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from <italic>P. minus</italic>. <italic>P. minus</italic> sesquiterpene synthase (<italic>PmSTS</italic>) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of <italic>PmSTS</italic>, we expressed this gene in <italic>Arabidopsis thaliana</italic>. Two transgenic lines, designated as <italic>OE3</italic> and <italic>OE7</italic>, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of <italic><italic>β</italic></italic>-sesquiphellandrene.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Zhao, J" uniqKey="Zhao J">J Zhao</name></author><author><name sortKey="Davis, Lc" uniqKey="Davis L">LC Davis</name></author><author><name sortKey="Verpoorte, R" uniqKey="Verpoorte R">R Verpoorte</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yonekura Sakakibara, K" uniqKey="Yonekura Sakakibara K">K Yonekura-Sakakibara</name></author><author><name sortKey="Saito, K" uniqKey="Saito K">K Saito</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wiart, C" uniqKey="Wiart C">C Wiart</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baharum, Sn" uniqKey="Baharum S">SN Baharum</name></author><author><name 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<front><journal-meta><journal-id journal-id-type="nlm-ta">ScientificWorldJournal</journal-id><journal-id journal-id-type="iso-abbrev">ScientificWorldJournal</journal-id><journal-id journal-id-type="publisher-id">TSWJ</journal-id><journal-title-group><journal-title>The Scientific World Journal</journal-title></journal-title-group><issn pub-type="epub">1537-744X</issn><publisher><publisher-name>Hindawi Publishing Corporation</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24678279</article-id><article-id pub-id-type="pmc">3942395</article-id><article-id pub-id-type="doi">10.1155/2014/840592</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Functional Characterization of Sesquiterpene Synthase from <italic>Polygonum minus</italic></article-title></title-group><contrib-group><contrib contrib-type="author"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-5159-940X</contrib-id><name><surname>Ee</surname><given-names>Su-Fang</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Mohamed-Hussein</surname><given-names>Zeti-Azura</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Othman</surname><given-names>Roohaida</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Shaharuddin</surname><given-names>Noor Azmi</given-names></name><xref ref-type="aff" rid="I3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-3576-9077</contrib-id><name><surname>Ismail</surname><given-names>Ismanizan</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zainal</surname><given-names>Zamri</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I2"><sup>2</sup></xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="I1"><sup>1</sup>School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</aff><aff id="I2"><sup>2</sup>Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia</aff><aff id="I3"><sup>3</sup>Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia</aff><author-notes><corresp id="cor1">*Zamri Zainal: <email>zz@ukm.my</email></corresp><fn fn-type="other"><p>Academic Editors: Y. H. Cheong and H. Verhoeven</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>11</day><month>2</month><year>2014</year></pub-date><volume>2014</volume><elocation-id>840592</elocation-id><history><date date-type="received"><day>25</day><month>10</month><year>2013</year></date><date date-type="accepted"><day>24</day><month>12</month><year>2013</year></date></history><permissions><copyright-statement>Copyright © 2014 Su-Fang Ee et al.</copyright-statement><copyright-year>2014</copyright-year><license license-type="open-access"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p><italic>Polygonum minus</italic> is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C<sub>15</sub>H<sub>24</sub>. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from <italic>P. minus</italic>. <italic>P. minus</italic> sesquiterpene synthase (<italic>PmSTS</italic>) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of <italic>PmSTS</italic>, we expressed this gene in <italic>Arabidopsis thaliana</italic>. Two transgenic lines, designated as <italic>OE3</italic> and <italic>OE7</italic>, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of <italic><italic>β</italic></italic>-sesquiphellandrene.</p></abstract></article-meta></front><floats-group><fig id="fig1" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Schematic diagram of the T-DNA region of pCAMSS vector. The pCAMSS recombinant vector contains the full length <italic>PmSTS</italic> gene, which is expressed under the control of the constitutive CaMV35S promoter.</p></caption><graphic xlink:href="TSWJ2014-840592.001"></graphic></fig><fig id="fig2" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Multiple sequence alignment. The deduced amino acid sequence of <italic>PmSTS</italic> was aligned with homologues identified from the BLASTX analysis. Sequences highlighted in black indicate identical residues, while those in grey indicate similar residues. The conserved motifs RXR, DDXXD, and NSE/DTE are marked. <italic>PmSTS</italic>: sesquiterpene synthase [<italic>P. minus</italic>]; <italic>TsSTS</italic>: sesquiterpene synthase [<italic>Toona sinensis</italic>]; <italic>CsTPS1</italic>: terpene synthase 1 [<italic>Citrus sinensis</italic>]; <italic>CsVS</italic>: valencene synthase [<italic>Citrus sinensis</italic>]; <italic>VvGAS</italic>: germacrene A synthase [<italic>Vitis vinifera</italic>]; <italic>VvBCS</italic>: (E-) beta-caryophyllene synthase [<italic>Vitis vinifera</italic>]; <italic>EtACS</italic>: alpha-copaene synthase [<italic>Eleutherococcus trifoliatus</italic>].</p></caption><graphic xlink:href="TSWJ2014-840592.002"></graphic></fig><fig id="fig3" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Predicted 3D model of PmSTS generated by the I-TASSER server. The two conserved domains are highlighted. The N-terminal domain is in blue and the C-terminal domain is in green. Both aspartate-rich motifs are displayed using ball and stick representation with CPK colour. The wire bonds represent the rest of the protein.</p></caption><graphic xlink:href="TSWJ2014-840592.003"></graphic></fig><fig id="fig4" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Phylogenetic tree of PmSTS with selected terpene synthases from other plants. Seven previously identified TPS subfamilies (Tps-a to Tps-g) were chosen based on Bohlmann et al. [<xref rid="B15" ref-type="bibr">15</xref>] and Danner et al. [<xref rid="B16" ref-type="bibr">16</xref>]. The Tps-c and Tps-e subfamilies, which are composed of the copalyl diphosphate (cdp) synthases and kaurene synthases and are involved in primary metabolism, were chosen as outgroups. The alignment was performed using the Clustal Omega algorithm. The tree was built using the neighbour joining method and 1000 replicates for bootstrapping. The numbers indicated are the actual bootstrap values of the branches.</p></caption><graphic xlink:href="TSWJ2014-840592.004"></graphic></fig><fig id="fig5" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Comparison of hygromycin-sensitive and hygromycin-resistant (shown in arrow) seedlings. The seedlings were screened for 20 days on selection plate containing MS media supplemented with 25 mg L<sup>−1</sup> hygromycin.</p></caption><graphic xlink:href="TSWJ2014-840592.005"></graphic></fig><fig id="fig6" orientation="portrait" position="float"><label>Figure 6</label><caption><p>Semiquantitative RT-PCR for sesquiterpene synthase (<italic>PmSTS</italic>). PCR for the 4HPPD gene was performed in parallel as a positive control. M: 100 bp DNA ladder; <italic>WT</italic>1 and <italic>WT</italic>2: wild type <italic>A. thaliana</italic> plants; <italic>OE</italic>3: <italic>OE</italic>3 transgenic plant; <italic>OE</italic>7: <italic>OE</italic>7 transgenic plant; <italic>N</italic>: no template control.</p></caption><graphic xlink:href="TSWJ2014-840592.006"></graphic></fig><fig id="fig7" orientation="portrait" position="float"><label>Figure 7</label><caption><p>Comparison of the phenotypes of the transgenic <italic>A. thaliana</italic> and wild type <italic>A. thaliana</italic>. (a) Plants (2-month-old) were grown on soil for 1 month. Upper panel: <italic>OE</italic>3 transgenic plant and wild type plant. Lower panel: <italic>OE</italic>7 transgenic plant and wild type plant. (b) Two 4-month-old <italic>OE</italic>3 and <italic>OE</italic>7 T<sub>2</sub> plants compared with a 3-month-old wild type plant.</p></caption><graphic xlink:href="TSWJ2014-840592.007"></graphic></fig><fig id="fig8" orientation="portrait" position="float"><label>Figure 8</label><caption><p>A simplified version of the sesquiterpene biosynthetic pathway. This pathway shows the predicted flux of substrate (IPP and FPP) to produce more sesquiterpenes (C<sub>15</sub>) in transgenic plants overexpressing the <italic>PmSTS </italic>gene. (+) indicates the increased flux of substrate and (−) indicates the decreased flux of substrate. GA3P: glyceraldehyde 3-phosphate. HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA. DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase. MVA: mevalonate acid. MEP: methyl erythritol phosphate. IPP: isopentyl pyrophosphate and its isomer DMAPP: dimethylallyl diphosphate. FPP: farnesyl diphosphate. GGPP: geranylgeranyl diphosphate (modified from Okada et al. [<xref rid="B31" ref-type="bibr">31</xref>] and Vickers et al. [<xref rid="B32" ref-type="bibr">32</xref>]).</p></caption><graphic xlink:href="TSWJ2014-840592.008"></graphic></fig><fig id="fig9" orientation="portrait" position="float"><label>Figure 9</label><caption><p>GC-MS chromatograms of the transgenic <italic>OE</italic>3 (b) and a wild type plant (a). The arrow (↓) indicates the peak at the retention time of 20.834 that shows high match with <italic>β</italic>-sesquiphellandrene in the NIST/EPA/NIH library.</p></caption><graphic xlink:href="TSWJ2014-840592.009"></graphic></fig><fig id="fig10" orientation="portrait" position="float"><label>Figure 10</label><caption><p>Mass spectra of <italic>β</italic>-sesquiphellandrene. (a) shows the mass spectrum for the sample compound, while (b) shows the mass spectrum of the highest hit in the NIST/EPA/NIH library.</p></caption><graphic xlink:href="TSWJ2014-840592.010"></graphic></fig><table-wrap id="tab1" orientation="portrait" position="float"><label>Table 1</label><caption><p>BLASTX analysis. <italic>PmSTS</italic> was compared with the NCBI protein database for gene identification purposes.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Description<sup>a</sup></th><th align="left" rowspan="1" colspan="1">Organism</th><th align="center" rowspan="1" colspan="1"><italic>E</italic> value</th><th align="center" rowspan="1" colspan="1">Identity (%)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Sesquiterpene synthase</td><td align="left" rowspan="1" colspan="1"><italic>Toona sinensis </italic></td><td align="center" rowspan="1" colspan="1">5<italic>e</italic><sup>−147</sup></td><td align="center" rowspan="1" colspan="1">43</td></tr><tr><td align="left" rowspan="1" colspan="1">Terpene synthase 1</td><td align="left" rowspan="1" colspan="1"><italic>Citrus sinensis </italic></td><td align="center" rowspan="1" colspan="1">2<italic>e</italic><sup>−135</sup></td><td align="center" rowspan="1" colspan="1">42</td></tr><tr><td align="left" rowspan="1" colspan="1">Valencene synthase</td><td align="left" rowspan="1" colspan="1"><italic>Citrus sinensis </italic></td><td align="center" rowspan="1" colspan="1">2<italic>e</italic><sup>−133</sup></td><td align="center" rowspan="1" colspan="1">42</td></tr><tr><td align="left" rowspan="1" colspan="1">Germacrene A synthase</td><td align="left" rowspan="1" colspan="1"><italic>Vitis vinifera </italic></td><td align="center" rowspan="1" colspan="1">4<italic>e</italic><sup>−141</sup></td><td align="center" rowspan="1" colspan="1">42</td></tr><tr><td align="left" rowspan="1" colspan="1">(<italic>E</italic>)-Beta-caryophyllene synthase</td><td align="left" rowspan="1" colspan="1"><italic>Vitis vinifera </italic></td><td align="center" rowspan="1" colspan="1">2<italic>e</italic><sup>−136</sup></td><td align="center" rowspan="1" colspan="1">42</td></tr><tr><td align="left" rowspan="1" colspan="1">Alpha-copaene synthase</td><td align="left" rowspan="1" colspan="1"><italic>Eleutherococcus trifoliatus </italic></td><td align="center" rowspan="1" colspan="1">2<italic>e</italic><sup>−136</sup></td><td align="center" rowspan="1" colspan="1">42</td></tr><tr><td align="left" rowspan="1" colspan="1">Germacrene D synthase</td><td align="left" rowspan="1" colspan="1"><italic>Actinidia deliciosa </italic></td><td align="center" rowspan="1" colspan="1">6<italic>e</italic><sup>−135</sup></td><td align="center" rowspan="1" colspan="1">41</td></tr><tr><td align="left" rowspan="1" colspan="1">Germacrene B synthase</td><td align="left" rowspan="1" colspan="1"><italic>Cistus creticus </italic>subsp. <italic>creticus </italic></td><td align="center" rowspan="1" colspan="1">8<italic>e</italic><sup>−130</sup></td><td align="center" rowspan="1" colspan="1">41</td></tr><tr><td align="left" rowspan="1" colspan="1">(+)-Delta-cadinene synthase</td><td align="left" rowspan="1" colspan="1"><italic>Gossypium arboreum </italic></td><td align="center" rowspan="1" colspan="1">4<italic>e</italic><sup>−136</sup></td><td align="center" rowspan="1" colspan="1">41</td></tr><tr><td align="left" rowspan="1" colspan="1">Terpene synthase</td><td align="left" rowspan="1" colspan="1"><italic>Camellia sinensis </italic></td><td align="center" rowspan="1" colspan="1">6<italic>e</italic><sup>−137</sup></td><td align="center" rowspan="1" colspan="1">41</td></tr><tr><td align="left" rowspan="1" colspan="1">Beta-curcumene <break></break>synthase</td><td align="left" rowspan="1" colspan="1"><italic>Vitis vinifera </italic></td><td align="center" rowspan="1" colspan="1">2<italic>e</italic><sup>−130</sup></td><td align="center" rowspan="1" colspan="1">41</td></tr></tbody></table><table-wrap-foot><fn><p><sup>a</sup>Description—homology search using BLASTX.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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