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Oxalate Oxidase Quantification and Relative Mrna Expression in Transgenic American Chestnut (Castanea Dentata)

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Oxalate Oxidase Quantification and Relative Mrna Expression in Transgenic American Chestnut (Castanea Dentata) SUNY College of Environmental Science and Forestry Digital Commons @ ESF Dissertations and Theses Spring 4-27-2020 Oxalate Oxidase Quantification and Relative Mrna Expression in Transgenic American Chestnut (Castanea Dentata) Dakota Matthews SUNY College of Environmental Science and Forestry, [email protected] Follow this and additional works at: https://digitalcommons.esf.edu/etds Part of the Forest Biology Commons Recommended Citation Matthews, Dakota, "Oxalate Oxidase Quantification and Relative Mrna Expression in Transgenic American Chestnut (Castanea Dentata)" (2020). Dissertations and Theses. 175. https://digitalcommons.esf.edu/etds/175 This Open Access Thesis is brought to you for free and open access by Digital Commons @ ESF. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of Digital Commons @ ESF. For more information, please contact [email protected], [email protected]. OXALATE OXIDASE QUANTIFICATION AND RELATIVE MRNA EXPRESSION IN TRANSGENIC AMERICAN CHESTNUT (CASTANEA DENTATA) by Dakota Matthews A thesis submitted in partial fulfillment of the requirements for the Masters of Science Degree State University of New York College of Environmental Science and Forestry Syracuse New York May 2020 Approved by: Dr. William A. Powell, Major Professor Dr. Stephen Shaw, Examining Committee Dr. Melissa K. Fierke, Department Chair S. Scott Shannon, Dean, The Graduate School i Acknowledgements I am extremely grateful to have been able to work on the American chestnut research and restoration project here at SUNY-ESF. This project would not have been possible without the support from many individuals. I would first like to thank my major professor Dr. William A. Powell, who has taught me so much and been extremely patient with me. I would also like to thank my steering committee Dr. Christopher Nomura, Dr. Susan Anagnost, my examiner Dr. John Drake, my defense chairperson Dr. Stephen Shaw, and all of the EFB department. I would also like to thank my coworkers and friends in the American chestnut research and restoration project; Andy Newhouse, Kristen Stewart, Linda McGuigan, Allison Oakes, Kaitlin Breda, Hannah Pilkey, Patricia Fernandes, and Erik Carlson. I would like to give a special thank you to Kathleen Baier who convinced me to go to grad school and taught me how to work in a molecular lab. I would also like to thank my family who have always supported me in completing my degree even if it took too long. This project was funded in part by the Brag program and the American Chestnut Foundation. ii Contents List of Tables ................................................................................................................................. v List of Figures............................................................................................................................... vi Abstract: ........................................................................................................................................ viii Chapter 1: Literature Review .......................................................................................................... 9 American Chestnut Background .................................................................................................. 9 RNA Expression and Quantitative Polymerase Chain Reactions .............................................. 11 Origin and Selection of Oxalate Oxidase as the Transgene ...................................................... 14 Oxalate oxidase defense mechanisms ...................................................................................... 17 Pathogen resistance in Cereal crops ......................................................................................... 19 Transgenic approaches to diseases resistance using OxO isoforms ......................................... 21 CaMV 35s promoter .................................................................................................................. 24 Possible causes for loss of transgene expression ..................................................................... 25 Literature Cited ......................................................................................................................... 27 Chapter II: Oxalate Oxidase Quantification in Transgenic American Chestnut Tissues and Natural Oxalate Oxidase Sources ............................................................................................................... 41 Abstract: .................................................................................................................................... 42 Introduction............................................................................................................................... 44 Methods .................................................................................................................................... 50 Transgenic American Chestnut Stem Tissue Preparation for RNA Extraction ...................... 50 RNA extraction protocol ........................................................................................................ 50 RNA expression ...................................................................................................................... 51 Transgenic nut core tissue preparation ................................................................................. 52 Coring of Chestnuts for Transgene inheritance testing ........................................................ 54 Histochemical assay to test for transgene inheritance in nut crosses .................................. 55 Transgenic American Chestnut Tissue Preparation............................................................... 58 Oxalate Oxidase colorimetric quantification assay ............................................................... 59 Statistical analysis .................................................................................................................. 61 Results ....................................................................................................................................... 62 Discussion .................................................................................................................................. 66 Literature Cited ......................................................................................................................... 72 Chapter III: Stability of Oxalate Oxidase Expression in Transgenic American Chestnuts and their Progeny ......................................................................................................................................... 81 iii Abstract: .................................................................................................................................... 83 Introduction............................................................................................................................... 84 Materials and Methods ............................................................................................................. 89 Transgenic American chestnut tissue prep for RNA extraction ............................................ 89 RNA extraction protocol ........................................................................................................ 90 RNA expression ...................................................................................................................... 90 Histochemical assay testing transgene inheritance .............................................................. 91 Growth per day of T2 generation .......................................................................................... 92 Statistical analysis .................................................................................................................. 92 Results ....................................................................................................................................... 93 Discussion .................................................................................................................................. 97 Literature Cited ....................................................................................................................... 102 Conclusions: ................................................................................................................................ 108 Appendices .................................................................................................................................. 112 Identifying Chestnut Pollen Baskets Collected by Italian Honeybees (Apis mellifera) ........... 113 Purpose ................................................................................................................................ 114 Materials and Methods ....................................................................................................... 114 Results: ................................................................................................................................ 115 Conclusions: ......................................................................................................................... 131 Literature Cited .................................................................................................................... 131 Reference gene optimization for qPCR figures ....................................................................... 132 iv List of Tables Table 1: Oxalate oxidase nucleotide
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