Throughout history, traditional plant breeding has been used to provide resistance to pests, disease and other forms of environmental stress, as well as to increase yield and improve upon quality and processing attributes. Over the last decade, the advancement in sequencing technology and bioinformatic analysis has unleashed a wealth of knowledge about chloroplast genetic organization and evolution. The lack of complete plastid genome sequences is one of the major limitations in advancing plastid genetic engineering to other useful crops. This is due to the fact that plastid genome sequences are essential for the identification of endogenous regulatory sequences and optimal sites for homologous recombination. Analysis of four Solanaceae genomes revealed significant genetic modifications in both coding and non-coding regions. Repeat analysis with Reputer revealed 33 to 45 direct and inverted repeats ≥ 30bp with at least 90% homology. All but five of the 42 repeats shared among all four genomes were located in the exact same genes or intergenic regions, suggesting a functional role. Intergenic analysis found four regions that are 100 percent identical in all four Solanaceae genomes. Such highly conserved intergenic regions are ideal targets for multi-species transformation cassettes. Protein disulfide isomerases (PDI) are a family of proteins known to function as molecular chaperones and aid in the formation of disulfide bonds during protein folding. They contain at least one thioredoxin domain used for the formation, isomerization, and reduction/oxidation of disulfide bonds. Bioinformatic analysis identified 13 PDI-like (PDIL) proteins found in Arabidopsis that contain at least one thioredoxin domain. In addition to the above-mentioned characteristics, PDIs have been shown to be directly involved in the translational regulation of the psbA mRNA in response to light and could potentially increase the efficiency of chloroplast engineering in plants. Human serum albumin (HSA) requires 17 disulfide bonds to be properly folded and is an ideal candidate for assessing the disulfide bond formation, protein folding, and other chaperone-like characteristics of PDIL proteins. Therefore, I have coexpressed HSA in order to further characterize an Arabidopsis PDIL protein, atPDIL5-4, and in particular, the redox control of the psbA 5'UTR. Interestingly, the polyclonal antibody used for identifying the PDIL protein cross-reacted and identified other proteins, but not the transgenic atPDIL5-4. Results of these investigations will be presented.
Master of Science (M.S.)
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
Length of Campus-only Access
Masters Thesis (Open Access)
Grevich, Justin James, "Bioinformatic Analysis Of Solanaceae Chloroplast Genomes And Characterization Of An Arabidopsis Protein Disulfide Isomerase In Transgenic Tobacco Chloroplasts" (2006). Electronic Theses and Dissertations. 6102.