| Christopher Donatelli | Scarlett Graham | Nathan Haines |
| Caitlin Holt | Katelyn Schultz | Nathan Shively |
Expression of ascorbate peroxidase in Brassica rapa in response to iron
Christopher Donatelli, 2007 (Advisor: Dr. Ann Kleinschmidt)
Iron is an essential nutrient for plants, but in excess amounts it can react with oxygen to form toxic reactive oxygen species (ROS) that can damage vital biomolecules within an organism. Plants therefore have multiple enzymes and antioxidant molecules that function to mediate oxidative stress and control the levels of ROS, including class III peroxidases, ascorbate peroxidase (APX), and ascorbate. This project investigated the effect of iron exposure on class III peroxidase expression, ascorbate peroxidase expression, and the concentration of ascorbate in Brassica rapa. Short term iron treatments (12h/24h) and long term iron treatments (1d, 3d, 7d) were performed, and leaf and stem tissue were assayed separately when possible. The results of native gel electrophoresis stained with H2O2 for peroxidase activity demonstrated that short term iron exposure generally caused a decrease in class III peroxidase activity but an increase in peroxidase activity in the long term. The amount of cytosolic APX mRNA was measured with RT-PCR. Short term iron treatment had no significant effect on cAPX gene expression. Long term iron treatment caused an increase in cAPX mRNA at 3 days but a decrease in the amount at 7 days. Ascorbate concentration was determined in response to iron exposure, and ascorbate concentration generally was greater in the iron-treated plants. The data from this project suggest that class III peroxidases, rather than cAPX, are more responsive to iron exposure in Brassica rapa.
Peroxidase Expression and Cadmium Accumulation in two Brassica rapa cultivars
Scarlett Graham, 2007 (Advisor: Dr. Ann Kleinschmidt)
In this study, two cultivars of Brassica rapa were analyzed for phytoremediation capabilities. The plants were treated with high concentrations of CdCl2 and then analyzed for peroxidase activity and Cd2+ accumulation. Overall Egypt had more peroxidase isoenzymes, and a novel peroxidase with an isoelectric point of 6.3 was observed in the Cd2+ treated leaf blade and petiole tissue. In addition, the Egypt cultivar had trichomes which are hypothesized to be a storage place for Cd2+ in the plant. The Egypt cultivar crop was observed to accumulate 277.97 µg g-1 of Cd2+ per dry weight over 3 weeks, and the Hong Kong cultivar accumulated 164.28 µg g-1 and 121.06 µg g-1 in two different samples. Explanations for Egypt’s ability to accumulate higher concentrations include the presence of trichomes and more peroxidase isoenyzmes. On average, the shoot of the Hong Kong cultivar was 2 g heavier than Egypt’s shoot after the 3 weeks. Biomasses of all plants were all significantly smaller when treated with Cd2+. The results of this study were consistent with previous findings indicating increased peroxidase activity in the presence of heavy metals, and the experiment contributes to the understanding of the potential for B. rapa as a phytoremediator species.
Progress Toward Generalization of the 2-Position of 2,4-Disubstituted Furans Synthesized from 2,3-dibromo-1-phenylsolfonyl-prop-1-ene and Acetyl Methyl Ketones
Nathan Haines, 2007 (Advisor: Dr. Shaun Murphree)
The 2,4-disubstituted furan moiety, while elusive in the laboratory, has been identified in several natural products of interest, including 5-(2,6-dimethyl-octa-5,7-dienyl)-furan-3-carboxylic acid 3, a known antifouling agent derived from finger leather coral. In light of recent motives toward environmentally friendly antifouling agents, synthesis of 3 has become of interest, and motivated this research. Progress towards its synthesis has focused on using a known methodology toward 2,4-disubstituted furans that exploits the acyclic precursor 2,3-dibromo-1-(phenylsulfonyl1)-1-propene (DBP). Previous efforts have attained the carboxylic acid moiety at the 4-position of the furan, and here, progress was made toward generalizing the 2-position. The proposed methodology utilized a variety of acetyl methyl ketones to selectively introduce chains that varied in length, substitution, and functionality at the 2-position (41-95% yields). The methodology relies on steric hindrance to selectively direct deacylation at a critical point along the mechanistic pathway. Ultimately, the methodology has shown promise for future efforts toward the synthesis of the natural product 3.
Optimizing PCR Conditions for the Cloning of a T-type Calcium Channel α1 Subunit
Caitlin Holt, 2007 (Advisor: Dr. Lauren French)
The purpose of this study was to manipulate and optimize degenerate PCR conditions for the cloning of the α1 subunit of a T-type calcium channel in the stomatogastric ganglion (STG) of the Panulirus interruptus. The STG contains several neuronal networks which produce rhythmic movements without sensory input, and are commonly known as a central pattern generator (CPG). This rhythmic pattern is produced by burst-firing of neurons which is initiated by low-voltage activated, or T-type, calcium channels. The α1 subunit acts as the channel pore and voltage sensor, thereby being of primary importance to the channel’s unique function. By cloning this subunit, further molecular and electrophysiological understanding can be applied to vertebrate neuronal networks and functions such as muscle movement, digestion, and respiration. The primary difficulty in cloning this subunit is the optimization of PCR conditions to amplify the cDNA sequence. Degenerate primers were designed based on the homologous conservation among species in particular regions of the α1 subunit gene. PCR optimization is generally determined empirically and requires adjustments in cycle specifications such as annealing temperatures and concentrations of reagents such as magnesium. The influence of these factors was investigated, and sub-optimal conditions were achieved. It was determined that other PCR techniques and changes in degenerate primer design may produce a higher amplification specificity.
Patterns of peroxidase expression in Alliaria petiolata shoot during development
Katelyn Schultz, 2007 (Advisor: Dr. Ann Kleinschmidt)
The patterns of peroxidase expression in the shoot of Alliaria petiolata were investigated at the protein and mRNA levels over a six week period from 3/30/06 to 5/3/06. Protein was isolated from shoot tissues, and an electrophoresis gel was run to separate the proteins based on their isoelectric points (IEPs). The A. petiolata actin sequence was determined through cloning, and primers were designed to serve as a control in the mRNA analysis. RNA was isolated from the shoots, and RT-PCR was performed using primers for GM10, GM20, GM22, and GM26, which are homologous to the Arabidopsis thaliana peroxidases AtPrx12, AtPrx67, AtPrx64, and AtPrx52, respectively. In the protein gel, bands appeared at IEPs of 10.4, 9.7, 9.0, 7.3, 6.2, and 5.9. The protein analysis showed relatively high levels of peroxidase present during the first three weeks, followed by an overall decrease in peroxidase during the remainder of the investigation. More peroxidase activity was present in the lower shoot than in the upper shoot. The mRNA analysis revealed an increase in peroxidase expression during the first three weeks, followed by unique expression patterns for each of the peroxidases investigated. The presence of peroxidases in the shoot throughout the development of A. petiolata corresponds to the dual roles of peroxidases in lignin synthesis for stability and in cell elongation for growth. The patterns may also reflect external factors in the natural environment, such as temperature, which are known to trigger peroxidase activity.
Progress Toward the Development of Felix 01 as a Reporter Phage for the Rapid Detection of Salmonella
Nathan Shively, 2007 (Advisor: Dr. Brandi Baros)
Nontyphoidal salmonellosis represents the most deadly form of food-related illness, and accounts for an estimated 1.4 million cases annually in the United States alone (Mead et al. 1999). Currently, most methods for detecting Salmonella spp., the bacteria responsible for salmonellosis, are slow, laborious, and/or expensive, and thus the development of a rapid, simple, and inexpensive means by which to detect the bacteria is desirable. The current project attempts to construct a recombinant bacteriophage for this purpose by inserting the gene for the fluorescent protein mCherry into the Felix 01 bacteriophage genome to act as a reporter. Genomic alterations were attempted in vitro followed by the transformation of genomic DNA into competent Salmonella cells. Attempts to recover a viable recombinant phage were unsuccessful, though significant progress was made toward its construction. One major limitation of the study was the inability to reduce or eliminate the presence of wild-type bacteriophage in the assays for the recombinant phage. Methods to overcome this limitation are suggested, along with methods to alter the mCherry-reporter fragments to increase chances of success.