Date of Award

1990

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Jesse M. Jaynes

Abstract

A novel protein (HEAAE II, High Essential Amino Acid Encoding Protein), rich in essential amino acids (75% of total), was designed and constructed in our laboratory. The monomeric form of the protein consists of 20 amino acid residues with four additional amino acids comprising a potential $\beta$-turn. Circular dichroism and size exclusion analysis demonstrated, that the monomer exists in a stable $\alpha$-helical conformation that self-aggregates in aqueous solution to form higher ordered multimeric structures, which are very reminiscent of natural plant storage proteins. The DNA encoding this amino acid sequence was synthesized, and from this monomeric gene fragment, the tetrameric form of the gene was generated by subcloning into the E. coli expression vector pKK223-3. The resultant DNA fragment was tested for in vitro and in vivo expression and then cloned into plant expression vector pBI121, under the control of the cauliflower mosaic virus 35S promoter. Agrobacterium tumefaciens, strain LBA4404, was subsequently transformed with this new construct and Nicotiana tabacum var. Xanthi transgenic plants were obtained. DNA analysis by Southern procedure confirmed the presence of the multi-copy gene in the transformed plants. Analysis of RNA and protein synthesized in these transgenic plants demonstrated the stable expression of this gene.

Pages

138

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