Masters Theses

Date of Award

12-2006

Degree Type

Thesis

Degree Name

Master of Science

Major

Plant Sciences

Major Professor

C. N. Stewart, Jr.

Committee Members

Steve Ripp, Janice Zale

Abstract

The use of antibiotic resistance markers is an important tool in the production and selection of transgenic plants. There have been increased concerns about the potential horizontal gene transfer (HGT) from transgenic plants to bacteria of medical and environmental importance. Until recently all antibiotic resistance genes used in transgenic studies have been bacterial in origin. An Arabidopsis thaliana ABC transporter, Atwbc19, was the first plant gene shown to confer kanamycin resistance when overexpressed in transgenic plants. The Atwbc19 gene was evaluated for its ability to transfer antibiotic resistance to Escherichia coli, which are found in the human gut and environment. Simulated HGT was staged by subcloning Atwbc19 under the control of a bacterial promoter, genetically transforming the bacteria and assessing if resistance was conferred as compared to the same treatment of the E. coli nptII gene. The nptII gene provided greater resistance to kanamycin in E. coli than that of the Atwbc19 gene and was significantly different from the no-plasmid control at higher concentrations of kanamycin (e.g., over 10 mg L -1) (p < 0.05). The Atwbc19 gene was not significantly different from the no-plasmid control at higher concentrations of kanamycin (e.g., over 25 mg L -1) (p < 0.05). E. coli transformed with Atwbc19 conferred little resistance to kanamycin at 100 mg L-1.

Results from Northern gel blot analysis indicated that expression levels for nptII were similar to that of Atwbc19 for the two concentrations of the antibiotic kanamycin tested. However, there was a slightly apparent decrease in the level of expression for Atwbc19 compared to the nptII gene that was most likely the result to the plant codon usage of the ABC gene or its large size (over two-fold greater than nptII). This research supports the use of the Atwbc19 gene in transgenic plants as a selectable marker and potential replacement of the nptII gene for kanamycin selection systems.

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