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dc.contributor.authorHegeman, Carla Elizabethen_US
dc.date.accessioned2014-03-14T20:19:43Z
dc.date.available2014-03-14T20:19:43Z
dc.date.issued1999-11-30en_US
dc.identifier.otheretd-120399-120335en_US
dc.identifier.urihttp://hdl.handle.net/10919/29906
dc.description.abstractThe objective of this research was to isolate and characterize soybean genes involved in phytic acid metabolism for use in genetic engineering strategies to improve phosphorus utilization. A soybean phytase from germinated cotyledons was purified 28,000-fold to apparent homogeneity and was determined to be a glycosylated homodimer with 70 kD subunits. Soybean phytase preferred phytate as substrate (Km = 60 mM) and was capable of removing of all six phosphate groups from phytate. The pH and temperature optima for soybean phytase activity were 4.5 and 58*C, respectively. The N-terminus and four internal peptides from the purified soybean phytase were sequenced by Edman degradation. The amino acid sequence data were used to design degenerate oligonucleotide primers for PCR amplification of the soybean phytase coding sequence. A protein 547 amino acids in length was predicted from the 1641 bp coding sequence. The phytase protein showed significant similarity to plant purple acid phosphatases (PAPs) and contained the conserved metallo-phosphomonoesterase active site motif. The soybean phytase coding sequence was placed under the control of a constitutive 35S CaMV promoter in a soybean biolistic transformation vector and was introduced into "Williams 82" suspension culture cells by particle bombardment. Stably transformed cell suspension lines were recovered. DNA blot analysis demonstrated that the recombinant soybean phytase coding sequence had integrated into the genomes of two cell lines. Expression of the transgene was confirmed by RNA blot analysis. Phytase activity was three to four fold higher in these two lines compared to control non-transformed cultures. A soybean L-myo-insoitol-1-phosphate synthase (MIPS) cDNA was isolated from total RNA from developing seeds. The protein encoded by the soybean MIPS cDNA showed 87-91% homology to MIPS protein sequences from other plant species. RNA blot analysis of staged developing soybean seeds revealed that MIPS is transcribed early in the cotyledonary stage of development. Compared to other soybean tissues, MIPS expression levels were highest in developing seeds. DNA blot analysis demonstrated that multiple copies of the MIPS gene are present within the soybean genome.en_US
dc.publisherVirginia Techen_US
dc.relation.haspart0chegeman.pdfen_US
dc.rightsI hereby grant to Virginia Tech or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University Libraries in all forms of media, now or hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.en_US
dc.subjectInositolen_US
dc.subjectPhytaseen_US
dc.subjectSoybeanen_US
dc.subjectPhytateen_US
dc.titleIsolation and Characterization of Soybean Genes Involved in Phytic Acid Metabolism: Phytase and 1-L-myo-Inositol-1-Phosphate Synthaseen_US
dc.typeDissertationen_US
dc.contributor.departmentPlant Pathology, Physiology, and Weed Scienceen_US
dc.description.degreePh. D.en_US
thesis.degree.namePh. D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
thesis.degree.disciplinePlant Pathology, Physiology, and Weed Scienceen_US
dc.contributor.committeechairGrabau, Elizabeth A.en_US
dc.contributor.committeememberCramer, Carole L.en_US
dc.contributor.committeememberDean, Dennis R.en_US
dc.contributor.committeememberRutherford, Charles L.en_US
dc.contributor.committeememberWinkel, Brenda S. J.en_US
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-120399-120335/en_US
dc.date.sdate1999-12-03en_US
dc.date.rdate2000-04-11
dc.date.adate1999-04-11en_US


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