Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition
Identifieur interne : 001159 ( Pmc/Curation ); précédent : 001158; suivant : 001160Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition
Auteurs : Anna Warzybok ; Magdalena MigockaSource :
- PLoS ONE [ 1932-6203 ] ; 2013.
Abstract
In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target
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DOI: 10.1371/journal.pone.0072887
PubMed: 24058446
PubMed Central: 3772881
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition</title><author><name sortKey="Warzybok, Anna" sort="Warzybok, Anna" uniqKey="Warzybok A" first="Anna" last="Warzybok">Anna Warzybok</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Migocka, Magdalena" sort="Migocka, Magdalena" uniqKey="Migocka M" first="Magdalena" last="Migocka">Magdalena Migocka</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24058446</idno><idno type="pmc">3772881</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772881</idno><idno type="RBID">PMC:3772881</idno><idno type="doi">10.1371/journal.pone.0072887</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">001160</idno><idno type="wicri:Area/Pmc/Curation">001159</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition</title><author><name sortKey="Warzybok, Anna" sort="Warzybok, Anna" uniqKey="Warzybok A" first="Anna" last="Warzybok">Anna Warzybok</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Migocka, Magdalena" sort="Migocka, Magdalena" uniqKey="Migocka M" first="Magdalena" last="Migocka">Magdalena Migocka</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target <italic>CsNRT1.1</italic> gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH<sub>3</sub>). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EFα proved to be the most suitable for normalization of <italic>CsNRT1.1</italic> expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bustin, Sa" uniqKey="Bustin S">SA Bustin</name></author><author><name sortKey="Benes, V" uniqKey="Benes V">V Benes</name></author><author><name sortKey="Nolan, T" uniqKey="Nolan T">T Nolan</name></author><author><name sortKey="Pfaffl, Mw" uniqKey="Pfaffl M">MW Pfaffl</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bustin, Sa" uniqKey="Bustin S">SA Bustin</name></author><author><name sortKey="Benes, V" uniqKey="Benes V">V Benes</name></author><author><name sortKey="Garson, Ja" uniqKey="Garson J">JA Garson</name></author><author><name sortKey="Hellemans, J" uniqKey="Hellemans J">J Hellemans</name></author><author><name sortKey="Huggett, J" uniqKey="Huggett J">J 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24058446</article-id><article-id pub-id-type="pmc">3772881</article-id><article-id pub-id-type="publisher-id">PONE-D-13-14044</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0072887</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition</article-title><alt-title alt-title-type="running-head">Selection of Reliable Reference Genes in Cucumber</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Warzybok</surname><given-names>Anna</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Migocka</surname><given-names>Magdalena</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib></contrib-group><aff id="aff1"><addr-line>Wrocław University, Institute of Experimental Biology, Department of Plant Molecular Physiology, Wroclaw, Poland</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Benos</surname><given-names>Panayiotis V.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>University of Pittsburgh, United States of America</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>anka.warzybok@biol.uni.wroc.pl</email></corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: MM. Performed the experiments: AW. Analyzed the data: AW MM. Contributed reagents/materials/analysis tools: AW MM. Wrote the paper: AW MM.</p></fn></author-notes><pub-date pub-type="collection"><year>2013</year></pub-date><pub-date pub-type="epub"><day>13</day><month>9</month><year>2013</year></pub-date><volume>8</volume><issue>9</issue><elocation-id>e72887</elocation-id><history><date date-type="received"><day>5</day><month>4</month><year>2013</year></date><date date-type="accepted"><day>20</day><month>7</month><year>2013</year></date></history><permissions><copyright-year>2013</copyright-year><copyright-holder>Warzybok, Migocka</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target <italic>CsNRT1.1</italic> gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH<sub>3</sub>). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EFα proved to be the most suitable for normalization of <italic>CsNRT1.1</italic> expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress.</p></abstract><funding-group><funding-statement>The research was mainly supported by the Polish National Science Centre (grant no. N N303 818740). AW was financially supported by the fellowship for young PhD researchers granted by European Social Fund of European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><page-count count="11"></page-count></counts></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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