Title page for ETD etd-08102001-144854


Type of Document Master's Thesis
Author Good, Laura Lee
Author's Email Address lgood@vt.edu
URN etd-08102001-144854
Title Isolation and Characterization of D-Myo-Inositol-3-Phosphate Synthase Gene Family Members in Soybean
Degree Master of Science
Department Plant Pathology, Physiology, and Weed Science
Advisory Committee
Advisor Name Title
Grabau, Elizabeth A. Committee Chair
Gillaspy, Glenda E. Committee Member
McDowell, John M. Committee Member
Keywords
  • MIPS
  • myo-inositol phosphate synthase
  • Glycine max
  • phytic acid
  • soybean seed development
Date of Defense 2003-07-18
Availability unrestricted
Abstract
The objective of this research was to isolate genes encoding isoforms of the enzyme D-myo-inositol 3-phosphate synthase (MIPS, E.C. 5.5.1.4) from soybean and to characterize their expression, especially with respect to their involvement in phytic acid biosynthesis. A MIPS-homologous cDNA, designated GmMIPS1, was isolated via PCR using total RNA from developing seeds. Southern blot analysis and examination of MIPS-homologous soybean EST sequences suggested that GmMIPS1 is part of a multigene family of at least four similar members. The sequences of promoter and genomic regions of GmMIPS1 and GmMIPS2 revealed a high degree of sequence conservation. Northern and western blot analyses showed that MIPS transcript and protein are abundantly expressed early in seed development. Immunolocalization of MIPS protein in developing seeds confirmed expression of MIPS early in seed development and correlated MIPS protein accumulation in soybean seed tissue with tissues in which phytic acid is known to accumulate. The promoter region of GmMIPS1 was isolated and analyzed for possible seed-specificity using promoter:GUS fusions. Two GmMIPS1 promoter fragments were capable of conferring GUS expression when bombarded directly into developing soybean seeds. However, preliminary bombardment experiments into soybean cell suspension culture indicated that both promoter fragments drove expression of GUS in undifferentiated tissue, indicating a potential lack of seed-specificity.
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