Expression of T7-based constructs in tobacco cells.
Identifieur interne : 000180 ( Main/Exploration ); précédent : 000179; suivant : 000181Expression of T7-based constructs in tobacco cells.
Auteurs : Hyukho Sheen [Canada] ; K Andrew White [Canada]Source :
- Biochemical and biophysical research communications [ 1090-2104 ] ; 2018.
Descripteurs français
- KwdFr :
- ARN des plantes (génétique), ARN des plantes (métabolisme), ARN messager (génétique), ARN messager (métabolisme), Agrobacterium (métabolisme), Bactériophage T7 (génétique), Biosynthèse des protéines (MeSH), Cellules végétales (métabolisme), Cytosol (métabolisme), Expression des gènes (MeSH), Noyau de la cellule (métabolisme), Protoplastes (métabolisme), Protéines à fluorescence verte (génétique), Protéines à fluorescence verte (métabolisme), Tabac (cytologie), Transcription génétique (MeSH), Éléments activateurs (génétique) (génétique).
- MESH :
- cytologie : Tabac.
- génétique : ARN des plantes, ARN messager, Bactériophage T7, Protéines à fluorescence verte, Éléments activateurs (génétique).
- métabolisme : ARN des plantes, ARN messager, Agrobacterium, Cellules végétales, Cytosol, Noyau de la cellule, Protoplastes, Protéines à fluorescence verte.
- Biosynthèse des protéines, Expression des gènes, Transcription génétique.
English descriptors
- KwdEn :
- Agrobacterium (metabolism), Bacteriophage T7 (genetics), Cell Nucleus (metabolism), Cytosol (metabolism), Enhancer Elements, Genetic (genetics), Gene Expression (MeSH), Green Fluorescent Proteins (genetics), Green Fluorescent Proteins (metabolism), Plant Cells (metabolism), Protein Biosynthesis (MeSH), Protoplasts (metabolism), RNA, Messenger (genetics), RNA, Messenger (metabolism), RNA, Plant (genetics), RNA, Plant (metabolism), Tobacco (cytology), Transcription, Genetic (MeSH).
- MESH :
- chemical , genetics : Green Fluorescent Proteins, RNA, Messenger, RNA, Plant.
- cytology : Tobacco.
- genetics : Bacteriophage T7, Enhancer Elements, Genetic.
- metabolism : Agrobacterium, Cell Nucleus, Cytosol, Green Fluorescent Proteins, Plant Cells, Protoplasts, RNA, Messenger, RNA, Plant.
- Gene Expression, Protein Biosynthesis, Transcription, Genetic.
Abstract
Bacteriophage T7 promoter and RNA polymerase (T7-Pol) are widely used for recombinant protein expression in bacteria. In plants, there exists conflicting results regarding the efficacy of protein expression from T7-Pol-derived mRNAs. To reconcile these contradictory observations, the expression of green fluorescent protein (GFP) from T7 constructs was evaluated in tobacco protoplasts. T7 constructs transcribed by a nuclearly targeted T7-Pol did not express GFP in plant protoplasts, however T7-Pol lacking a nuclear targeting signal was able to translate cytosolically transcribed mRNAs, but only if the messages contained a viral translation enhancer. GFP expression was further evaluated at the plant level by using agroinfiltration-mediated transient expression system. Unlike for cytosolic expression, nuclear T7 transcripts containing a viral translation enhancer element did not express GFP, and modifications designed to stabilize and facilitate export of T7 transcripts to the cytosol did not improve the expression. We conclude that expression of nuclear T7 constructs is not feasible in tobacco cells, but cytosolic transcription provides an alternative means to over-express RNAs directly in the cytosol.
DOI: 10.1016/j.bbrc.2018.03.123
PubMed: 29555475
Affiliations:
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Electronic address: kawhite@yorku.ca.</nlm:affiliation><country xml:lang="fr">Canada</country><wicri:regionArea>Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3</wicri:regionArea><wicri:noRegion>M3J 1P3</wicri:noRegion></affiliation></author></analytic><series><title level="j">Biochemical and biophysical research communications</title><idno type="eISSN">1090-2104</idno><imprint><date when="2018" type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Agrobacterium (metabolism)</term><term>Bacteriophage T7 (genetics)</term><term>Cell Nucleus (metabolism)</term><term>Cytosol (metabolism)</term><term>Enhancer Elements, Genetic (genetics)</term><term>Gene Expression (MeSH)</term><term>Green Fluorescent Proteins (genetics)</term><term>Green Fluorescent Proteins (metabolism)</term><term>Plant Cells (metabolism)</term><term>Protein Biosynthesis (MeSH)</term><term>Protoplasts (metabolism)</term><term>RNA, Messenger (genetics)</term><term>RNA, Messenger (metabolism)</term><term>RNA, Plant (genetics)</term><term>RNA, Plant (metabolism)</term><term>Tobacco (cytology)</term><term>Transcription, Genetic (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN des plantes (génétique)</term><term>ARN des plantes (métabolisme)</term><term>ARN messager (génétique)</term><term>ARN messager (métabolisme)</term><term>Agrobacterium (métabolisme)</term><term>Bactériophage T7 (génétique)</term><term>Biosynthèse des protéines (MeSH)</term><term>Cellules végétales (métabolisme)</term><term>Cytosol (métabolisme)</term><term>Expression des gènes (MeSH)</term><term>Noyau de la cellule (métabolisme)</term><term>Protoplastes (métabolisme)</term><term>Protéines à fluorescence verte (génétique)</term><term>Protéines à fluorescence verte (métabolisme)</term><term>Tabac (cytologie)</term><term>Transcription génétique (MeSH)</term><term>Éléments activateurs (génétique) (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Green Fluorescent Proteins</term><term>RNA, Messenger</term><term>RNA, Plant</term></keywords><keywords scheme="MESH" qualifier="cytologie" xml:lang="fr"><term>Tabac</term></keywords><keywords scheme="MESH" qualifier="cytology" xml:lang="en"><term>Tobacco</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Bacteriophage T7</term><term>Enhancer Elements, Genetic</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN des plantes</term><term>ARN messager</term><term>Bactériophage T7</term><term>Protéines à fluorescence verte</term><term>Éléments activateurs (génétique)</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Agrobacterium</term><term>Cell Nucleus</term><term>Cytosol</term><term>Green Fluorescent Proteins</term><term>Plant Cells</term><term>Protoplasts</term><term>RNA, Messenger</term><term>RNA, Plant</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ARN des plantes</term><term>ARN messager</term><term>Agrobacterium</term><term>Cellules végétales</term><term>Cytosol</term><term>Noyau de la cellule</term><term>Protoplastes</term><term>Protéines à fluorescence verte</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Gene Expression</term><term>Protein Biosynthesis</term><term>Transcription, Genetic</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Biosynthèse des protéines</term><term>Expression des gènes</term><term>Transcription génétique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Bacteriophage T7 promoter and RNA polymerase (T7-Pol) are widely used for recombinant protein expression in bacteria. In plants, there exists conflicting results regarding the efficacy of protein expression from T7-Pol-derived mRNAs. To reconcile these contradictory observations, the expression of green fluorescent protein (GFP) from T7 constructs was evaluated in tobacco protoplasts. T7 constructs transcribed by a nuclearly targeted T7-Pol did not express GFP in plant protoplasts, however T7-Pol lacking a nuclear targeting signal was able to translate cytosolically transcribed mRNAs, but only if the messages contained a viral translation enhancer. GFP expression was further evaluated at the plant level by using agroinfiltration-mediated transient expression system. Unlike for cytosolic expression, nuclear T7 transcripts containing a viral translation enhancer element did not express GFP, and modifications designed to stabilize and facilitate export of T7 transcripts to the cytosol did not improve the expression. We conclude that expression of nuclear T7 constructs is not feasible in tobacco cells, but cytosolic transcription provides an alternative means to over-express RNAs directly in the cytosol.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">29555475</PMID><DateCompleted><Year>2018</Year><Month>10</Month><Day>09</Day></DateCompleted><DateRevised><Year>2018</Year><Month>10</Month><Day>09</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1090-2104</ISSN><JournalIssue CitedMedium="Internet"><Volume>499</Volume><Issue>2</Issue><PubDate><Year>2018</Year><Month>05</Month><Day>05</Day></PubDate></JournalIssue><Title>Biochemical and biophysical research communications</Title><ISOAbbreviation>Biochem Biophys Res Commun</ISOAbbreviation></Journal><ArticleTitle>Expression of T7-based constructs in tobacco cells.</ArticleTitle><Pagination><MedlinePgn>196-201</MedlinePgn></Pagination><ELocationID EIdType="pii" ValidYN="Y">S0006-291X(18)30615-6</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.bbrc.2018.03.123</ELocationID><Abstract><AbstractText>Bacteriophage T7 promoter and RNA polymerase (T7-Pol) are widely used for recombinant protein expression in bacteria. In plants, there exists conflicting results regarding the efficacy of protein expression from T7-Pol-derived mRNAs. To reconcile these contradictory observations, the expression of green fluorescent protein (GFP) from T7 constructs was evaluated in tobacco protoplasts. T7 constructs transcribed by a nuclearly targeted T7-Pol did not express GFP in plant protoplasts, however T7-Pol lacking a nuclear targeting signal was able to translate cytosolically transcribed mRNAs, but only if the messages contained a viral translation enhancer. GFP expression was further evaluated at the plant level by using agroinfiltration-mediated transient expression system. Unlike for cytosolic expression, nuclear T7 transcripts containing a viral translation enhancer element did not express GFP, and modifications designed to stabilize and facilitate export of T7 transcripts to the cytosol did not improve the expression. We conclude that expression of nuclear T7 constructs is not feasible in tobacco cells, but cytosolic transcription provides an alternative means to over-express RNAs directly in the cytosol.</AbstractText><CopyrightInformation>Copyright © 2018 Elsevier Inc. All rights reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Sheen</LastName><ForeName>Hyukho</ForeName><Initials>H</Initials><AffiliationInfo><Affiliation>Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada. Electronic address: hyukhos@yorku.ca.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>White</LastName><ForeName>K Andrew</ForeName><Initials>KA</Initials><AffiliationInfo><Affiliation>Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada. Electronic address: kawhite@yorku.ca.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2018</Year><Month>03</Month><Day>22</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biochem Biophys Res Commun</MedlineTA><NlmUniqueID>0372516</NlmUniqueID><ISSNLinking>0006-291X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D012333">RNA, Messenger</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D018749">RNA, Plant</NameOfSubstance></Chemical><Chemical><RegistryNumber>147336-22-9</RegistryNumber><NameOfSubstance UI="D049452">Green Fluorescent Proteins</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D060054" MajorTopicYN="N">Agrobacterium</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D017123" MajorTopicYN="N">Bacteriophage T7</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D002467" MajorTopicYN="N">Cell Nucleus</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D003600" MajorTopicYN="N">Cytosol</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004742" MajorTopicYN="N">Enhancer Elements, Genetic</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015870" MajorTopicYN="Y">Gene Expression</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D049452" MajorTopicYN="N">Green Fluorescent Proteins</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D059828" MajorTopicYN="N">Plant Cells</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014176" MajorTopicYN="N">Protein Biosynthesis</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011523" MajorTopicYN="N">Protoplasts</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D012333" MajorTopicYN="N">RNA, Messenger</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D018749" MajorTopicYN="N">RNA, Plant</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014026" MajorTopicYN="N">Tobacco</DescriptorName><QualifierName UI="Q000166" MajorTopicYN="Y">cytology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014158" MajorTopicYN="N">Transcription, Genetic</DescriptorName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">Plant</Keyword><Keyword MajorTopicYN="Y">Recombinant protein expression</Keyword><Keyword MajorTopicYN="Y">T7 RNA polymerase</Keyword><Keyword MajorTopicYN="Y">T7 promoter</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2018</Year><Month>03</Month><Day>13</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2018</Year><Month>03</Month><Day>15</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2018</Year><Month>3</Month><Day>21</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2018</Year><Month>10</Month><Day>10</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2018</Year><Month>3</Month><Day>21</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">29555475</ArticleId><ArticleId IdType="pii">S0006-291X(18)30615-6</ArticleId><ArticleId IdType="doi">10.1016/j.bbrc.2018.03.123</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Canada</li></country></list><tree><country name="Canada"><noRegion><name sortKey="Sheen, Hyukho" sort="Sheen, Hyukho" uniqKey="Sheen H" first="Hyukho" last="Sheen">Hyukho Sheen</name></noRegion><name sortKey="White, K Andrew" sort="White, K Andrew" uniqKey="White K" first="K Andrew" last="White">K Andrew White</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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