M.S. (Master of Science)
Department of Biological Sciences
Lipoxygenase is a ubiquitous protein which plays a significant role in plant lipid metabolism. The enzyme is involved in the production of volatile compounds which may be involved in pathogen and disease resistance, plant/pest signaling, plant stress response, and the production of flavors and odors. The goal of this project was to isolate a lipoxygenase sequence from alfalfa and use this information to identify ribozymes which may be used to modulate the expression of this enzyme. The nucleic acid sequences of nine known lipoxygenases were examined for common regions of homology. Two regions were selected for use as templates and degenerate oligonucleotides were designed to anneal to these regions. These oligonucleotides were used as primers in the polymerase chain reaction ( PCR ) to amplify alfalfa cDNA which could be used as a probe for lipoxygenase. An alfalfa cDNA library was screened with the PCR-generated probe. Five clones, with inserts ranging in size from 700 base pairs to 2,200 base pairs, were isolated from the library. The largest of these clones was selected for restriction enzyme mapping, nucleic acid sequencing, and expression analysis. The nucleic acid and amino acid sequence of the cDNA clone was 64% homologous to Soybean LOX 1 and 74% homologous to Soybean LOX 2 and LOX 3. The cDNA clone contained eight of nine homology domains common to other known plant lipoxygenases. Expression analysis by northern blot of RNA isolated from various alfalfa tissues revealed the greatest expression of this sequence to be in alfalfa leaf tissue. This result and the conserved homology domains are evidence that the isolated clone is an alfalfa leaf lipoxygenase. ABSTRACT The nucleic acid sequence of the cDNA clone was examined for potential ribozyme target sites. A total of twenty-five GTC cleavage sites were found. Fourteen of these sites had the necessary nucleic acid sequences in the flanking regions to make potential ribozyme targets which may be useful for regulating the expression of lipoxygenase.
White, James M., "Isolation of a lipoxygenase cDNA clone from Medicago sativa" (1994). Graduate Research Theses & Dissertations. 3681.
Northern Illinois University
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