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A Thesis Submitted to the College of Graduate Studies and Research In MOLECULAR CLONING AND FUNCTIONAL CHARACTERIZATION OF GENES INVOLVED IN THE BIOSYNTHESIS OF POLYUNSATURATED FATTY ACIDS IN OAT (AVENA SATIVA L.) A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Master of Science In the Department of Food and Bioproduct Sciences College of Agriculture and Bioresources University of Saskatchewan Saskatoon, Saskatchewan Canada by MATTHEW HUNTER BERNARD © Matthew Hunter Bernard, May 2014. All Rights Reserved. PERMISSION TO USE In presenting this thesis/dissertation in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis/dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis/dissertation or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis/dissertation. DISCLAIMER Reference in this thesis/dissertation to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favoring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan, and shall not be used for advertising or product endorsement purposes. Requests for permission to copy or to make other uses of materials in this thesis/dissertation in whole or part should be addressed to: The Head of the Department of Food and Bioproduct Sciences 51 Campus Drive, University of Saskatchewan Saskatoon, Saskatchewan S7N 5A8 Canada OR Dean College of Graduate Studies and Research University of Saskatchewan 107 Administration Place Saskatoon, Saskatchewan S7N 5A2 Canada i ABSTRACT This thesis research started with analysis of oat fatty acids by using three different transmethylation methods. Basic sodium methoxide was compared with traditional acidic methanol for the total fatty acid analysis, while diazomethane was used to analyze free fatty acids. Epoxy FAs were readily hydrolyzed to dihydroxy fatty acids under the acidic condition, which suggest an overestimation of hydroxyl fatty acids and underestimation of epoxy fatty acids in previous analyses. The sodium methoxide method proved more reliable to quantify the oat seed fatty acid composition. CDC Dancer oat seed analyzed here was comprised mostly of palmitic acid (PA), oleic acid (OA) and the polyunsaturated fatty acid (PUFA) linoleic acid (LA) in quantities of 23%, 32%, and 37% of total seed FA, respectively. As well, the seed contained small quantities of another PUFA, α-linolenic (ALA) and several unusual oxygenated fatty acids (UFAs), Δ15-hydroxy fatty acid (15HFA) and epoxy fatty acids in quantities of 0.85%, 0.68%, and 2.3%, respectively. This thesis further aimed to identify and assemble all FAD2-like genes from an oat Expressed-Sequence Tag (EST) database using FAD2 and FAD2-like proteins from other organisms as query sequences in order to clone all putative FAD2-like genes-of-interest (GOIs) from oat. From the contig assemblies of retrieved oat ESTs, four distinct, putative genes were identified. From the Δ12-desaturase (FAD2) queries, a putative FAD2-like (AsFAD2) gene was identified; the Δ15-desaturase (FAD3) queries revealed two putative oat FAD3-like (AsFAD3-1 and AsFAD3-2) genes, while an ω-3 desaturase (FAD7) query identified a fourth putative full-length FAD6-like coding sequence of two possible lengths, AsFADX and AsFADX+. The GOIs were then subcloned into a yeast expression vector and functionally characterized. AsFAD2a and AsFAD2b both demonstrated Δ12 desaturation on 18:1-9c substrate. AsFAD3-1 had no activity on any substrates present, while AsFAD3-2 exhibited weak Δ15-desaturation activity specifically on 18:2-9c,12c. Finally, AsFADX converted 18:1-9c to 18:2-9c,12c, while AsFADX+ had no activity. Then, a comparative analysis of transcript levels of these GOIs via quantitative real-time PCR (qRT-PCR) was performed across oat germinating seed, root, leaf, and developing seed. AsFAD2 transcript abundance was generally much higher than AsFAD3-1 and AsFAD3-2 in all tissues. AsFAD3-1 mRNA level was highest in developing seed tissue, slightly lower in leaf tissue, and lowest in root. AsFAD3-2 mRNA was highest in germinating seed, and lowest in leaf tissue. In summary, the data produced from this thesis could be used to enhance breeding efforts for establishing oat cultivars with healthier oil content. ii ACKNOWLEDGEMENTS I would like to take this opportunity to acknowledge everyone without whom this thesis would not have been possible. First I would like to thank my supervisor Dr. Xiao Qiu for recognizing my potential and offering me the opportunity to enhance my education at this level, as well as for his support and guidance throughout this project. His depth of knowledge and patience as a mentor has been instrumental in preparation for my professional career. For technical assistance and additional guidance throughout this project, I would also like to thank Dr. Dauenpen Meesapyodsuk. Her extensive knowledge and experience were invaluable, and her leadership was very much appreciated. For their diverse expertise and counsel, I would also like to thank my committee members, Dr. Aaron Beattie, Dr. Mark Smith, Dr. Robert Tyler, and Dr. Darren Korber. I am thankful to all the advice and guidance offered to ensure the success of the project. Working with other labs within the college and sharing lab equipment made some days much more efficient; for this, I would like to thank all my colleagues in the Department of Food & Bioproduct Sciences, the Department of Plant Sciences and the Department of Soil Sciences for their willingness to work together. I would also like to thank Dallas Kessler and Agriculture and Agri-Food Canada for providing oat seed. Much was learned in discussions with my colleagues; I wish to thank all my fellow students and professors in the College of Agriculture & Bioresources, who have since become good friends, for their insightful discussions either within the discipline of molecular biology, or in those areas less familiar to me. I would especially like to thank my labmates, without whom this project would have been incredibly challenging. Our discussions covering everything from molecular biology, agriculture, weather, travel, culture, and language added much amusement and enjoyment over the duration of this project; these things learned from one another are the unofficial results of the degree and the memories will be cherished. Finally, I cannot continue without acknowledging the patience and understanding of my family and friends from my personal life. Their support throughout my academic journey has been vital in helping me achieve this level of success, and I cannot properly put into words how meaningful that support has been. iii This convergence of agriculture and dietary nutrition is dedicated to my parents, whom have always offered their unwavering support and guidance through every trial, tribulation, and triumph that I have encountered. “There are more things in heaven and earth…than are dreamt of in our philosophy.” (Shakespeare, 1.5.167-8) iv TABLE OF CONTENTS Permission to Use ............................................................................................................................ i Disclaimer ........................................................................................................................................ i Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Table of Contents ............................................................................................................................ v List of Tables ................................................................................................................................ vii List of Figures ................................................................................................................................ ix List of Abbreviations .................................................................................................................... xii 1. Introduction ............................................................................................................................. 1 2. Literature Survey .................................................................................................................... 3 2.1. Dietary Oil and Human Health......................................................................................... 3 2.2. Oat .................................................................................................................................... 6 2.3. Oat Oil .............................................................................................................................
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