Silencing of the CaCP Gene Delays Salt- and Osmotic-Induced Leaf Senescence in Capsicum annuum L.
Identifieur interne : 000F86 ( Pmc/Curation ); précédent : 000F85; suivant : 000F87Silencing of the CaCP Gene Delays Salt- and Osmotic-Induced Leaf Senescence in Capsicum annuum L.
Auteurs : Huai-Juan Xiao [République populaire de Chine] ; Yan-Xu Yin [République populaire de Chine] ; Wei-Guo Chai ; Zhen-Hui Gong [République populaire de Chine]Source :
- International Journal of Molecular Sciences [ 1422-0067 ] ; 2014.
Abstract
Cysteine proteinases have been known to participate in developmental processes and in response to stress in plants. Our present research reported that a novel CP gene,
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DOI: 10.3390/ijms15058316
PubMed: 24823878
PubMed Central: 4057733
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Silencing of the <italic>CaCP</italic> Gene Delays Salt- and Osmotic-Induced Leaf Senescence in <italic>Capsicum annuum</italic> L.</title><author><name sortKey="Xiao, Huai Juan" sort="Xiao, Huai Juan" uniqKey="Xiao H" first="Huai-Juan" last="Xiao">Huai-Juan Xiao</name><affiliation><nlm:aff id="af1-ijms-15-08316">College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China; E-Mails:<email>yuanyi041ban@126.com</email> (H.-J.X.);<email>yinyanxu2008@126.com</email> (Y.-X.Y.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijms-15-08316">State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi</wicri:regionArea></affiliation></author><author><name sortKey="Yin, Yan Xu" sort="Yin, Yan Xu" uniqKey="Yin Y" first="Yan-Xu" last="Yin">Yan-Xu Yin</name><affiliation><nlm:aff id="af1-ijms-15-08316">College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China; E-Mails:<email>yuanyi041ban@126.com</email> (H.-J.X.);<email>yinyanxu2008@126.com</email> (Y.-X.Y.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijms-15-08316">State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi</wicri:regionArea></affiliation></author><author><name sortKey="Chai, Wei Guo" sort="Chai, Wei Guo" uniqKey="Chai W" first="Wei-Guo" last="Chai">Wei-Guo Chai</name><affiliation><nlm:aff id="af3-ijms-15-08316">Institute of Vegetables, Hangzhou Academy of Agricultural Sciences, Hangzhou 311104, Zhejiang, China; E-Mail:<email>kuni@21cn.com</email></nlm:aff></affiliation></author><author><name sortKey="Gong, Zhen Hui" sort="Gong, Zhen Hui" uniqKey="Gong Z" first="Zhen-Hui" last="Gong">Zhen-Hui Gong</name><affiliation><nlm:aff id="af1-ijms-15-08316">College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China; E-Mails:<email>yuanyi041ban@126.com</email> (H.-J.X.);<email>yinyanxu2008@126.com</email> (Y.-X.Y.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijms-15-08316">State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24823878</idno><idno type="pmc">4057733</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057733</idno><idno type="RBID">PMC:4057733</idno><idno type="doi">10.3390/ijms15058316</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000F87</idno><idno type="wicri:Area/Pmc/Curation">000F86</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Silencing of the <italic>CaCP</italic> Gene Delays Salt- and Osmotic-Induced Leaf Senescence in <italic>Capsicum annuum</italic> L.</title><author><name sortKey="Xiao, Huai Juan" sort="Xiao, Huai Juan" uniqKey="Xiao H" first="Huai-Juan" last="Xiao">Huai-Juan Xiao</name><affiliation><nlm:aff id="af1-ijms-15-08316">College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China; E-Mails:<email>yuanyi041ban@126.com</email> (H.-J.X.);<email>yinyanxu2008@126.com</email> (Y.-X.Y.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijms-15-08316">State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi</wicri:regionArea></affiliation></author><author><name sortKey="Yin, Yan Xu" sort="Yin, Yan Xu" uniqKey="Yin Y" first="Yan-Xu" last="Yin">Yan-Xu Yin</name><affiliation><nlm:aff id="af1-ijms-15-08316">College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China; E-Mails:<email>yuanyi041ban@126.com</email> (H.-J.X.);<email>yinyanxu2008@126.com</email> (Y.-X.Y.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijms-15-08316">State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi</wicri:regionArea></affiliation></author><author><name sortKey="Chai, Wei Guo" sort="Chai, Wei Guo" uniqKey="Chai W" first="Wei-Guo" last="Chai">Wei-Guo Chai</name><affiliation><nlm:aff id="af3-ijms-15-08316">Institute of Vegetables, Hangzhou Academy of Agricultural Sciences, Hangzhou 311104, Zhejiang, China; E-Mail:<email>kuni@21cn.com</email></nlm:aff></affiliation></author><author><name sortKey="Gong, Zhen Hui" sort="Gong, Zhen Hui" uniqKey="Gong Z" first="Zhen-Hui" last="Gong">Zhen-Hui Gong</name><affiliation><nlm:aff id="af1-ijms-15-08316">College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China; E-Mails:<email>yuanyi041ban@126.com</email> (H.-J.X.);<email>yinyanxu2008@126.com</email> (Y.-X.Y.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijms-15-08316">State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi</wicri:regionArea></affiliation></author></analytic><series><title level="j">International Journal of Molecular Sciences</title><idno type="eISSN">1422-0067</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>Cysteine proteinases have been known to participate in developmental processes and in response to stress in plants. Our present research reported that a novel CP gene, <italic>CaCP</italic>, was involved in leaf senescence in pepper (<italic>Capsicum annuum</italic> L.). The full-length <italic>CaCP</italic> cDNA is comprised of 1316 bp, contains 1044 nucleotides in open reading frame (ORF), and encodes a 347 amino acid protein. The deduced protein belongs to the papain-like cysteine proteases (CPs) superfamily, containing a highly conserved ERFNIN motif, a GCNGG motif and a conserved catalytic triad. This protein localized to the vacuole of plant cells. Real-time quantitative PCR analysis revealed that the expression level of <italic>CaCP</italic> gene was dramatically higher in leaves and flowers than that in roots, stems and fruits. Moreover, <italic>CaCP</italic> transcripts were induced upon during leaf senescence. <italic>CaCP</italic> expression was upregulated by plant hormones, especially salicylic acid. <italic>CaCP</italic> was also significantly induced by abiotic and biotic stress treatments, including high salinity, mannitol and <italic>Phytophthora capsici.</italic> Loss of function of <italic>CaCP</italic> using the virus-induced gene-silencing technique in pepper plants led to enhanced tolerance to salt- and osmotic-induced stress. Taken together, these results suggest that <italic>CaCP</italic> is a senescence-associated gene, which is involved in developmental senescence and regulates salt- and osmotic-induced leaf senescence in pepper.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Smart, C" uniqKey="Smart C">C. Smart</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lim, P O" uniqKey="Lim P">P.O. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Mol Sci</journal-id><journal-id journal-id-type="iso-abbrev">Int J Mol Sci</journal-id><journal-id journal-id-type="publisher-id">ijms</journal-id><journal-title-group><journal-title>International Journal of Molecular Sciences</journal-title></journal-title-group><issn pub-type="epub">1422-0067</issn><publisher><publisher-name>Molecular Diversity Preservation International (MDPI)</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24823878</article-id><article-id pub-id-type="pmc">4057733</article-id><article-id pub-id-type="doi">10.3390/ijms15058316</article-id><article-id pub-id-type="publisher-id">ijms-15-08316</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Silencing of the <italic>CaCP</italic> Gene Delays Salt- and Osmotic-Induced Leaf Senescence in <italic>Capsicum annuum</italic> L.</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Xiao</surname><given-names>Huai-Juan</given-names></name><xref ref-type="aff" rid="af1-ijms-15-08316">1</xref><xref ref-type="aff" rid="af2-ijms-15-08316">2</xref></contrib><contrib contrib-type="author"><name><surname>Yin</surname><given-names>Yan-Xu</given-names></name><xref ref-type="aff" rid="af1-ijms-15-08316">1</xref><xref ref-type="aff" rid="af2-ijms-15-08316">2</xref></contrib><contrib contrib-type="author"><name><surname>Chai</surname><given-names>Wei-Guo</given-names></name><xref ref-type="aff" rid="af3-ijms-15-08316">3</xref></contrib><contrib contrib-type="author"><name><surname>Gong</surname><given-names>Zhen-Hui</given-names></name><xref ref-type="aff" rid="af1-ijms-15-08316">1</xref><xref ref-type="aff" rid="af2-ijms-15-08316">2</xref><xref rid="c1-ijms-15-08316" ref-type="corresp">*</xref></contrib></contrib-group><aff id="af1-ijms-15-08316"><label>1</label>College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China; E-Mails:<email>yuanyi041ban@126.com</email> (H.-J.X.);<email>yinyanxu2008@126.com</email> (Y.-X.Y.)</aff><aff id="af2-ijms-15-08316"><label>2</label>State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China</aff><aff id="af3-ijms-15-08316"><label>3</label>Institute of Vegetables, Hangzhou Academy of Agricultural Sciences, Hangzhou 311104, Zhejiang, China; E-Mail:<email>kuni@21cn.com</email></aff><author-notes><corresp id="c1-ijms-15-08316"><label>*</label>Author to whom correspondence should be addressed; E-Mail: <email>zhgong@nwsuaf.edu.cn</email>; Tel.: +86-29-8708-2102; Fax: +86-29-8708-2613.</corresp></author-notes><pub-date pub-type="collection"><month>5</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>12</day><month>5</month><year>2014</year></pub-date><volume>15</volume><issue>5</issue><fpage>8316</fpage><lpage>8334</lpage><history><date date-type="received"><day>28</day><month>3</month><year>2014</year></date><date date-type="rev-recd"><day>23</day><month>4</month><year>2014</year></date><date date-type="accepted"><day>28</day><month>4</month><year>2014</year></date></history><permissions><copyright-statement>© 2014 by the authors; licensee MDPI, Basel, Switzerland</copyright-statement><copyright-year>2014</copyright-year><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">http://creativecommons.org/licenses/by/3.0/</ext-link>).</license-p></license></permissions><abstract><p>Cysteine proteinases have been known to participate in developmental processes and in response to stress in plants. Our present research reported that a novel CP gene, <italic>CaCP</italic>, was involved in leaf senescence in pepper (<italic>Capsicum annuum</italic> L.). The full-length <italic>CaCP</italic> cDNA is comprised of 1316 bp, contains 1044 nucleotides in open reading frame (ORF), and encodes a 347 amino acid protein. The deduced protein belongs to the papain-like cysteine proteases (CPs) superfamily, containing a highly conserved ERFNIN motif, a GCNGG motif and a conserved catalytic triad. This protein localized to the vacuole of plant cells. Real-time quantitative PCR analysis revealed that the expression level of <italic>CaCP</italic> gene was dramatically higher in leaves and flowers than that in roots, stems and fruits. Moreover, <italic>CaCP</italic> transcripts were induced upon during leaf senescence. <italic>CaCP</italic> expression was upregulated by plant hormones, especially salicylic acid. <italic>CaCP</italic> was also significantly induced by abiotic and biotic stress treatments, including high salinity, mannitol and <italic>Phytophthora capsici.</italic> Loss of function of <italic>CaCP</italic> using the virus-induced gene-silencing technique in pepper plants led to enhanced tolerance to salt- and osmotic-induced stress. Taken together, these results suggest that <italic>CaCP</italic> is a senescence-associated gene, which is involved in developmental senescence and regulates salt- and osmotic-induced leaf senescence in pepper.</p></abstract><kwd-group><kwd>pepper</kwd><kwd>cysteine proteinase</kwd><kwd>leaf senescence</kwd><kwd>osmotic stresses</kwd></kwd-group></article-meta></front><floats-group><fig id="f1-ijms-15-08316" position="float"><label>Figure 1.</label><caption><p>Gene structure of <italic>CaCP</italic> and multiple sequence alignment of the CaCP protein and other plant cysteine proteases (CPs). (<bold>a</bold>) Schematic representation the full-length DNA sequence of <italic>CaCP</italic>. The <italic>CaCP</italic> gene consists of two introns and three exons. Orang boxes represent exon sequences, blue lines between the exons indicate introns and both ends of the lines correspond to 5′- and 3′-UTR; (<bold>b</bold>) Multiple sequence alignment of CaCP and other plant CPs using DNAMAN software (Lynnon Biosoft, Vaudreuil, QC, Canada). Dark-blue shading and gray shading reflect 100% and 75% amino acid residues conservation, respectively. The arrow indicates the cleavage site of the signal peptide; the double line indicates the ERFNIN motif; the single line indicates the GCNGG motif, and the catalytic triad Cys, His, and Asn and also the Glu active site residue are indicated by the asterisks. Besides CaCP, other amino acid sequences included in this alignment were NtCP1 (ADV41672.1), NtSAG12 (AAW78661.2), TcCP (EOY28020.1), RcCP (XP_002523447.1), MtCP (XP_003612210.1), AgCP (AAA50755.1) and AtSAG12 (AAC49135.1). Ca, <italic>Capsicum annuum</italic> L; Nt, <italic>Nicotiana tabacum</italic>; Tc, <italic>Theobroma cacao</italic>; Rc, <italic>Ricinus communis</italic>; Dc, <italic>Daucus carota</italic>; Mt, <italic>Medicago truncatula</italic>; Ag, <italic>Alnus glutinosa</italic>; At, <italic>Arabidopsis thaliana</italic>.</p></caption><graphic xlink:href="ijms-15-08316f1"></graphic></fig><fig id="f2-ijms-15-08316" position="float"><label>Figure 2.</label><caption><p>Phylogenetic analysis of CaCP protein and CP proteins of other plant species. The phylogenetic tree was constructed by the neighbor-joining method using MEGA5.05 software (The Biodesign Institute). Branches were labelled with the protein names and GenBank accession numbers. The tree was displayed as a phylogram in which branch lengths are proportional to distance. Numbers on the branches represent bootstrap values (for 1000 replicates). Ca, <italic>Capsicum annuum</italic> L; Nt, <italic>Nicotiana tabacum</italic>; Tc, <italic>Theobroma cacao</italic>; Rc, <italic>Ricinus communis</italic>; Dc, <italic>Daucus carota</italic>; Mt, <italic>Medicago truncatula</italic>; Ag, <italic>Alnus glutinosa</italic>; At, <italic>Arabidopsis thaliana</italic>.</p></caption><graphic xlink:href="ijms-15-08316f2"></graphic></fig><fig id="f3-ijms-15-08316" position="float"><label>Figure 3.</label><caption><p>Schematic representation of the vector PVBG2307-<italic>GFP</italic> and PVBG2307-<italic>CaCP-GFP</italic> used for CaCP subcellular localization (<bold>a</bold>,<bold>b</bold>); and vector TRV2-<italic>CaCP</italic> used for <italic>CaCP</italic> gene silencing (<bold>c</bold>). LB: left borders of the T-DNA; RB: right borders of the T-DNA; CaMV35S: CaMV35S promoter; <italic>CaCP: Capsicum annuum</italic> cysteine proteinase; GFP: green fluorescent protein; nptII: neomycin phosphotransferase; N: nos-terminator; 2 × 35S: two copies of the cauliflower mosaic virus 35S promoter; CP: coat protein; R: ribozyme.</p></caption><graphic xlink:href="ijms-15-08316f3"></graphic></fig><fig id="f4-ijms-15-08316" position="float"><label>Figure 4.</label><caption><p>Subcellular localization of the GFP protein (<bold>upper</bold>) and CaCP-GFP fusion protein (<bold>lower</bold>) in onion epidermal cells. (<bold>a</bold>) The fluorescence images of non-plasmolysed cells; (<bold>b</bold>) The fluorescence images of plasmolysed cells.</p></caption><graphic xlink:href="ijms-15-08316f4"></graphic></fig><fig id="f5-ijms-15-08316" position="float"><label>Figure 5.</label><caption><p>(<bold>a</bold>) Expression pattern of <italic>CaCP</italic> in different pepper tissues. Samples were collected from roots, stems, green leaves, flowers and mature fruits. Relative expression levels of the <italic>CaCP</italic> transcripts were determined in different tissues in comparison to that in roots; (<bold>b</bold>) The expression profiles of <italic>CaCP</italic> during pepper leaf developmental stage. RNA was extracted from young leaves, fully expanded leaves and senescent leaves, respectively. Error bars represent SD for three biological replicates. Asterisks indicate a significant difference (<italic>p</italic> < 0.05) from the roots.</p></caption><graphic xlink:href="ijms-15-08316f5"></graphic></fig><fig id="f6-ijms-15-08316" position="float"><label>Figure 6.</label><caption><p>Expression profiles of <italic>CaCP</italic> in response to biotic and abiotic stresses including salt stress (<bold>a</bold>); osmotic stress (<bold>b</bold>); exogenous phytohormones (<bold>c</bold>) and pathogen infection (<bold>d</bold>). The relative transcriptional expression of <italic>CaCP</italic> was calculated in various treated leaves in comparison to that in the mock controls (0 h). Error bars represent SD for three biological replicates. Asterisks indicate a significant difference (<italic>p</italic> < 0.05) from 0 h p.t. or 0 h p.i.</p></caption><graphic xlink:href="ijms-15-08316f6"></graphic></fig><fig id="f7-ijms-15-08316" position="float"><label>Figure 7.</label><caption><p>Efficiency of <italic>CaCP</italic> in pepper plants using a TRV-based VIGS system. (<bold>a</bold>) Phenotypes of gene-silenced pepper plants 45 days after inoculation. <bold>Left</bold>: <italic>CaCP</italic>-silenced plant (TRV2:<italic>CaCP</italic>); <bold>middle</bold>: <italic>CaPDS</italic>-silenced plant (TRV2:<italic>CaPDS</italic>); <bold>right</bold>: control plant (TRV2:00); (<bold>b</bold>) Quantitative real time-PCR analysis of <italic>CaCP</italic> expression levels in leaves of <italic>CaCP</italic>-silenced plants (TRV2:<italic>CaCP1</italic>, TRV2:<italic>CaCP2</italic> and TRV2:<italic>CaCP3</italic> indicates three biological levels of efficiency of <italic>CaCP</italic> gene silencing) and control plants (TRV2:00) 45 days after inoculation. Error bars represent SD for three biological replicates. Asterisks indicate a significant difference (<italic>p</italic> < 0.05) compared to TRV2:00.</p></caption><graphic xlink:href="ijms-15-08316f7"></graphic></fig><fig id="f8-ijms-15-08316" position="float"><label>Figure 8.</label><caption><p>Enhance tolerance of <italic>CaCP</italic>-silenced pepper plants to salt stress. Phenotypes (<bold>a</bold>); chlorophyll contents (<bold>b</bold>) and MDA contents (<bold>c</bold>) of leaf discs of the gene-silenced plants in response to high salt stress. Leaf discs from the gene-silenced pepper leaves were floated in different concentrations of NaCl solutions (0, 100, 200, 300, 400 and 500 mM) for 3 days at 26 °C with continuous fluorescent lighting. The scale bar represents 1.0 cm. Error bars represent SD for three biological replicates. Asterisks indicate a significant difference (<italic>p</italic> < 0.05) compared to TRV2:00 leaves.</p></caption><graphic xlink:href="ijms-15-08316f8"></graphic></fig><fig id="f9-ijms-15-08316" position="float"><label>Figure 9.</label><caption><p>Enhanced tolerance of <italic>CaCP</italic>-silenced pepper plants to osmotic stress. Phenotypes (<bold>a</bold>); chlorophyll contents (<bold>b</bold>) and MDA contents (<bold>c</bold>) of leaf discs of the gene-silenced plants in response to high osmotic stress. Leaf discs from the gene-silenced pepper leaves were floated in different concentrations of mannitol solutions (0, 100, 200, 300, 400, and 500 mM) for 3 days at 26 °C with continuous fluorescent lighting. The scale bar represents 1.0 cm. Error bars represent SD for three biological replicates. Asterisks indicate a significant difference (<italic>p</italic> < 0.05) compared to TRV2:00 leaves.</p></caption><graphic xlink:href="ijms-15-08316f9"></graphic></fig><table-wrap id="t1-ijms-15-08316" position="float"><label>Table 1.</label><caption><p>Primer sequences in this study.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="bottom" rowspan="1" colspan="1">Primers</th><th align="center" valign="bottom" rowspan="1" colspan="1">Sequence(5′–3′)</th></tr></thead><tbody><tr><td colspan="2" align="center" valign="top" rowspan="1"><italic>Cloning of CaCP cDNA sequence</italic></td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">EST-<italic>CaCP</italic>F</td><td align="center" valign="top" rowspan="1" colspan="1">TACAGGATATGAAGATGTCCCAGC</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">EST-<italic>CaCP</italic>R</td><td align="center" valign="top" rowspan="1" colspan="1">AAGCCTCCATGGCAAGTCC</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">5′RACE-<italic>CaCP</italic>GSP</td><td align="center" valign="top" rowspan="1" colspan="1">GTTTTCACCCCATTTACTGCCCC</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">5′RACE-<italic>CaCP</italic>NGSP</td><td align="center" valign="top" rowspan="1" colspan="1">CCACTGACACTGGTTGATTTGCC</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">3′RACE-<italic>CaCP</italic>GSP</td><td align="center" valign="top" rowspan="1" colspan="1">GATATGAAGATGTCCCAGCCAAC</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">3′RACE-<italic>CaCP</italic>NGSP</td><td align="center" valign="top" rowspan="1" colspan="1">GTGGTGTATTCAGTGGATCATGC</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td colspan="2" align="center" valign="top" rowspan="1"><italic>Cloning of CaCP DNA sequence</italic></td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">DNA-<italic>CaCP</italic>F</td><td align="center" valign="top" rowspan="1" colspan="1">TAGTTGTTCTATAATGGCCTTTGCA</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">DNA-<italic>CaCP</italic>R</td><td align="center" valign="top" rowspan="1" colspan="1">ACAATTTAGAAGCTGGCACCATT</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td colspan="2" align="center" valign="top" rowspan="1"><italic>quantitative real-time PCR</italic></td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">RT-<italic>CaCP</italic>F</td><td align="center" valign="top" rowspan="1" colspan="1">TTGTTCTATAATGGCCTTTGCA</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">RT-<italic>CaCP</italic>R</td><td align="center" valign="top" rowspan="1" colspan="1">TCATAGTGAATTGGACGTGGTG</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"><italic>CaUBI3</italic>F</td><td align="center" valign="top" rowspan="1" colspan="1">TGTCCATCTGCTCTCTGTTG</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"><italic>CaUBI3R</italic></td><td align="center" valign="top" rowspan="1" colspan="1">CACCCCAAGCACAATAAGAC</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td colspan="2" align="center" valign="top" rowspan="1"><italic>Virus-induced gene-silencing (VIGS) vector construction</italic></td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">TRV-<italic>CaCP</italic>F</td><td align="center" valign="top" rowspan="1" colspan="1">GAGTAGCTTCGATTTCCAGTTC</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">TRV-<italic>CaCP</italic>R</td><td align="center" valign="top" rowspan="1" colspan="1">CAAATCCACAACGTATTCACAT</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"><italic>CaPDS</italic>F</td><td align="center" valign="top" rowspan="1" colspan="1">TGTTGTCAAAACTCCAAGGTCTGTA</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"><italic>CaPDS</italic>R</td><td align="center" valign="top" rowspan="1" colspan="1">TTTCTCCCACTTGGTTCACTCTTGT</td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td colspan="2" align="center" valign="top" rowspan="1"><italic>PVBG2307-CaCP-GFP vector construction</italic></td></tr><tr><td colspan="2" align="left" valign="top" rowspan="1"><hr></hr></td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">GFP-<italic>CaCP</italic>F</td><td align="center" valign="top" rowspan="1" colspan="1">TTCTATAATGGCCTTTGCAAACC</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">GFP-<italic>CaCP</italic>R</td><td align="center" valign="top" rowspan="1" colspan="1">GGCAGTGGGATAAGAAGCCTC</td></tr></tbody></table></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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