Development of antibodies for characterizing the Arabidopsis flavonoid biosynthetic pathway

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dc.contributor.authorCain, Cody Christopheren
dc.contributor.committeechairWinkel, Brenda S. J.en
dc.contributor.committeememberCramer, Carole L.en
dc.contributor.committeememberWalker, Richard A.en
dc.contributor.committeememberRutherford, Charles L.en
dc.contributor.departmentBiologyen
dc.date.accessioned2014-03-14T21:50:05Zen
dc.date.adate2008-11-18en
dc.date.available2014-03-14T21:50:05Zen
dc.date.issued1995-12-09en
dc.date.rdate2008-11-18en
dc.date.sdate2008-11-18en
dc.description.abstractPolyclonal antibodies against the first two enzymes of the Arabidopsis thaliana flavonoid biosynthetic pathway were developed using conventional and phage antibody technology. cDNAs from Arabidopsis coding regions of chalcone synthase (CHS) and chalcone isomerase (CHI) were sub-cloned in frame into a bacterial expression vector as fusions with glutathione Stransferase (GST) using standard directional cloning techniques. Analysis of crude extracts of Escherichia coli containing GST .. CHS or GST .. CHI fusion protein indicated that the cells expressed equivalent amounts per volume of culture. CHS and CHI were purified to near homogeneity, yielding approximately 100 micrograms of GST .. CHS and 1 milligram of GST-CHI per liter of culture. The purified fusion proteins were injected into chickens and polyclonal lgY·s were purified from egg yolk Accumulation of CHS and CHI, as well as products of the pathway, were compared during the first eight days of Arabidopsis development. CHS and CHI are sequentially induced and reach maximal accumulation levels by day 5. Anthocyanidin levels are offset by one reaching maximal levels at day 6. The fusion proteins were also used to screen a phage-display library for Fabl fragments that recognize CHS and CHI epitopes. Preliminary data indicated that enrichment of phage displaying antibodies against CHS and CHI was successful. Phage-derived antibodies against CHS and CHI provide valuable tools for future experiments addressing Western blot analysis, immunolocalization experiments, and disruption of the flavonoid biosynthetic pathway by introduction of the corresponding genes into transgenic Arabidopsis plants.en
dc.description.degreeMaster of Scienceen
dc.format.extentviii, 81 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-11182008-063141en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-11182008-063141/en
dc.identifier.urihttp://hdl.handle.net/10919/45841en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V855_1995.C356.pdfen
dc.relation.isformatofOCLC# 34563186en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectArabidopsis thalianaen
dc.subjectchalcone synthaseaen
dc.subjectchalcone isomeraseen
dc.subjectflavonoidsen
dc.subjectphage display technologyen
dc.subject.lccLD5655.V855 1995.C356en
dc.titleDevelopment of antibodies for characterizing the Arabidopsis flavonoid biosynthetic pathwayen
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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