A versatile zero background T-vector system for gene cloning and functional genomics.
Identifieur interne : 000662 ( Main/Exploration ); précédent : 000661; suivant : 000663A versatile zero background T-vector system for gene cloning and functional genomics.
Auteurs : Songbiao Chen [États-Unis] ; Pattavipha Songkumarn ; Jianli Liu ; Guo-Liang WangSource :
- Plant physiology [ 0032-0889 ] ; 2009.
Descripteurs français
- KwdFr :
- ADN des plantes (composition chimique), Agrobacterium tumefaciens (MeSH), Arabidopsis (génétique), Clonage moléculaire (méthodes), Données de séquences moléculaires (MeSH), Fragmentation de l'ADN (MeSH), Génomique (méthodes), Interférence par ARN (MeSH), Oryza (génétique), Protéines végétales (composition chimique), Protéines végétales (génétique), Protéines végétales (métabolisme), Réaction de polymérisation en chaîne (MeSH), Régions promotrices (génétique) (MeSH), Vecteurs génétiques (MeSH), microARN (MeSH).
- MESH :
- composition chimique : ADN des plantes, Protéines végétales.
- génétique : Arabidopsis, Oryza, Protéines végétales.
- métabolisme : Protéines végétales.
- méthodes : Clonage moléculaire, Génomique.
- Agrobacterium tumefaciens, Données de séquences moléculaires, Fragmentation de l'ADN, Interférence par ARN, Réaction de polymérisation en chaîne, Régions promotrices (génétique), Vecteurs génétiques, microARN.
English descriptors
- KwdEn :
- Agrobacterium tumefaciens (MeSH), Arabidopsis (genetics), Cloning, Molecular (methods), DNA Fragmentation (MeSH), DNA, Plant (chemistry), Genetic Vectors (MeSH), Genomics (methods), MicroRNAs (MeSH), Molecular Sequence Data (MeSH), Oryza (genetics), Plant Proteins (chemistry), Plant Proteins (genetics), Plant Proteins (metabolism), Polymerase Chain Reaction (MeSH), Promoter Regions, Genetic (MeSH), RNA Interference (MeSH).
- MESH :
- chemical , chemistry : DNA, Plant, Plant Proteins.
- genetics : Arabidopsis, Oryza, Plant Proteins.
- chemical , metabolism : Plant Proteins.
- methods : Cloning, Molecular, Genomics.
- Agrobacterium tumefaciens, DNA Fragmentation, Genetic Vectors, MicroRNAs, Molecular Sequence Data, Polymerase Chain Reaction, Promoter Regions, Genetic, RNA Interference.
Abstract
With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.
DOI: 10.1104/pp.109.137125
PubMed: 19403729
PubMed Central: PMC2705043
Affiliations:
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wicri:level="1"><nlm:affiliation>Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210</wicri:regionArea><wicri:noRegion>Ohio 43210</wicri:noRegion></affiliation></author><author><name sortKey="Songkumarn, Pattavipha" sort="Songkumarn, Pattavipha" uniqKey="Songkumarn P" first="Pattavipha" last="Songkumarn">Pattavipha Songkumarn</name></author><author><name sortKey="Liu, Jianli" sort="Liu, Jianli" uniqKey="Liu J" first="Jianli" last="Liu">Jianli Liu</name></author><author><name sortKey="Wang, Guo Liang" sort="Wang, Guo Liang" uniqKey="Wang G" first="Guo-Liang" last="Wang">Guo-Liang Wang</name></author></analytic><series><title level="j">Plant physiology</title><idno type="ISSN">0032-0889</idno><imprint><date when="2009" 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séquences moléculaires (MeSH)</term><term>Fragmentation de l'ADN (MeSH)</term><term>Génomique (méthodes)</term><term>Interférence par ARN (MeSH)</term><term>Oryza (génétique)</term><term>Protéines végétales (composition chimique)</term><term>Protéines végétales (génétique)</term><term>Protéines végétales (métabolisme)</term><term>Réaction de polymérisation en chaîne (MeSH)</term><term>Régions promotrices (génétique) (MeSH)</term><term>Vecteurs génétiques (MeSH)</term><term>microARN (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA, Plant</term><term>Plant Proteins</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>ADN des plantes</term><term>Protéines végétales</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Arabidopsis</term><term>Oryza</term><term>Plant Proteins</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Arabidopsis</term><term>Oryza</term><term>Protéines végétales</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Plant Proteins</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Cloning, Molecular</term><term>Genomics</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Protéines végétales</term></keywords><keywords scheme="MESH" qualifier="méthodes" xml:lang="fr"><term>Clonage moléculaire</term><term>Génomique</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Agrobacterium tumefaciens</term><term>DNA Fragmentation</term><term>Genetic Vectors</term><term>MicroRNAs</term><term>Molecular Sequence Data</term><term>Polymerase Chain Reaction</term><term>Promoter Regions, Genetic</term><term>RNA Interference</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Agrobacterium tumefaciens</term><term>Données de séquences 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Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">19403729</PMID><DateCompleted><Year>2009</Year><Month>09</Month><Day>14</Day></DateCompleted><DateRevised><Year>2019</Year><Month>12</Month><Day>10</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0032-0889</ISSN><JournalIssue CitedMedium="Print"><Volume>150</Volume><Issue>3</Issue><PubDate><Year>2009</Year><Month>Jul</Month></PubDate></JournalIssue><Title>Plant physiology</Title><ISOAbbreviation>Plant Physiol</ISOAbbreviation></Journal><ArticleTitle>A versatile zero background T-vector system for gene cloning and functional genomics.</ArticleTitle><Pagination><MedlinePgn>1111-21</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1104/pp.109.137125</ELocationID><Abstract><AbstractText>With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. 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IdType="pubmed">20507457</ArticleId></ArticleIdList></Reference></ReferenceList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country></list><tree><noCountry><name sortKey="Liu, Jianli" sort="Liu, Jianli" uniqKey="Liu J" first="Jianli" last="Liu">Jianli Liu</name><name sortKey="Songkumarn, Pattavipha" sort="Songkumarn, Pattavipha" uniqKey="Songkumarn P" first="Pattavipha" last="Songkumarn">Pattavipha Songkumarn</name><name sortKey="Wang, Guo Liang" sort="Wang, Guo Liang" uniqKey="Wang G" first="Guo-Liang" last="Wang">Guo-Liang Wang</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Chen, Songbiao" sort="Chen, Songbiao" uniqKey="Chen S" first="Songbiao" last="Chen">Songbiao Chen</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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