Date of Award

1990

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Jesse M. Jaynes

Abstract

A 38 amino acid long peptide, Shiva-1, was designed and chemically synthesized in our laboratory. Shiva-1 has only 46% amino acid homology with the naturally occurring lytic peptide from Hyalophora cecropia, known as Cecropin B. However, hydrophobic properties and charge density of the natural molecule were conserved at 100% in the synthetic peptide. This novel peptide was shown to be capable of killing a number of different species of plant pathogenic bacteria at nanomolar concentrations. Comparative studies show that Shiva-1 is more effective in this bacteriolytic activity than Cecropin B. The gene for Shiva-1 was chemically synthesized and cloned into the binary vector Agrobacterium tumefaciens LBA4404/pBI121 under the control of a wound-inducible plant promoter. Tobacco leaf discs were transformed using this binary system containing the gene encoding Shiva-1 and transgenic plants were obtained. These plants were shown to be kanamycin-resistant and displayed $\beta$-glucoronidase activity indicating effective plant transformation. Southern blots confirmed the presence of a single copy Shiva-1 gene integrated into the genomes of individual transgenic plants. Southern analyses also showed stable inheritance of the Shiva-1 gene. Northern analyses verified that the expression of the gene could be triggered by mechanical and pathogen-induced wounding. Northern blots also showed that a mRNA with the expected size of Shiva-1 transcript hybridized to the Shiva-1 probe. Western blots indicated the presence of a distinct band hybridizing to antisera raised against Shiva-1. R1 plants from self-crossed mother transgenic plants demonstrated a 3:1 segregation pattern for kanamycin resistance gene. Plant bacterial challenge suggested that the R1 transgenic plants exhibit delayed symptoms, reduced disease severity and mortality after infection with Pseudomonas solenacearum, when compared to control plants.

Pages

148

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