published_or_final_version / Oral Biology / Doctoral / Doctor of Philosophy

The expression of two cotton osmotin genes was evaluated in terms of the mRNA and protein expression patterns in response to chemical inducers such as ethylene, hydrogen peroxide, and sodium chloride. Reverse transcriptase-polymerase chain reactions (RT-PCR) indicated that osmotin mRNAs are expressed constitutively in root tissues of cotton plants, and that they are rapidly induced in leaf and stem tissues upon ethylene treatment. Real time RT-PCR indicated that osmotin transcript levels were induced 2 to 4 h after treatment with ethephon. The osmotin mRNA levels appear to increase 12 h after treatment, decrease, and then increase again. The osmotin protein expression patterns were analyzed in Western blot analyses using an anti-osmotin antibody preparation. A 24-KDa protein band was detected from cotton plants treated with the inducers. The 24-KDa osmotin proteins were induced 4 h after treatment with ethephon, while down-regulated 96 h after treatment. Multiple osmotin isoforms were observed to be induced in cotton plants upon treatment with ethephon by two-dimensional gel electrophoresis. One goal of this dissertation research was to genetically engineer two cotton osmotin genes to routinely overproduce their antifungal proteins in transgenic Arabidopsis and cotton plants as a natural defense against fungal infections, using co-cultivation with Agrobacterium tumefaciens cells harboring pCAMBIA 2301 vector constructs containing the osmotin genes. Many transgenic Arabidopsis and cotton plants were generated. However, genomic blotting analyses indicated the absence of the osmotin transgenes, but the presence of GUS genes from the vector cassette. Alkaline blot analyses of the vector DNAs from transformed Agrobacterium cells confirmed that an anomalous DNA structural rearrangement or aberrant recombination event probably occurred in the Agrobacterium cells, interdicting the integration of osmotin transgenes into the Arabidopsis and cotton plants. This research provides crucial baseline information on expression of cotton osmotin mRNAs and proteins.

Three overlapping genomic clones covering 29.0 kilobases of cotton DNA were found to encompass a cluster of two presumptive osmotin genes (OSMI and OSMII) and two osmotin pseudogenes (OSMIII and OSMIV). A segment of 16,007 basepairs of genomic DNA was sequenced from the overlapping genomic clones (GenBank Accessions AY303690 and AF304007). The two cotton osmotin genes were found to have open reading frames of 729 basepairs without any introns, and would encode presumptive osmotin preproteins of 242 amino acids. The open reading frames of the genes are identical in sequence to two corresponding cDNA clones (GenBank Accessions AF192271 and AY301283). The two cDNA inserts are almost full-length, since one lacks codons for the four N-terminal amino acids, and the other cDNA insert lacks the coding region for the 34 N-terminal amino acids. The cotton osmotin preproteins can be identified as PR5 proteins from their similarities to the deduced amino acid sequences of other plant osmotin PR5 preproteins. The preproteins would have N-terminal signal sequences of 24 amino acids, and the mature 24 kilodalton isoforms would likely be targeted for extracellular secretion. Prospective promoter elements, including two ethylene response elements, implicated as being positive regulatory elements in the expression of a number of PR-proteins, occur in the 5'-flanking regions. The mature osmotin proteins accumulate in cotton plants treated with the inducers ethephon and hydrogen peroxide. Thus, the two cotton osmotin genes encode osmotin proteins. The coding regions of the two genes have been expressed and isolated as fusion polypeptides in a bacterial expression system. Binary constructs containing the open reading frames of the two osmotin genes under the control of the 35S CaMV promoter have been generated for eventual production of transgenic Arabidopsis and cotton plants for potential constitutive expression of the osmotin proteins for increased resistance against fungal pathogens.

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: IAA; S. cerevisiae; quorum sensing. Includes bibliographical references (p.57-59).

Graduation date: 2000

Thesis (Ph. D.)(Botany)--University of Pretoria, 2001. / Acrobat Adobe Reder needed to open files.

No description available.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: G.D. Wright. Includes bibliographical references.

Thesis (M.S.)--University of Tennessee Health Science Center, 2009. / Title from title page screen (viewed on August 11, 2009). Research advisor: Jegdish P. Babu, Ph.D. Document formatted into pages (ix, 32 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 28-31).

Thesis (M. Sc.)--Carleton University, 2001. / Includes bibliographical references (p. 85-94). Also available in electronic format on the Internet.