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Functional Genomics and Metabolite Profiling As Tools for Alkaloid Biosynthetic Gene Discovery University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2015-12-16 Functional Genomics and Metabolite Profiling as Tools for Alkaloid Biosynthetic Gene Discovery Dinsmore, Donald Reed Dinsmore, D. R. (2015). Functional Genomics and Metabolite Profiling as Tools for Alkaloid Biosynthetic Gene Discovery (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26246 http://hdl.handle.net/11023/2686 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Functional Genomics and Metabolite Profiling as Tools for Alkaloid Biosynthetic Gene Discovery by Donald Reed Dinsmore A THESIS SUMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA NOVEMBER, 2015 © Donald Reed Dinsmore 2015 ABSTRACT The benzylisoquinoline alkaloids (BIAs) are diverse group of plant specialized metabolites found in the families Papaveracea, Ranunculaceae, Berberidaceae and Menispermaceae. Plants remain the only commercial source for BIAs and their biosynthesis is poorly understood. O-methyltransferases (OMTs) are wide spread in BIA biosynthesis. Putative OMTs were found in stem and root Next-Generation Sequencing transcriptomic databases. Putative OMT cDNAs were isolated from Papaver somniferum and commercially synthesized. Recombinant protoberberine 2-O-methyltransferase (2OMT) was heterologously expressed in Escherichia coli and assayed. 2OMT demonstrated the 2-O-methylation of protoberberine alkaloids and the 7-O-methylation of simple BIAs. The substrate range and tissue specific expression of 2OMT suggest its in vivo role is converting (S)-cheilanthifoline to (S)-sinactine. A LC-MS based targeted alkaloid profiling of twenty BIA producing species from the families Papaveracea, Ranunculaceae, Berberidaceae and Menispermaceae was conducted. i ACKNOWLEDGEMENTS The completion of this thesis would not have been possible without the continued support and help from my fiancé, Alexis Greene, my parents Murray and Debra Dinsmore, my co- workers Scott Farrow and Guillaume Beaudoin and finally my supervisor Dr. Peter Facchini. I would also like to thank my committee members, Dr. Greg Moorhead and Dr. David Schriemer for their guidance throughout the duration of my program. ii TABLE OF CONTENTS ABSTRACT............................................................................................................................................................... I ACKNOWLEDGEMENTS......................................................................................................................................II TABLE OF CONTENTS ....................................................................................................................................... III LIST OF TABLES................................................................................................................................................. VII LIST OF FIGURES ..............................................................................................................................................VIII LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE................................................................IX CHAPTER ONE: INTRODUCTION .................................................................................................................... 1 1.1 SECONDARY OR SPECIALIZED METABOLISM IN PLANTS ..............................................................................................1 1.2 ALKALOID BIOSYNTHESIS IN PLANTS..............................................................................................................................4 1.3 BENZYLISOQUINOLINE ALKALOID BIOSYNTHESIS ..................................................................................................... 12 1.3.1 Structural diversity of benzylisoquinoline alkaloids ............................................................................... 12 1.3.2 Molecular biology and biochemistry of benzylisoquinoline alkaloids............................................. 14 1.3.3 The catalytic mechanism of OMTs................................................................................................................... 22 1.4 APPROACHES TO GENE DISCOVERY AND FUNCTIONAL CHARACTERIZATION OF ALKALOID BIOSYNTHETIC GENES........................................................................................................................................................................................ 24 1.4.1 Traditional approaches to gene identification .......................................................................................... 24 1.4.2 Genomics-based gene discovery approaches .............................................................................................. 24 1.4.3 Integration of targeted metabolomics and transcriptomics for gene discovery ...................... 26 1.4 OBJECTIVES ...................................................................................................................................................................... 27 CHAPTER TWO: MATERIALS AND METHODS..........................................................................................29 2.1 NUCLEIC ACID ISOLATION AND ANALYSIS .................................................................................................................. 29 iii 2.1.1 Isolation of cDNAs encoding putative OMTs from opium poppy stem............................................. 29 2.1.2 Isolation of cDNAs encoding putative OMTs from opium poppy root.............................................. 29 2.2 PHYLOGENETIC ANALYSIS.............................................................................................................................................. 30 2.3 CONSTRUCTION OF EXPRESSION VECTORS AND HETEROLOGOUS EXPRESSION.................................................... 32 2.3.1 Stem OMT gene candidates IzOMT1 through IzOMT6 ........................................................................... 32 2.3.2 Root OMT gene candidates DDOMT1-4......................................................................................................... 34 2.4 PURIFICATION OF RECOMBINANT PROTEINS AND ANALYSIS .................................................................................. 35 2.4.1 Purification of recombinant IzOMT 1-6 and DDOMT 1-4 ..................................................................... 35 2.4.2 SDS-PAGE.................................................................................................................................................................... 36 2.4.3 Western Blot Analysis ........................................................................................................................................... 36 2.5 RECOMBINANT OMT CANDIDATE ASSAYS ................................................................................................................ 36 2.5.1 Routine Enzyme assays......................................................................................................................................... 36 2.5.2 DDOMT1 Temperature and pH Optima assay............................................................................................ 37 2.5.3 DDOMT1 Km Assays ................................................................................................................................................ 37 2.6 LC-MS CONDITIONS FOR ENZYME ASSAY ANALYSIS................................................................................................. 38 2.6.1 LC conditions for enzyme assays ...................................................................................................................... 38 2.6.2 Mass analyzer conditions .................................................................................................................................... 38 2.7 REAL-TIME QUANTITATIVE PCR (QPCR).................................................................................................................. 39 2.8 LC-MS BASED TARGETED ALKALOID PROFILING OF 20 BIA PRODUCING PLANTS SPECIES.............................. 40 2.8.1 Plants............................................................................................................................................................................ 40 2.8.2 Chemical Standards ............................................................................................................................................... 40 2.8.3 Extraction of alkaloids from BIA producing plant species.................................................................... 41 2.8.4 LC conditions for targeted alkaloid profiling ............................................................................................. 42 2.5.2 Mass analyzer conditions for targeted alkaloids profiling................................................................... 42 iv CHAPTER THREE: THE ISOLATION AND FUNCTIONAL EXPRESSION OF A MOLECULAR CLONE ENCODING 2-PROTOBERBERINE O-METHYLTRANSFERASE...............................................................44
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