Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco by Justin Kirke A Thesis Submitted to the Faculty of The Charles E. Schmidt College of Science In Partial Fulfillment of the Requirements for the Degree of Master of Science Florida Atlantic University Boca Raton, FL December 2019 Copyright 2019 by Justin Kirke ii Abstract Author: Justin Kirke Title: Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco Institution: Florida Atlantic University Thesis Advisor: Dr. Xing-Hai Zhang Degree: Master of Science Year: 2019 DNA damage is one of the most harmful stress inducers in living organisms. Studies have shown that exposure to high doses of various types of radiation cause DNA sequence changes (mutation) and disturb protein synthesis, hormone balance, leaf gas exchange and enzyme activity. Recent discovery of a protein called Damage Suppressor Protein (Dsup), found in the tardigrade species Ramazzotius varieornatus, has shown to reduce the effects of radiation damage in human cell lines. We have generated multiple lines of tobacco plants expressing the Dsup gene and preformed numerous tests to show viability and response of these transgenic plants when exposed to mutagenic chemicals, UV radiation and ionizing radiation. We have also investigated Dsup function in association to DNA damage and repair in plants by analyzing the expression of related genes using RT-qPCR. We have also analyzed DNA damage from X-ray and UV treatments using an Alkaline Comet Assay. This project has the potential to help generate plants that are tolerant to more extreme stress environments, particularly DNA damage and iv mutation, unshielded by our atmosphere. The possibility of growing plants accompanying human space travel and extraterrestrial colonization inspires our imagination. Extremotolerant tardigrade genes such as Dsup may be a valuable avenue in helping to cultivate crops in these future endeavors. v Acknowledgements I would like to start by acknowledging my thesis advisor and mentor Dr. Xing-Hai Zhang. Without his insight and guidance, I would not be where I am today. I would also like to thank my other committee members Dr. Mary Jane Sunders and Dr. David Binninger for their additional guidance and thoughtful input into my project. I would also like to thank my coworkers and students who volunteered to help during the completion of this work. My fellow lab mates, Paveena Vichyavichien, and Xiao-Lu Jin. My direct independent research students, Tahoe Albergo, Andrew Balsamo, Nicholas Nifakos, Milove Jeannot, Amanda Lam, Ronscardy Mondesir, Andrew Adeyiga, Mohamed Abutineh, and Nicholas Pizzo. Next, I would like to thank the Department of Biology, the college of medicine, and the FAU high school with all of their help to complete this achievement, including technical support and allowing me the use of equipment require to conduct my study. I would like to thank my friends and family. Without the emotional support and encouragement of my parents and my life partner Jennifer Castignoli, I would not be where I am at today. Lastly, I would also like to thank my scholarly Dungeons and Dragons crew, Amy Makler, Josh Disatham, Douglas Holmes, Gurtejpal Ghuman, Sean Paz, Anthony Muscarella, and Erick España for helping distract me from the stress of this journey with another one. vi Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco List of Tables ..................................................................................................................... ix List of Figures ..................................................................................................................... x Introduction ......................................................................................................................... 1 DNA Damage and Repair in plants ................................................................................ 1 Damage Suppressor Protein (Dsup) from Tardigrade .................................................... 3 Single Cell Gel Electrophoresis (Alkaline Comet Assay) .............................................. 4 Dsup expression in plants ............................................................................................... 5 Methods and Materials ........................................................................................................ 6 Generation of Transgenic Plants ..................................................................................... 6 Growth Analysis ............................................................................................................. 8 Histochemical Staining analysis ..................................................................................... 8 DNA and RNA extraction, cDNA synthesis, and qPCR analysis .................................. 8 Analysis of DNA Damage Targets through RT-qPCR (Damage Assay) ....................... 9 Single Cell Gel Electrophoresis (Alkaline Comet Assay) ............................................ 10 Results ............................................................................................................................... 12 Bioinformatics Analysis................................................................................................ 12 Genotyping and Expression of Dsup Transgenic Plants ............................................... 14 Histochemical Staining analysis ................................................................................... 15 vii Growth Assay................................................................................................................ 16 Analysis of DNA Damage Targets by RT-qPCR (Damage Assay) ............................. 19 Single Cell Gel Electrophoresis (Alkaline Comet Assay) ............................................ 27 Discussion ......................................................................................................................... 31 References ......................................................................................................................... 36 viii List of Tables Table 1: Target genes tested for expression level changes in Dsup vs Control plants treated with genomutagens. ↓ showed downregulation, ↑ showed upregulation, and ≌ showed no change in gene expression. ............................................................ 20 ix List of Figures Figure 1: Engineered Ti plasmid containing Dsup and NPTII genes driven by NOS and CaMV-35S promoters. ........................................................................................... 7 Figure 2: Amino acid sequence for Dsup protein based off ascension “P0DOW4”. Highlighted in yellow is the N-terminus, underlined is the α-helical region, and highlighted in green is the C-terminus. Boxed in Red is a possible nuclear localization signal (NLS) ............................................................................................ 12 Figure 3:I-TASSER Predicted 3D structure of the Dsup Protein ..................................... 13 Figure 4: pBlast results for α-helical region of the Dsup protein ..................................... 13 Figure 5: Relative gene dosage of Dsup gene for T0 plants based on reference gene EFα. ............................................................................................................................. 14 Figure 6: Relative expression of Dsup mRNA in T0 plants based on reference gene EFα. ............................................................................................................................. 14 Figure 7: Relative gene dosage of Dsup gene for T1 plants based on reference gene EFα. ............................................................................................................................. 15 Figure 8: GUS stain analysis of T0 tissue regeneration on EMS media. .......................... 16 Figure 9: Average fresh weight of EMS treated samples. *Significant difference found between treatment samples with p-value < 0.05. p-value = 0.0001262 ........... 17 Figure 10: Average root length of EMS treated samples. *Significant difference found between treatment samples with p-value > 0.05. p-value = 7.465e-05. ........... 18 x Figure 11: Growth assay plates for RM1 under 0 mM, 1 mM and 3 mM EMS treatment. .................................................................................................................... 19 Figure 12: Growth assay plates for R5-3 under 0 mM, 1 mM and 3 mM EMS treatment. .................................................................................................................... 19 Figure 13: Relative gene expression of ATR. Example of upregulation between treated samples. ........................................................................................................... 21 Figure 14: Relative gene expression of TGA. Example of downregulation between treated samples. ........................................................................................................... 21 Figure 15:Relative gene expression of CASP5/6. Example of downregulation to normal levels between treated samples. ...................................................................... 22 Figure 16: Relative gene expression of Ku70. Example of no change in regulation between treated samples. ............................................................................................ 22 Figure 17: Relative gene expression of ERCC. Example of downregulation to normal levels between