Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.
Identifieur interne : 000644 ( Main/Corpus ); précédent : 000643; suivant : 000645Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.
Auteurs : Yun-Qing Cheng ; Jun Yang ; Feng-Ping Xu ; Li-Jia An ; Jian-Feng Liu ; Zhi-Wen ChenSource :
- Applied biochemistry and biotechnology [ 1559-0291 ] ; 2009.
English descriptors
- KwdEn :
- MESH :
- cytology : Onions.
- genetics : Genetic Vectors, Onions, Rhizobium.
- metabolism : Onions.
- Microscopy, Polymerase Chain Reaction, Transformation, Bacterial.
Abstract
A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.
DOI: 10.1007/s12010-009-8554-7
PubMed: 19255730
Links to Exploration step
pubmed:19255730Le document en format XML
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Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">19255730</PMID><DateCompleted><Year>2010</Year><Month>01</Month><Day>12</Day></DateCompleted><DateRevised><Year>2009</Year><Month>10</Month><Day>23</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1559-0291</ISSN><JournalIssue CitedMedium="Internet"><Volume>159</Volume><Issue>3</Issue><PubDate><Year>2009</Year><Month>Dec</Month></PubDate></JournalIssue><Title>Applied biochemistry and biotechnology</Title><ISOAbbreviation>Appl Biochem Biotechnol</ISOAbbreviation></Journal><ArticleTitle>Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.</ArticleTitle><Pagination><MedlinePgn>739-49</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1007/s12010-009-8554-7</ELocationID><Abstract><AbstractText>A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Cheng</LastName><ForeName>Yun-Qing</ForeName><Initials>YQ</Initials><AffiliationInfo><Affiliation>Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, Liaoning, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Yang</LastName><ForeName>Jun</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Xu</LastName><ForeName>Feng-Ping</ForeName><Initials>FP</Initials></Author><Author ValidYN="Y"><LastName>An</LastName><ForeName>Li-Jia</ForeName><Initials>LJ</Initials></Author><Author ValidYN="Y"><LastName>Liu</LastName><ForeName>Jian-Feng</ForeName><Initials>JF</Initials></Author><Author ValidYN="Y"><LastName>Chen</LastName><ForeName>Zhi-Wen</ForeName><Initials>ZW</Initials></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>2009</Year><Month>03</Month><Day>03</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Appl Biochem Biotechnol</MedlineTA><NlmUniqueID>8208561</NlmUniqueID><ISSNLinking>0273-2289</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D005822" MajorTopicYN="N">Genetic Vectors</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D008853" MajorTopicYN="N">Microscopy</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D019697" MajorTopicYN="N">Onions</DescriptorName><QualifierName UI="Q000166" MajorTopicYN="Y">cytology</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D016133" MajorTopicYN="N">Polymerase Chain Reaction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D012231" MajorTopicYN="N">Rhizobium</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014169" MajorTopicYN="Y">Transformation, Bacterial</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2008</Year><Month>07</Month><Day>24</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2009</Year><Month>02</Month><Day>02</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2009</Year><Month>3</Month><Day>4</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2009</Year><Month>3</Month><Day>4</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2010</Year><Month>1</Month><Day>13</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">19255730</ArticleId><ArticleId IdType="doi">10.1007/s12010-009-8554-7</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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