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Horizontal Gene Transfer to Bacteria of an Arabidopsis Thaliana ABC Transporter That Confers Kanamycin Resistance in Transgenic Plants

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Horizontal Gene Transfer to Bacteria of an Arabidopsis Thaliana ABC Transporter That Confers Kanamycin Resistance in Transgenic Plants University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Masters Theses Graduate School 12-2006 Horizontal Gene Transfer to Bacteria of an Arabidopsis Thaliana ABC Transporter That Confers Kanamycin Resistance in Transgenic Plants Kellie Parks Burris University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes Part of the Plant Sciences Commons Recommended Citation Burris, Kellie Parks, "Horizontal Gene Transfer to Bacteria of an Arabidopsis Thaliana ABC Transporter That Confers Kanamycin Resistance in Transgenic Plants. " Master's Thesis, University of Tennessee, 2006. https://trace.tennessee.edu/utk_gradthes/1518 This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Kellie Parks Burris entitled "Horizontal Gene Transfer to Bacteria of an Arabidopsis Thaliana ABC Transporter That Confers Kanamycin Resistance in Transgenic Plants." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Master of Science, with a major in Plant Sciences. C. N. Stewart, Jr., Major Professor We have read this thesis and recommend its acceptance: Steve Ripp, Janice Zale Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a thesis written by Kellie Parks Burris entitled “Horizontal gene transfer to bacteria of an Arabidopsis thaliana ABC transporter that confers kanamycin resistance in transgenic plants.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Plant Sciences. C. N. Stewart, Jr.______ Major Professor We have read this thesis and recommend its acceptance: Steve Ripp________________ Janice Zale________________ Accepted for the council: Linda Painter __ Interim Dean of Graduate Studies (Original signatures are on file with official student records). Horizontal gene transfer to bacteria of an Arabidopsis thaliana ABC transporter that confers kanamycin resistance in transgenic plants A Thesis presented for the Masters of Science degree The University of Tennessee, Knoxville Kellie Parks Burris December, 2006 Acknowledgements I would like to offer my sincere thanks and appreciation to Dr. Neal Stewart, my major professor, who provided me the opportunity to gain new skills and knowledge in the area of Plant Biotechnology. It is through his dedication and support that my research was completed. I would also like to thank Dr. Steve Ripp, who was helpful and encouraging with anything in projects relating to microbiology and to Dr. Janice Zale, who served as a committee member and provided suggestions on my research and writing. I would like to especially thank my husband, friend and colleague, Jason Burris, who with his love, support, understanding and patience guided me through the most difficult times. Without his help and encouragement, I would not have been able to complete this project. Also, I would like to thank my parents, Tom and Gail Parks, who with their love and support encouraged me to fulfill my educational goals, and to my very special sister, Kim Parks, who I looked to for advice and support and who I could count on for the comfort and encouragement I so needed. And a special thanks to my fellow colleagues for their assistance and advice: Mentewab Ayalew, Laura Good, Reggie Millwood, Hong-Seok Moon, Jaime Shockley, Joshua Yuan, and all other members of the Stewart lab. ii 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 iii 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. iv Table of Contents 1. Introduction.................................................................................................................1 2. Literature Review........................................................................................................6 2.1 Aminoglycoside antibiotics................................................................................ 6 2.1.1 Introduction................................................................................................. 6 2.1.2 Prevalence of antibiotic resistance in nature............................................... 7 2.1.3 Aminoglycosidic antibiotics and their uses ................................................ 8 2.1.4 Resistance mechanisms............................................................................. 10 2.1.5 Importance in human health...................................................................... 11 2.2 ABC transporters.............................................................................................. 12 2.2.1 Introduction............................................................................................... 12 2.2.2 White-Brown Complex subfamily............................................................ 13 2.2.3 Discovery and functional characterization of Arabidopsis thaliana WBC transporters................................................................................................................ 13 2.3 Horizontal gene transfer.................................................................................... 15 2.3.1 Introduction............................................................................................... 15 2.3.2 Mechanisms of HGT................................................................................. 16 2.3.3 Barriers to HGT........................................................................................ 18 2.3.4 HGT detection and its limitations............................................................. 20 2.4 Markers used in plant biotechnology................................................................ 23 2.4.1 Introduction............................................................................................... 23 2.4.2 Antibiotic resistance markers.................................................................... 24 2.4.3 Alternative markers to antibiotic resistance genes.................................... 26 2.4.4 Implications of ARMs on human and animal health ................................ 27 2.4.5 Regulations regarding the use of ARMs in transgenic plants................... 28 3. Objectives ................................................................................................................. 30 4. Materials and Methods.............................................................................................. 31 4.1 Bacterial strains and plasmids........................................................................... 31 4.2 Determining susceptibility of Escherichia coli to kanamycin.......................... 31 4.3 Determining potential usage of other bacteria in HGT studies......................... 32 4.4 Transformation.................................................................................................. 32 4.5 Assessment of new antibiotic resistance through natural transformation......... 32 4.6 Statistical analysis............................................................................................. 33 4.7 Reverse transcription polymerase chain
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