Title page for ETD etd-120399-120335


Type of Document Dissertation
Author Hegeman, Carla Elizabeth
Author's Email Address chegeman@vt.edu
URN etd-120399-120335
Title Isolation and Characterization of Soybean Genes Involved in Phytic Acid Metabolism: Phytase and 1-L-myo-Inositol-1-Phosphate Synthase
Degree PhD
Department Plant Pathology, Physiology, and Weed Science
Advisory Committee
Advisor Name Title
Grabau, Elizabeth A. Committee Chair
Cramer, Carole L. Committee Member
Dean, Dennis R. Committee Member
Rutherford, Charles L. Committee Member
Winkel, Brenda S. J. Committee Member
Keywords
  • Inositol
  • Phytase
  • Soybean
  • Phytate
Date of Defense 1999-11-30
Availability unrestricted
Abstract
The 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.

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