417 lines
51 KiB
XML
417 lines
51 KiB
XML
<document id="EE4A6C4A6A5D3FE34CCA464C7C093E50" ID-DOI="10.1016/j.phytochem.2021.112932" ID-ISSN="1873-3700" ID-Zenodo-Dep="8258180" IM.bibliography_approvedBy="juliana" IM.illustrations_approvedBy="juliana" IM.materialsCitations_approvedBy="juliana" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="juliana" IM.taxonomicNames_approvedBy="juliana" IM.treatments_approvedBy="juliana" checkinTime="1692305192677" checkinUser="felipe" docAuthor="Zhang, Haihua, Xu, Jinfeng, Chen, Haimin, Jin, Weibo & Liang, Zongsuo" docDate="2021" docId="03F987D2FFFCFFF7AD2DFF66FF68FD12" docLanguage="en" docName="Phytochemistry.191.112932.pdf" docOrigin="Phytochemistry (112932) 191" docSource="http://dx.doi.org/10.1016/j.phytochem.2021.112932" docStyle="DocumentStyle:F36D69FC8B198FBE91029DF9C24697D3.5:Phytochemistry.2020-.journal_article" docStyleId="F36D69FC8B198FBE91029DF9C24697D3" docStyleName="Phytochemistry.2020-.journal_article" docStyleVersion="5" docTitle="Salvia miltiorrhiza Bunge" docType="treatment" docVersion="1" lastPageNumber="7" masterDocId="FFC0FFAAFFF9FFF1AD49FFADFFA1FFD1" masterDocTitle="Characterization of NAC family genes in Salvia miltiorrhiza and NAC 2 potentially involved in the biosynthesis of tanshinones" masterLastPageNumber="8" masterPageNumber="1" pageNumber="6" updateTime="1692630214032" updateUser="juliana">
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<mods:title id="807C1FFDF123E71076A02B0E21E35F26">Characterization of NAC family genes in Salvia miltiorrhiza and NAC 2 potentially involved in the biosynthesis of tanshinones</mods:title>
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<mods:namePart id="806E0CA9545915F7AF554E02DE7A42FB">Zhang, Haihua</mods:namePart>
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<mods:namePart id="A5A92F25F3E57460BA2F021F826D3FEF">Chen, Haimin</mods:namePart>
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<mods:namePart id="83EAFDC340C36609086C006F0E0F4FAC">Jin, Weibo</mods:namePart>
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<treatment id="03F987D2FFFCFFF7AD2DFF66FF68FD12" LSID="urn:lsid:plazi:treatment:03F987D2FFFCFFF7AD2DFF66FF68FD12" httpUri="http://treatment.plazi.org/id/03F987D2FFFCFFF7AD2DFF66FF68FD12" lastPageId="6" lastPageNumber="7" pageId="5" pageNumber="6">
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<heading id="D0A781A8FFFCFFF4AD2DFF66FF45FF2B" bold="true" centered="true" fontSize="36" level="1" pageId="5" pageNumber="6" reason="1">
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<emphasis id="B924EAD6FFFCFFF4AD2DFF66FD4EFF0F" bold="true" box="[100,751,203,223]" italics="true" pageId="5" pageNumber="6">
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5.1. Identification and phylogenetic analysis of the
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<collectionCode id="ED41AE01FFFCFFF4AF7EFF66FDC3FF0F" box="[567,610,203,222]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
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family genes in
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</emphasis>
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<taxonomicName id="4C504D47FFFCFFF4AD2DFF4AFF45FF2B" ID-CoL="6XH2C" ID-ENA="226208" authority="Bunge" authorityName="Bunge" box="[100,228,231,250]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B924EAD6FFFCFFF4AD2DFF4AFF45FF2B" bold="true" box="[100,228,231,250]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
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</taxonomicName>
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</heading>
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<paragraph id="8BEF36C4FFFCFFF4ADCDFEB2FD0AFD04" blockId="5.[100,770,287,948]" pageId="5" pageNumber="6">
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The assembly and annotation data of
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<taxonomicName id="4C504D47FFFCFFF4AF46FEB2FCA3FEE2" authority="Bunge" authorityName="Bunge" box="[527,770,287,307]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B924EAD6FFFCFFF4AF46FEB2FD1BFEE3" bold="true" box="[527,698,287,306]" italics="true" pageId="5" pageNumber="6">Salvia miltiorrhiza</emphasis>
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Bunge
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</taxonomicName>
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(
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<taxonomicName id="4C504D47FFFCFFF4AD23FE96FF70FE9F" box="[106,209,315,334]" class="Magnoliopsida" family="Lamiaceae" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="family">Lamiaceae</taxonomicName>
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) in the Genome Warehouse in BIG Data Center under Project numbers PRJCA003150, which are accessible at https://bigd.big.ac.cn/ gwh (
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<bibRefCitation id="EFC14B35FFFCFFF4ADD0FEDEFE93FE57" author="Song, Z. & Lin, C. & Xing, P. & Fen, Y. & Li, X." box="[153,306,371,390]" pageId="5" pageNumber="6" pagination="1" refId="ref10111" refString="Song, Z., Lin, C., Xing, P., Fen, Y., Li, X., 2020. A high quality reference genome sequence of Salvia miltiorrhiza provides insights into tanshinone synthesis in its red rhizomes. Plant Genome 13 (1). https: // doi. org / 10.1002 / tpg 2.20041." type="journal article" year="2020">Song et al., 2020</bibRefCitation>
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). The
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<taxonomicName id="4C504D47FFFCFFF4AC25FEDFFDA2FE57" authority="NAC" authorityName="NAC" box="[364,515,370,390]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="genus">
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<emphasis id="B924EAD6FFFCFFF4AC25FEDFFE72FE54" bold="true" box="[364,467,370,389]" italics="true" pageId="5" pageNumber="6">Arabidopsis</emphasis>
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NAC
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</taxonomicName>
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amino acid sequences were obtained from TAIR (http://www.arabidopsis.org) and were used as query in searches against the
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<taxonomicName id="4C504D47FFFCFFF4AC34FE06FE5DFE6C" box="[381,508,426,446]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B924EAD6FFFCFFF4AC34FE06FE5DFE6C" bold="true" box="[381,508,426,446]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
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</taxonomicName>
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genome database using the BLASTP program to obtain homologous sequences (
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<bibRefCitation id="EFC14B35FFFCFFF4AF0BFE6AFD6DFE0B" author="Jin, J. F. & Wang, Z. Q. & He, Q. Y. & Wang, J. Y. & Li, P. F. & Xu, J. M. & Zheng, S. J. & Fan, W. & Yang, J. L." box="[578,716,455,474]" pageId="5" pageNumber="6" pagination="288" refId="ref7738" refString="Jin, J. F., Wang, Z. Q., He, Q. Y., Wang, J. Y., Li, P. F., Xu, J. M., Zheng, S. J., Fan, W., Yang, J. L., 2020. Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress. BMC Genom. 21 (1), 288. https: // doi. org / 10.1186 / s 12864 - 020 - 6689 - 7." type="journal article" year="2020">Jin et al., 2020</bibRefCitation>
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). The Hidden Markov Model (HMM) corresponding to the
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<collectionCode id="ED41AE01FFFCFFF4AFCAFE4EFD0FFE27" box="[643,686,483,502]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
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domain (PF02365) was downloaded from the pfam protein database, and HMMER 3.2 was used to examine the
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<collectionCode id="ED41AE01FFFCFFF4ACACFDB7FDB1FDFC" box="[485,528,538,557]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
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genes from the BLASTP aligned sequences. Default parameters were employed, and the cutoff value was set to 0.01 (
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<bibRefCitation id="EFC14B35FFFCFFF4AC7CFDFFFDABFDB4" author="Letunic, I. & Bork, P." box="[309,522,594,613]" pageId="5" pageNumber="6" pagination="1" refId="ref8076" refString="Letunic, I., Bork, P., 2018. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 46 (D 1), D 493 - d 496. https: // doi. org / 10.1093 / nar / gkx 922." type="journal article" year="2018">Letunic and Bork 2018</bibRefCitation>
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). Genes encoding proteins containing
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<collectionCode id="ED41AE01FFFCFFF4AD84FDC3FF59FD50" box="[205,248,622,641]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
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domains were identified as
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<collectionCode id="ED41AE01FFFCFFF4ACB3FDC3FD84FD50" box="[506,549,622,641]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
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genes. All the Sm
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<emphasis id="B924EAD6FFFCFFF4AF81FDC3FCA3FD50" bold="true" box="[712,770,622,641]" italics="true" pageId="5" pageNumber="6">-NACs</emphasis>
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were mapped to the eight chromosomes and one scaffold of
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<taxonomicName id="4C504D47FFFCFFF4AD2DFD0BFF46FD69" box="[100,231,677,697]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B924EAD6FFFCFFF4AD2DFD0BFF46FD69" bold="true" box="[100,231,677,697]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
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</taxonomicName>
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using the TBtools program and the physical locational information from the
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<taxonomicName id="4C504D47FFFCFFF4AC7BFD6CFE10FD05" box="[306,433,705,724]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B924EAD6FFFCFFF4AC7BFD6CFE10FD05" bold="true" box="[306,433,705,724]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
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</taxonomicName>
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genome (
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<bibRefCitation id="EFC14B35FFFCFFF4AF46FD6CFD3CFD04" author="Li, B. & Fan, R. & Yang, Q. & Hu, C. & Sheng, O. & Deng, G. & Dong, T. & Li, C. & Peng, X. & Bi, F. & Yi, G." box="[527,669,705,725]" pageId="5" pageNumber="6" pagination="2" refId="ref8128" refString="Li, B., Fan, R., Yang, Q., Hu, C., Sheng, O., Deng, G., Dong, T., Li, C., Peng, X., Bi, F., Yi, G., 2020 a. Genome-wide identification and characterization of the NAC transcription factor family in Musa acuminata and expression analysis during fruit ripening. Int. J. Mol. Sci. 21 (2) https: // doi. org / 10.3390 / ijms 21020634." type="journal article" year="2020">Li et al., 2020a</bibRefCitation>
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||
).
|
||
</paragraph>
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<paragraph id="8BEF36C4FFFCFFF4ADCDFD73FD00FC65" blockId="5.[100,770,287,948]" pageId="5" pageNumber="6">
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A multi-sequence alignment of
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<collectionCode id="ED41AE01FFFCFFF4ACFAFD73FE7FFD20" box="[435,478,734,753]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
|
||
proteins from
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<taxonomicName id="4C504D47FFFCFFF4AF26FD70FD77FD21" box="[623,726,733,752]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B924EAD6FFFCFFF4AF26FD70FD77FD21" bold="true" box="[623,726,733,752]" italics="true" pageId="5" pageNumber="6">Arabidopsis</emphasis>
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</taxonomicName>
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and
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<taxonomicName id="4C504D47FFFCFFF4AD2DFD54FF45FCDD" box="[100,228,761,780]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
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<emphasis id="B924EAD6FFFCFFF4AD2DFD54FF45FCDD" bold="true" box="[100,228,761,780]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
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||
was performed using ClustalW in MEGA7.0 with default parameters (https://www.megasoftware.net/). Because the Sm-NAC family sequence lengths varied greatly, the alignment results were used to construct a phylogenetic tree using the Maximum Likelihood method with 1000 bootstrap replicates (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4ACACFCC4FD22FCAD" author="Felsenstein, J." box="[485,643,873,892]" pageId="5" pageNumber="6" pagination="783 - 791" refId="ref7461" refString="Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39 (4), 783 - 791. https: // doi. org / 10.1111 / j. 1558 - 5646.1985. tb 00420. x." type="journal article" year="1985">Felsenstein 1985</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFC14B35FFFCFFF4AFD9FCC4FF35FC49" author="Jones, D. T. & Taylor, W. R. & Thornton, J. M." pageId="5" pageNumber="6" pagination="275 - 282" refId="ref7850" refString="Jones, D. T., Taylor, W. R., Thornton, J. M., 1992. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8 (3), 275 - 282. https: // doi. org / 10.1093 / bioinformatics / 8.3.275." type="journal article" year="1992">Jones et al., 1992</bibRefCitation>
|
||
). Additionally, Evolview (http://www.evolgenius.info/) was used to beautify the evolutionary tree (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4ACECFC0CFD35FC65" author="Subramanian, B. & Gao, S. & Lercher, M. J. & Hu, S. & Chen, W. H." box="[421,660,929,948]" pageId="5" pageNumber="6" pagination="275" refId="ref10176" refString="Subramanian, B., Gao, S., Lercher, M. J., Hu, S., Chen, W. H., 2019. Evolview v 3: a webserver for visualization, annotation, and management of phylogenetic trees. Nucleic Acids Res. 47 (W 1), W 270 - W 275. https: // doi. org / 10.1093 / nar / gkz 357." type="journal article" year="2019">Subramanian et al., 2019</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4AD2DFC74FD0CFC3D" blockId="5.[100,685,985,1004]" box="[100,685,985,1004]" pageId="5" pageNumber="6">
|
||
<heading id="D0A781A8FFFCFFF4AD2DFC74FD0CFC3D" bold="true" box="[100,685,985,1004]" fontSize="36" level="1" pageId="5" pageNumber="6" reason="1">
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||
<emphasis id="B924EAD6FFFCFFF4AD2DFC74FD0CFC3D" bold="true" box="[100,685,985,1004]" italics="true" pageId="5" pageNumber="6">5.2. Gene structure and protein motif analyses of Sm-NAC genes</emphasis>
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</heading>
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</paragraph>
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<paragraph id="8BEF36C4FFFCFFF4ADCDFBBCFF7CFAD2" blockId="5.[100,770,1041,1283]" pageId="5" pageNumber="6">
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An online program of the gene structure display server (GSDS2.0) (http://gsds.cbi.pku.edu.cn/index.php) was used to draw the exonintron distribution of each Sm
|
||
<emphasis id="B924EAD6FFFCFFF4ACCDFBE4FE17FB8D" bold="true" box="[388,438,1097,1116]" italics="true" pageId="5" pageNumber="6">
|
||
-
|
||
<collectionCode id="ED41AE01FFFCFFF4ACC3FBE4FE17FB8D" box="[394,438,1097,1116]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
|
||
</emphasis>
|
||
gene by comparing predicted coding sequences (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4ADB1FBC8FE2BFBA6" author="Hu, B. & Jin, J. & Guo, A. Y. & Zhang, H. & Luo, J. & Gao, G." box="[248,394,1124,1144]" pageId="5" pageNumber="6" pagination="1296 - 1297" refId="ref7509" refString="Hu, B., Jin, J., Guo, A. Y., Zhang, H., Luo, J., Gao, G., 2015. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics 31 (8), 1296 - 1297. https: // doi. org / 10.1093 / bioinformatics / btu 817." type="journal article" year="2015">Hu et al., 2015</bibRefCitation>
|
||
). Conserved motifs of Sm-NAC protein sequences were investigated using the online software MEME5.0.4 (htt p://meme-suite.org/tools/meme) with default values for the motif parameters and the number of motifs searched set as 20 (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4AFCEFB15FF35FB36" author="Bailey, T. L. & Boden, M. & Buske, F. A. & Frith, M. & Grant, C. E. & Clementi, L. & Ren, J. & Li, W. W. & Noble, W. S." pageId="5" pageNumber="6" pagination="208" refId="ref6792" refString="Bailey, T. L., Boden, M., Buske, F. A., Frith, M., Grant, C. E., Clementi, L., Ren, J., Li, W. W., Noble, W. S., 2009. Meme suite: tools for motif discovery and searching. Nucleic Acids Res. 37, W 202 - W 208. https: // doi. org / 10.1093 / nar / gkp 335. Web Server issue." type="journal article" year="2009">Bailey et al., 2009</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFC14B35FFFCFFF4ADD7FB79FEE5FB36" author="Munir, N. & Yukun, C. & Xiaohui, C. & Nawaz, M. A. & Iftikhar, J. & Rizwan, H. M. & Xu, S. & Yuling, L. & Xuhan, X. & Zhongxiong, L." box="[158,324,1236,1256]" pageId="5" pageNumber="6" pagination="169 - 184" refId="ref9113" refString="Munir, N., Yukun, C., Xiaohui, C., Nawaz, M. A., Iftikhar, J., Rizwan, H. M., Xu, S., Yuling, L., Xuhan, X., Zhongxiong, L., 2020. Genome-wide identification and comprehensive analyses of NAC transcription factor gene family and expression patterns during somatic embryogenesis in Dimocarpus longan Lour. Plant Physiol. Biochem.: PPB (Plant Physiol. Biochem.) 157, 169 - 184. https: // doi. org / 10.1016 / j. plaphy. 2020.10.009." type="journal article" year="2020">Munir et al., 2020</bibRefCitation>
|
||
). TBtools was used to visualize the results (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4AF9BFB79FF71FAD2" author="Chen, C. & Chen, H. & Zhang, Y. & Thomas, H. R. & Frank, M. H. & He, Y. & Xia, R." pageId="5" pageNumber="6" pagination="1194 - 1202" refId="ref6983" refString="Chen, C., Chen, H., Zhang, Y., Thomas, H. R., Frank, M. H., He, Y., Xia, R., 2020. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 13 (8), 1194 - 1202. https: // doi. org / 10.1016 / j. molp. 2020.06.009." type="journal article" year="2020">Chen et al., 2020</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection id="C34A654FFFFCFFF4AD2DFA85FD43F8C7" pageId="5" pageNumber="6" type="materials_examined">
|
||
<paragraph id="8BEF36C4FFFCFFF4AD2DFA85FDC5FAEA" blockId="5.[100,612,1320,1339]" box="[100,612,1320,1339]" pageId="5" pageNumber="6">
|
||
<heading id="D0A781A8FFFCFFF4AD2DFA85FDC5FAEA" bold="true" box="[100,612,1320,1339]" fontSize="36" level="1" pageId="5" pageNumber="6" reason="1">
|
||
<emphasis id="B924EAD6FFFCFFF4AD2DFA85FDC5FAEA" bold="true" box="[100,612,1320,1339]" italics="true" pageId="5" pageNumber="6">5.3. Expression profile analysis using transcriptome data</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4ADCDFACCFD43F8C7" blockId="5.[100,770,1376,1814]" pageId="5" pageNumber="6">
|
||
To understand
|
||
<emphasis id="B924EAD6FFFCFFF4AC59FACDFE9AFAA2" bold="true" box="[272,315,1376,1395]" italics="true" pageId="5" pageNumber="6">
|
||
<collectionCode id="ED41AE01FFFCFFF4AC59FACDFE9AFAA2" box="[272,315,1376,1395]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
|
||
</emphasis>
|
||
gene expression changes after MeJA exposure in
|
||
<taxonomicName id="4C504D47FFFCFFF4AD2DFAD1FF46FA5E" box="[100,231,1404,1423]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B924EAD6FFFCFFF4AD2DFAD1FF46FA5E" bold="true" box="[100,231,1404,1423]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
and the expression levels in different tissue, the transcriptome data was retrieved from the NCBI Sequence Read Archive.
|
||
<materialsCitation id="3B383C99FFFCFFF4AFABFA35FE0AF977" pageId="5" pageNumber="6" specimenCount="1">
|
||
For the different tissues, we selected three tissues in the same batch of Sequence Read Archive (SRA) data: flower, leaf, and root (accession numbers:
|
||
<accessionNumber id="9403AB27FFFCFFF4AD8DFA41FEE1FA2E" box="[196,320,1516,1535]" pageId="5" pageNumber="6">SRR1020591</accessionNumber>
|
||
,
|
||
<accessionNumber id="9403AB27FFFCFFF4AC05FA41FE68FA2E" box="[332,457,1516,1535]" pageId="5" pageNumber="6">SRR1043998</accessionNumber>
|
||
, and
|
||
<accessionNumber id="9403AB27FFFCFFF4AF49FA41FDDDFA2E" box="[512,636,1516,1535]" pageId="5" pageNumber="6">SRR1045051</accessionNumber>
|
||
) (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4AFDAFA41FF35F9CA" author="Chen, H. & Zhang, J. & Yuan, G. & Liu, C." pageId="5" pageNumber="6" refId="ref7065" refString="Chen, H., Zhang, J., Yuan, G., Liu, C., 2014. Complex interplay among DNA modification, noncoding RNA expression and protein-coding RNA expression in Salvia miltiorrhiza chloroplast genome. PloS One 9. https: // doi. org / 10.1371 / journal. pone. 0099314." type="journal volume" year="2014">Chen et al., 2014</bibRefCitation>
|
||
). The transcriptome data after the MeJA treatment was selected from two-month-old sterile seedlings grown on 0.5
|
||
<emphasis id="B924EAD6FFFCFFF4AF02F989FDF8F9E6" box="[587,601,1572,1591]" italics="true" pageId="5" pageNumber="6">×</emphasis>
|
||
MS medium containing 100 μM MeJA or the simulated solution (ethanol). The roots of the treated seedlings were collected from three biological replicates (accession numbers:
|
||
<accessionNumber id="9403AB27FFFCFFF4AC63F9DAFDE1F95B" box="[298,576,1655,1674]" pageId="5" pageNumber="6">SRR11484256-SRR11484259</accessionNumber>
|
||
,
|
||
<accessionNumber id="9403AB27FFFCFFF4AF02F9DAFD75F95B" box="[587,724,1655,1674]" pageId="5" pageNumber="6">SRR11484266</accessionNumber>
|
||
, and
|
||
<accessionNumber id="9403AB27FFFCFFF4AD2DF93EFF4CF977" box="[100,237,1683,1702]" pageId="5" pageNumber="6">SRR11484271</accessionNumber>
|
||
) (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4AC49F93EFE3CF977" author="Zhou, Y. & Feng, J. & Li, Q. & Huang, D. & Chen, X. & Du, Z. & Xiao, Y. & Han, Y. & Chen, J. & Chen, W." box="[256,413,1683,1702]" pageId="5" pageNumber="6" pagination="1415" refId="ref11407" refString="Zhou, Y., Feng, J., Li, Q., Huang, D., Chen, X., Du, Z., Lv, Z., Xiao, Y., Han, Y., Chen, J., Chen, W., 2020. SmMYC 2 b enhances tanshinone accumulation in Salvia miltiorrhiza by activating pathway genes and promoting lateral root development. Front. Plant Sci. 11 (1415) https: // doi. org / 10.3389 / fpls. 2020.559438." type="journal article" year="2020">Zhou et al., 2020</bibRefCitation>
|
||
).
|
||
</materialsCitation>
|
||
In accordance with the TopHat BAM files and the reference GTF file, cuffdiff was used to calculate fragments per kb per million reads values (FPKM) of different tissue samples and MeJA-treated samples, and the expression differences between different samples were determined at the same time (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4AF4FF8AEFD74F8C7" author="Sulayman, A. & Tian, K. & Huang, X. & Tian, Y. & Tulafu, H." box="[518,725,1795,1814]" pageId="5" pageNumber="6" pagination="1" refId="ref10254" refString="Sulayman, A., Tian, K., Huang, X., Tian, Y., Tulafu, H., 2019. Genome-wide identification and characterization of long non-coding RNAs expressed during sheep fetal and postnatal hair follicle development. Sci. Rep. 9 (1) https: // doi. org / 10.1038 / s 41598 - 019 - 44600 - w." type="journal article" year="2019">Sulayman et al., 2019</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection id="C34A654FFFFCFFF7AD2DF896FF68FD12" lastPageId="6" lastPageNumber="7" pageId="5" pageNumber="6" type="description">
|
||
<paragraph id="8BEF36C4FFFCFFF4AD2DF896FE34F89F" blockId="5.[100,405,1851,1870]" box="[100,405,1851,1870]" pageId="5" pageNumber="6">
|
||
<heading id="D0A781A8FFFCFFF4AD2DF896FE34F89F" bold="true" box="[100,405,1851,1870]" fontSize="36" level="1" pageId="5" pageNumber="6" reason="1">
|
||
<emphasis id="B924EAD6FFFCFFF4AD2DF896FE34F89F" bold="true" box="[100,405,1851,1870]" italics="true" pageId="5" pageNumber="6">5.4. RNA isolation and qRT-PCR</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4ADCDF8DEFCDFFEC7" blockId="5.[100,771,1907,1982]" lastBlockId="5.[818,1488,148,585]" pageId="5" pageNumber="6">
|
||
Total RNA was extracted from the rhizome, leaves, and transgenic roots of
|
||
<taxonomicName id="4C504D47FFFCFFF4ADFCF822FE94F870" box="[181,309,1934,1954]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B924EAD6FFFCFFF4ADFCF822FE94F870" bold="true" box="[181,309,1934,1954]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
in accordance with the instructions of the polysaccharide and polyphenol plant RNAprep Pure Plant Kit (TIANGEN,
|
||
<collectingCountry id="F3477654FFFCFFF4AE7BFF39FCCCFF76" box="[818,877,148,167]" name="China" pageId="5" pageNumber="6">China</collectingCountry>
|
||
). The RNA from the rhizome, leaves, and transgenic roots of
|
||
<taxonomicName id="4C504D47FFFCFFF4AE7BFF1DFC12FF13" box="[818,947,175,195]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B924EAD6FFFCFFF4AE7BFF1DFC12FF13" bold="true" box="[818,947,175,195]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
were mixed and reverse transcribed into cDNA using a PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian,
|
||
<collectingCountry id="F3477654FFFCFFF4A8C3FF66FA64FF0F" box="[1418,1477,203,222]" name="China" pageId="5" pageNumber="6">China</collectingCountry>
|
||
). The cDNA was used as the template to amplify the
|
||
<emphasis id="B924EAD6FFFCFFF4A874FF4AFAD2FF2B" bold="true" box="[1341,1395,231,250]" italics="true" pageId="5" pageNumber="6">
|
||
<collectionCode id="ED41AE01FFFCFFF4A874FF4AFACBFF2B" box="[1341,1386,231,250]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
|
||
2
|
||
</emphasis>
|
||
gene for cloning.
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4AE18FEB2FB54FD98" blockId="5.[818,1488,148,585]" pageId="5" pageNumber="6">
|
||
The cDNA for qRT-PCR was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa,
|
||
<collectingRegion id="4994F826FFFCFFF4A8C6FE96FA6DFE9F" box="[1423,1484,315,334]" country="Japan" name="Tokyo" pageId="5" pageNumber="6">Tokyo</collectingRegion>
|
||
,
|
||
<collectingCountry id="F3477654FFFCFFF4AE7BFEFAFCCAFEBB" box="[818,875,343,362]" name="Japan" pageId="5" pageNumber="6">Japan</collectingCountry>
|
||
) with oligo dT. The qRT-PCR was performed in accordance with the instructions of the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa on a QuantStudio™ 6 Flex (Life Technologies, Carlsbad, CA,
|
||
<collectingCountry id="F3477654FFFCFFF4AEA6FE06FBBCFE6F" box="[1007,1053,427,446]" name="United States of America" pageId="5" pageNumber="6">USA</collectingCountry>
|
||
)). The procedure was as follows: 95
|
||
<emphasis id="B924EAD6FFFCFFF4A823FE0AFAD3FE65" box="[1386,1394,423,436]" italics="true" pageId="5" pageNumber="6">
|
||
<superScript id="7C259B8CFFFCFFF4A823FE0AFAD3FE65" attach="right" box="[1386,1394,423,436]" fontSize="6" pageId="5" pageNumber="6">◦</superScript>
|
||
</emphasis>
|
||
<collectionCode id="ED41AE01FFFCFFF4A83BFE06FADEFE6F" box="[1394,1407,427,446]" country="Denmark" name="University of Copenhagen" pageId="5" pageNumber="6" type="Herbarium">C</collectionCode>
|
||
for 30 s, then 40 cycles of 95
|
||
<emphasis id="B924EAD6FFFCFFF4AEB4FE6EFBA4FE01" box="[1021,1029,451,464]" italics="true" pageId="5" pageNumber="6">
|
||
<superScript id="7C259B8CFFFCFFF4AEB4FE6EFBA4FE01" attach="right" box="[1021,1029,451,464]" fontSize="6" pageId="5" pageNumber="6">◦</superScript>
|
||
</emphasis>
|
||
<collectionCode id="ED41AE01FFFCFFF4A94FFE6AFBB2FE0B" box="[1030,1043,455,474]" country="Denmark" name="University of Copenhagen" pageId="5" pageNumber="6" type="Herbarium">C</collectionCode>
|
||
for 5 s and 59
|
||
<emphasis id="B924EAD6FFFCFFF4A9E2FE6EFB12FE01" box="[1195,1203,451,464]" italics="true" pageId="5" pageNumber="6">
|
||
<superScript id="7C259B8CFFFCFFF4A9E2FE6EFB12FE01" attach="right" box="[1195,1203,451,464]" fontSize="6" pageId="5" pageNumber="6">◦</superScript>
|
||
</emphasis>
|
||
<collectionCode id="ED41AE01FFFCFFF4A9FDFE6AFB60FE0B" box="[1204,1217,455,474]" country="Denmark" name="University of Copenhagen" pageId="5" pageNumber="6" type="Herbarium">C</collectionCode>
|
||
for 30 s. Each reaction was repeated three times. The reference gene was the
|
||
<emphasis id="B924EAD6FFFCFFF4A868FE4FFAECFE24" bold="true" box="[1313,1357,482,501]" italics="true" pageId="5" pageNumber="6">actin</emphasis>
|
||
(
|
||
<bibRefCitation id="EFC14B35FFFCFFF4A817FE4EFCC3FDC0" author="Yang, Y. & Hou, S. & Cui, G. & Chen, S. & Wei, J. & Huang, L." pageId="5" pageNumber="6" pagination="507 - 513" refId="ref10991" refString="Yang, Y., Hou, S., Cui, G., Chen, S., Wei, J., Huang, L., 2010. Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Salvia miltiorrhiza. Mol. Biol. Rep. 37 (1), 507 - 513. https: // doi. org / 10.1007 / s 11033 - 009 - 9703 - 3." type="journal article" year="2010">Yang et al., 2010</bibRefCitation>
|
||
). The primers used are list in Supplementary
|
||
<tableCitation id="C6D2037FFFFCFFF4A843FE53FAFBFDC0" box="[1290,1370,510,529]" captionStart="Table 2" captionStartId="3.[818,868,150,166]" captionTargetPageId="3" captionText="Table 2 FPKM values of 10 genes screened by MeJA in different tissues." pageId="5" pageNumber="6">Table S2</tableCitation>
|
||
. The
|
||
<superScript id="7C259B8CFFFCFFF4A8C7FE54FA1BFDD9" attach="right" box="[1422,1466,505,520]" fontSize="6" pageId="5" pageNumber="6">2 ΔΔ</superScript>
|
||
Ct method was used to analyze the qRT-PCR data and calculate the relative expression levels (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4AE97FD9BFB46FD98" author="Livak, K J & Schmittgen, T" box="[990,1255,566,585]" pageId="5" pageNumber="6" refId="ref8837" refString="Livak, K J, Schmittgen, T, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 - DDCt method. Methods. https: // doi. org / 10.1006 / meth. 2001.1262." type="book" year="2001">Livak and Schmittgen, 2001</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4AE7BFDC3FB02FD50" blockId="5.[818,1187,622,641]" box="[818,1187,622,641]" pageId="5" pageNumber="6">
|
||
<heading id="D0A781A8FFFCFFF4AE7BFDC3FB02FD50" bold="true" box="[818,1187,622,641]" fontSize="36" level="1" pageId="5" pageNumber="6" reason="1">
|
||
<emphasis id="B924EAD6FFFCFFF4AE7BFDC3FB02FD50" bold="true" box="[818,1187,622,641]" italics="true" pageId="5" pageNumber="6">5.5. Plant expression vector construction</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4AE18FD0BFA90FCB1" blockId="5.[818,1487,678,1060]" pageId="5" pageNumber="6">
|
||
To construct the Sm
|
||
<emphasis id="B924EAD6FFFCFFF4A958FD0BFBEFFD68" bold="true" box="[1041,1102,678,697]" italics="true" pageId="5" pageNumber="6">
|
||
-
|
||
<collectionCode id="ED41AE01FFFCFFF4A95EFD0BFBE5FD68" box="[1047,1092,678,697]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
|
||
2
|
||
</emphasis>
|
||
-overexpression vector, the gene-specific primers Sm-NAC2-OE-F and Sm-NAC2-OE-R were used to amplify the complete ORF of Sm
|
||
<emphasis id="B924EAD6FFFCFFF4AEBAFD70FB96FD21" bold="true" box="[1011,1079,733,752]" italics="true" pageId="5" pageNumber="6">
|
||
-
|
||
<collectionCode id="ED41AE01FFFCFFF4AEB0FD70FB89FD21" box="[1017,1064,733,752]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
|
||
2.
|
||
</emphasis>
|
||
Gateway technology was used to construct the expression vector. First, the ORF of Sm
|
||
<emphasis id="B924EAD6FFFCFFF4A987FD54FAAAFCDD" bold="true" box="[1230,1291,761,780]" italics="true" pageId="5" pageNumber="6">
|
||
-
|
||
<collectionCode id="ED41AE01FFFCFFF4A99DFD54FAA0FCDD" box="[1236,1281,761,780]" country="Japan" httpUri="http://biocol.org/urn:lsid:biocol.org:col:13686" lsid="urn:lsid:biocol.org:col:13686" name="Nagano Environmental Conservation Research Institute" pageId="5" pageNumber="6" type="Herbarium">NAC</collectionCode>
|
||
2
|
||
</emphasis>
|
||
was cloned into the pDONR207 entry vector using the BP Clonase Enzyme Kit, and then, it was cloned into the pK7WG2R destination vector using an LR Clonase Enzyme Kit (Invitrogen, MA,
|
||
<collectingCountry id="F3477654FFFCFFF4A90EFCE0FBD3FCB1" box="[1095,1138,845,864]" name="United States of America" pageId="5" pageNumber="6">USA</collectingCountry>
|
||
) (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4A9CFFCE0FA85FCB1" author="Ding, K. & Pei, T. & Bai, Z. & Jia, Y. & Ma, P. & Liang, Z." box="[1158,1316,845,864]" pageId="5" pageNumber="6" refId="ref7371" refString="Ding, K., Pei, T., Bai, Z., Jia, Y., Ma, P., Liang, Z., 2017. SmMYB 36, a novel R 2 R 3 - MYB transcription factor, enhances tanshinone accumulation and decreases phenolic acid content in Salvia miltiorrhiza transgenic roots. Sci. Rep. 7 (1) https: // doi. org / 10.1038 / s 41598 - 017 - 04909 - w, 5104 - 5104." type="journal volume" year="2017">Ding et al., 2017</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4AE18FCC4FC28FBF5" blockId="5.[818,1487,678,1060]" pageId="5" pageNumber="6">
|
||
A 116-bp sequence was amplified using the primers Sm-NAC2-RNAi- F and Sm-NAC2-RNAi-R to construct the plant RNAi vector. The amplified fragment was cloned into the pDONR207 entry vector, and then cloned into the pK7GWIWG2R binary vector, as described by (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4A8EAFC10FC3BFC3D" author="Ding, K. & Pei, T. & Bai, Z. & Jia, Y. & Ma, P. & Liang, Z." pageId="5" pageNumber="6" refId="ref7371" refString="Ding, K., Pei, T., Bai, Z., Jia, Y., Ma, P., Liang, Z., 2017. SmMYB 36, a novel R 2 R 3 - MYB transcription factor, enhances tanshinone accumulation and decreases phenolic acid content in Salvia miltiorrhiza transgenic roots. Sci. Rep. 7 (1) https: // doi. org / 10.1038 / s 41598 - 017 - 04909 - w, 5104 - 5104." type="journal volume" year="2017">Ding et al., 2017</bibRefCitation>
|
||
). The recombinant vector was confirmed by sequencing. The primers used in this experiment are provided in Supplementary
|
||
<tableCitation id="C6D2037FFFFCFFF4AE7BFBBDFC22FBF5" box="[818,899,1040,1060]" captionStart="Table 2" captionStartId="3.[818,868,150,166]" captionTargetPageId="3" captionText="Table 2 FPKM values of 10 genes screened by MeJA in different tissues." pageId="5" pageNumber="6">Table S2</tableCitation>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4AE7BFBE5FAF8FB8A" blockId="5.[818,1369,1096,1115]" box="[818,1369,1096,1115]" pageId="5" pageNumber="6">
|
||
<heading id="D0A781A8FFFCFFF4AE7BFBE5FAF8FB8A" bold="true" box="[818,1369,1096,1115]" fontSize="36" level="1" pageId="5" pageNumber="6" reason="1">
|
||
<emphasis id="B924EAD6FFFCFFF4AE7BFBE5FAF8FB8A" bold="true" box="[818,1369,1096,1115]" italics="true" pageId="5" pageNumber="6">
|
||
5.6. Acquisition of
|
||
<taxonomicName id="4C504D47FFFCFFF4AEACFBE5FBC4FB8A" box="[997,1125,1096,1115]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="subSpecies" species="miltiorrhiza" subSpecies="transgenic">S. miltiorrhiza</taxonomicName>
|
||
transgenic transgenic roots
|
||
</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4AE18FB2DFA85F910" blockId="5.[818,1488,1152,1897]" pageId="5" pageNumber="6">
|
||
The leaves of the sterile seedlings of
|
||
<taxonomicName id="4C504D47FFFCFFF4A9D6FB2DFABDFB42" box="[1183,1308,1152,1171]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B924EAD6FFFCFFF4A9D6FB2DFABDFB42" bold="true" box="[1183,1308,1152,1171]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
were cut into small pieces of 1
|
||
<emphasis id="B924EAD6FFFCFFF4AEEFFB31FC15FB7E" box="[934,948,1180,1199]" italics="true" pageId="5" pageNumber="6">×</emphasis>
|
||
<quantity id="4CA89B21FFFCFFF4AE89FB31FC53FB7E" box="[960,1010,1180,1199]" metricMagnitude="-2" metricUnit="m" metricValue="1.0" pageId="5" pageNumber="6" unit="cm" value="1.0">1 cm</quantity>
|
||
and placed them on a 1/2MS solid medium for cultivating in the dark for 2–3 days at 25
|
||
<emphasis id="B924EAD6FFFCFFF4A99EFB18FB7EFB13" box="[1239,1247,1205,1218]" italics="true" pageId="5" pageNumber="6">
|
||
<superScript id="7C259B8CFFFCFFF4A99EFB18FB7EFB13" attach="right" box="[1239,1247,1205,1218]" fontSize="6" pageId="5" pageNumber="6">◦</superScript>
|
||
</emphasis>
|
||
C. Single colonies of the
|
||
<taxonomicName id="4C504D47FFFCFFF4AE7BFB79FC08FB37" box="[818,937,1235,1255]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="rhizogenes">
|
||
<emphasis id="B924EAD6FFFCFFF4AE7BFB79FC08FB37" bold="true" box="[818,937,1235,1255]" italics="true" pageId="5" pageNumber="6">A. rhizogenes</emphasis>
|
||
</taxonomicName>
|
||
cells harboring the recombinant plasmid were inoculated into 50 ml of liquid YEB medium with
|
||
<quantity id="4CA89B21FFFCFFF4A9D7FB42FB78FAD2" box="[1182,1241,1263,1283]" metricMagnitude="-5" metricUnit="kg" metricValue="5.0" pageId="5" pageNumber="6" unit="mg" value="50.0">50 mg</quantity>
|
||
l
|
||
<superScript id="7C259B8CFFFCFFF4A9BBFB47FB5AFB29" attach="right" box="[1266,1275,1258,1272]" fontSize="6" pageId="5" pageNumber="6">1</superScript>
|
||
of spectinomycin, and grown on a shaker at 28
|
||
<emphasis id="B924EAD6FFFCFFF4A95DFAA5FBBDFAC4" box="[1044,1052,1288,1301]" italics="true" pageId="5" pageNumber="6">
|
||
<superScript id="7C259B8CFFFCFFF4A95DFAA5FBBDFAC4" attach="right" box="[1044,1052,1288,1301]" fontSize="6" pageId="5" pageNumber="6">◦</superScript>
|
||
</emphasis>
|
||
C for 16–18 h until the OD
|
||
<quantity id="4CA89B21FFFCFFF4A85AFAA6FAF6FACE" box="[1299,1367,1291,1311]" metricMagnitude="-7" metricUnit="m" metricValue="6.0" pageId="5" pageNumber="6" unit="nm" value="600.0">600 nm</quantity>
|
||
reached 0.6. Cells were collected by centrifugation, and re-suspended in 50 ml of liquid 1/2MS medium. Next, the leaf discs were submerged and shaken in the suspension for 30 min with 100 rpm at 25
|
||
<emphasis id="B924EAD6FFFCFFF4A9B3FAF1FAA3FAB8" box="[1274,1282,1372,1385]" italics="true" pageId="5" pageNumber="6">
|
||
<superScript id="7C259B8CFFFCFFF4A9B3FAF1FAA3FAB8" attach="right" box="[1274,1282,1372,1385]" fontSize="6" pageId="5" pageNumber="6">◦</superScript>
|
||
</emphasis>
|
||
C. Then, the leaf discs were taken out and cultured on 1/2 MS solid medium for 3 d in the dark. The leaf discs were moved onto 1/2 MS selection solid medium with
|
||
<quantity id="4CA89B21FFFCFFF4A8FEFA3AFCEEFA17" metricMagnitude="-5" metricUnit="kg" metricValue="5.0" pageId="5" pageNumber="6" unit="mg" value="50.0">50 mg</quantity>
|
||
l
|
||
<superScript id="7C259B8CFFFCFFF4AE23FA00FCD2FA6A" attach="right" box="[874,883,1453,1467]" fontSize="6" pageId="5" pageNumber="6">1</superScript>
|
||
kanamycin and reduced cefotaxime. The sterilization medium was changed once in 10–15 d, and the concentration of cefotaxime in the medium was gradually reduced: from
|
||
<quantity id="4CA89B21FFFCFFF4A9EEFA47FB50FA2F" box="[1191,1265,1514,1534]" metricMagnitude="-4" metricUnit="kg" metricValue="5.0" pageId="5" pageNumber="6" unit="mg" value="500.0">500 mg</quantity>
|
||
l
|
||
<superScript id="7C259B8CFFFCFFF4A844FA48FAB7FA22" attach="right" box="[1293,1302,1509,1523]" fontSize="6" pageId="5" pageNumber="6">1</superScript>
|
||
to zero. When the transgenic roots grew to
|
||
<quantity id="4CA89B21FFFCFFF4A950F9AAFBFEF9CB" box="[1049,1119,1542,1562]" metricMagnitude="-2" metricUnit="m" metricValue="4.5" metricValueMax="5.0" metricValueMin="4.0" pageId="5" pageNumber="6" unit="cm" value="4.5" valueMax="5.0" valueMin="4.0">4–5 cm</quantity>
|
||
, and a single root was cut from the leaf and placed on a sterile medium for individual culture. The rapidly growing kanamycin-resistant and agrobacterium-free transgenic roots were transferred to 50 ml of liquid 1/2 MS medium and maintained by transferring
|
||
<quantity id="4CA89B21FFFCFFF4AEEEF9DBFC76F958" box="[935,983,1654,1673]" metricMagnitude="-4" metricUnit="kg" metricValue="3.0" pageId="5" pageNumber="6" unit="g" value="0.3">0.3 g</quantity>
|
||
of root material into fresh 1/2 MS medium every 30 d (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4AE03F93FFC71F974" author="Ru, M. & An, Y. & Wang, K. & Peng, L. & Li, B. & Bai, Z. & Wang, B. & Liang, Z." box="[842,976,1682,1701]" pageId="5" pageNumber="6" pagination="494 - 502" refId="ref9764" refString="Ru, M., An, Y., Wang, K., Peng, L., Li, B., Bai, Z., Wang, B., Liang, Z., 2016. Prunella vulgaris L. transgenic roots: culture, growth, and elicitation by ethephon and salicylic acid. Eng. Life Sci. 16 (5), 494 - 502. https: // doi. org / 10.1002 / elsc. 201600001." type="journal article" year="2016">Ru et al., 2016</bibRefCitation>
|
||
). The WT control was transgenic roots developed using
|
||
<taxonomicName id="4C504D47FFFCFFF4AE7BF903FC09F911" authority="ATCC" authorityName="ATCC" box="[818,936,1709,1729]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="rhizogenes">
|
||
<emphasis id="B924EAD6FFFCFFF4AE7BF903FC09F911" bold="true" box="[818,936,1709,1729]" italics="true" pageId="5" pageNumber="6">A. rhizogenes</emphasis>
|
||
</taxonomicName>
|
||
ATCC15834 not harboring the plasmid.
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFCFFF4AE18F967FB4AF8B9" blockId="5.[818,1488,1152,1897]" pageId="5" pageNumber="6">
|
||
The genomic DNA from fresh transgenic roots was isolated use the cetyltrimethylammonium bromide (CTAB) method (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4A809F94BFC05F8C4" author="Sambrock, J. & Russel, D. W." pageId="5" pageNumber="6" refId="ref9935" refString="Sambrock, J., Russel, D. W., 2001. Molecular Cloning, third ed. A Laboratory Manual. https: // doi. org / 10.2307 / 1309366." type="book" year="2001">Sambrock and Russel 2001</bibRefCitation>
|
||
). Four pairs of specific primers were used to identify positive transgenic strains (Supplementary
|
||
<tableCitation id="C6D2037FFFFCFFF4A9EFF8B0FB59F8E0" box="[1190,1272,1821,1841]" captionStart="Table 2" captionStartId="3.[818,868,150,166]" captionTargetPageId="3" captionText="Table 2 FPKM values of 10 genes screened by MeJA in different tissues." pageId="5" pageNumber="6">Table S2</tableCitation>
|
||
). The identified transgenic transgenic roots were cultured as described previously to further study Sm
|
||
<emphasis id="B924EAD6FFFCFFF4AEC1F8F8FC64F8B9" bold="true" box="[904,965,1877,1896]" italics="true" pageId="5" pageNumber="6">-NAC2</emphasis>
|
||
functions (
|
||
<bibRefCitation id="EFC14B35FFFCFFF4A978F8F8FB7CF8B9" author="Zhang, C. & Xing, B. & Yang, D. & Ren, M. & Guo, H. & Yang, S. & Liang, Z." box="[1073,1245,1877,1897]" pageId="5" pageNumber="6" pagination="112183" refId="ref11247" refString="Zhang, C., Xing, B., Yang, D., Ren, M., Guo, H., Yang, S., Liang, Z., 2020. SmbHLH 3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza transgenic roots. Phytochemistry 169, 112183. https: // doi. org / 10.1016 / j. phytochem. 2019.112183." type="journal article" year="2020">Zhang et al., 2020</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFFFFF7AD2DFF39FD82FF77" blockId="6.[100,547,147,167]" box="[100,547,147,167]" pageId="6" pageNumber="7">
|
||
<heading id="D0A781A8FFFFFFF7AD2DFF39FD82FF77" bold="true" box="[100,547,147,167]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B924EAD6FFFFFFF7AD2DFF39FD82FF77" bold="true" box="[100,547,147,167]" italics="true" pageId="6" pageNumber="7">5.7. Extraction and determination of tanshinones</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFFFFF7ADCDFF66FDD5FE0B" blockId="6.[100,770,200,474]" pageId="6" pageNumber="7">
|
||
The sampled transgenic roots were placed in an oven at 45
|
||
<emphasis id="B924EAD6FFFFFFF7AFF0FF65FD60FF04" box="[697,705,200,213]" italics="true" pageId="6" pageNumber="7">
|
||
<superScript id="7C259B8CFFFFFFF7AFF0FF65FD60FF04" attach="right" box="[697,705,200,213]" fontSize="6" pageId="6" pageNumber="7">◦</superScript>
|
||
</emphasis>
|
||
<collectionCode id="ED41AE01FFFFFFF7AF8BFF61FD6EFF0E" box="[706,719,204,223]" country="Denmark" name="University of Copenhagen" pageId="6" pageNumber="7" type="Herbarium">C</collectionCode>
|
||
until they were completely dehydrated, and then, the dried
|
||
<taxonomicName id="4C504D47FFFFFFF7AFC9FF4AFCA3FF2B" box="[640,770,231,250]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="6" pageNumber="7" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B924EAD6FFFFFFF7AFC9FF4AFCA3FF2B" bold="true" box="[640,770,231,250]" italics="true" pageId="6" pageNumber="7">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
samples were crushed into a powder using a grinder. In total,
|
||
<quantity id="4CA89B21FFFFFFF7AFE7FEAEFD48FEC6" box="[686,745,259,279]" metricMagnitude="-5" metricUnit="kg" metricValue="2.0" pageId="6" pageNumber="7" unit="g" value="0.02">0.02 g</quantity>
|
||
of sample powder was placed into 2 mL of 70% methanol. After soaking overnight in the dark, the sample was subjected to ultrasound for 45 min and then centrifuged at
|
||
<quantity id="4CA89B21FFFFFFF7AC17FEFAFE04FEBB" box="[350,421,343,362]" metricMagnitude="0" metricUnit="kg" metricValue="8.0" pageId="6" pageNumber="7" unit="g" value="8000.0">8000 g</quantity>
|
||
for 10 min. The supernatant was removed and filtered through a 0.45 μm membrane. Afterward, 10 μL of the sample was used for HPLC detection on a Waters HPLC e2695system (Waters, Milford, MA,
|
||
<collectingCountry id="F3477654FFFFFFF7AC0BFE06FED1FE6F" box="[322,368,427,446]" name="United States of America" pageId="6" pageNumber="7">USA</collectingCountry>
|
||
). The HPLC conditions were those established previously in our laboratory (
|
||
<bibRefCitation id="EFC14B35FFFFFFF7ACF3FE6AFDC6FE0B" author="Zhang, C. & Xing, B. & Yang, D. & Ren, M. & Guo, H. & Yang, S. & Liang, Z." box="[442,615,455,474]" pageId="6" pageNumber="7" pagination="112183" refId="ref11247" refString="Zhang, C., Xing, B., Yang, D., Ren, M., Guo, H., Yang, S., Liang, Z., 2020. SmbHLH 3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza transgenic roots. Phytochemistry 169, 112183. https: // doi. org / 10.1016 / j. phytochem. 2019.112183." type="journal article" year="2020">Zhang et al., 2020</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFFFFF7AD2DFDA5FE29FDCA" blockId="6.[100,392,520,539]" box="[100,392,520,539]" pageId="6" pageNumber="7">
|
||
<heading id="D0A781A8FFFFFFF7AD2DFDA5FE29FDCA" bold="true" box="[100,392,520,539]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B924EAD6FFFFFFF7AD2DFDA5FE29FDCA" bold="true" box="[100,392,520,539]" italics="true" pageId="6" pageNumber="7">5.8. Data statistics and analysis</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BEF36C4FFFFFFF7ADCDFDEDFF68FD12" blockId="6.[100,770,576,707]" pageId="6" pageNumber="7">
|
||
All the experiments were performed three times, and summary statistics are presented as means
|
||
<emphasis id="B924EAD6FFFFFFF7ACC6FDF1FE02FDBE" box="[399,419,604,623]" italics="true" pageId="6" pageNumber="7">±</emphasis>
|
||
standard deviations (SDs). One-way ANOVAs (followed by a Tukey’ s comparisons) were used to test for significant differences among the means (indicated by different letters at
|
||
<emphasis id="B924EAD6FFFFFFF7AD2DFD1DFF1AFD13" bold="true" box="[100,187,687,707]" italics="true" pageId="6" pageNumber="7">P <0.05</emphasis>
|
||
).
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |