Promoter fusion with Green Fluorescent Protein (GFP) is often used to determine a gene’s transcriptional activity in vivo. To transform Arabidopsis plant with promoter-GFP transgenes, Agrobacterium inserts a transfer DNA (T-DNA) containing promoter-GFP and a kanamycin-resistant gene into the plant genome. T-DNA insertion can happen at a single locus or multiple loci. The identification of a single insertion line is necessary because it simplifies downstream genetic analysis. A transgenic line is likely a single insertion line if 75% of the progenies from a heterozygous plant are kanamycin-resistant. The goal of this study is to identify single insertion lines for InvINH1 and InvINH2 promoter-GFP fusions. For each transgenic line, 300 T2 seeds were sterilized and plated on MS medium containing 35ug/ml kanamycin. The number of kanamycin-resistant seedlings was determined two weeks after germination. In total, 11 lines were analyzed for both transgenes. For each transgene, at least two single insertion lines had been identified, and their GFP expression pattern will later be validated. In addition, these lines will be used to determine whether both the paternal and maternal InvINHs alleles are expressed in the endosperm. Confirmed lines will be deposited into a stock center and made available to the scientific community.

The endosperm and embryo are the results of double fertilization in flowering plants. These structures rely on each other for successful seed development. In the early stages of Arabidopsis seed development, the endosperm grows rapidly while the embryo grows slowly. After endosperm cellularization, this growth pattern changes so that the endosperm grows slowly while the embryo grows quickly. This shift in growth is likely due to nutrients shifting from the endosperm to the embryo. We hypothesized that invertase, an enzyme that breaks down sucrose, is one of the primary mechanisms for this nutrient shift. Invertase Inhibitor 1 (InvINH1) was identified in our lab as being specifically expressed before endosperm cellularization. Therefore, the presence of InvINH1 is correlated with slow embryo growth. To further investigate the effects of InhINH1 on embryo growth rate, KRS promoter was selected to ectopically express InvINH1 after endosperm cellularization. The 2048bp KRS promoter region was amplified and cloned in front of InvINH1 coding region, creating the chimeric gene pKRS-InvINH1. For transgenic plants carrying pKRS-InvINH1, we expect to see a delay in embryo growth, which will support our hypothesis that the function of InvINH1 is to suppress embryo growth before endosperm cellularization. 

Appropriate seed development in Arabidopsis involves the coordinated growth between embryo and endosperm. After endosperm cellularization, embryo growth accelerates, which suggests that sugar is reallocated from endosperm to embryo. Invertase, an enzyme that breaks down sucrose, has been shown to regulate the movement of sugar from maternal tissue to the endosperm. Our lab identified an invertase inhibitor (InvINH1) expressed in the endosperm before cellularization. When ZOU promoter was used to express InvINH1 in the cellularized endosperm, a subtle delay in embryo growth was observed. To validate this phenotype, a stronger promoter from RGP3 was used to drive the expression of InvINH1. RGP3 is an enzyme essential for the formation of the plant cell wall. To ectopically express InvINH1, RGP3 promoter was the first cloned in front of the InvINH1 coding region to create construct pRGP3-InvINH1. To confirm the expression pattern of RGP3 promoter, the promoter was also cloned in front of the coding region of a green flourescent protein. Both constructs were transformed into Arabidopsis. After transgenic plants carrying pRGP3-InvINH1 transgene are identified, detailed phenotypic analysis will be carried out to determine whether pRGP3-InvINH1 could enhance the delayed embryo growth phenotype.
 

Salmonella enterica is a member of the Enterobacteriaceae family of gram-negative, non-spore forming, rod bacteria that are found in soil, water, and animals. There are 6 subspecies and over 2,600 serotypes. Salmonella species can cause many different diseases with gastroenteritis being the most common as it is associated with over 70% of foodborne infections. S. enterica is associated with food poisoning and characterized by vomiting, abdominal pains, diarrhea and headaches. Antibiotic resistance in Salmonella enterica is becoming widespread as a result of mechanisms including horizontal gene transfer of antibiotic resistance genes and mutations that can confer resistance to antibiotics. In order to characterize antibiotic resistance acquisition in S. enterica we studied the genomes of 20 environmental strains isolated from farmland in southeastern Georgia that were potentially exposed to antibiotics used from nearby farms raising livestock. We used bioinformatic methods to identify and characterize antibiotic resistance genes and mutations in these isolates, and infered the source and significance of antibiotic resistance acquired by these isolates. We identify antibiotic resistance mechanisms that were conferred via horizontal gene transfer or mutations via this analysis. Future work incoporate laboratory methods to verify antibiotic resistance identified using bioinformatic approaches, and compare antibiotic resistance profiles to clinical strains of S. enterica.