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Development of Functional Genomic Resources in Oat for Identification of Genes Associated with Beta-Glucan

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Development of Functional Genomic Resources in Oat for Identification of Genes Associated with Beta-Glucan DEVELOPMENT OF FUNCTIONAL GENOMIC RESOURCES IN OAT FOR IDENTIFICATION OF GENES ASSOCIATED WITH BETA-GLUCAN ANNIS FATMAWATI 260816959 DEPARTMENT OF PLANT SCIENCE, MACDONALD CAMPUS, MCGILL UNIVERSITY, MONTREAL, QUEBEC, CANADA NOVEMBER, 2020 A THESIS SUBMITTED TO MCGILL UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE © Annis Fatmawati 2020 I Table of content List of Figures ............................................................................................................................VI List of Table ................................................................................................................................ X Abstract ..................................................................................................................................... XI Resume..................................................................................................................................... XII Acknowledgment .................................................................................................................... XIII Preface ................................................................................................................................... XIV Contribution of Authors ......................................................................................................... XIV CHAPTER I: GENERAL INTRODUCTION ....................................................................................... 1 1.1 Introduction................................................................................................................. 1 1.2 Hypotheses .................................................................................................................. 3 1.3 Studies ......................................................................................................................... 3 1.3.1 Study 1: Functional Characterization of a candidate Gene (TLP8) Associated with β- glucan in Oat .......................................................................................................................... 3 1.3.2 Study 2: Standardizing the Tissue Culture and Transformation System and Developing Transposon Functional Genomic Resources in Avena strigosa ............................................. 3 CHAPTER II: LITERATURE REVIEW .............................................................................................. 4 2.1 History and Importance of Oat ........................................................................................ 4 2.2 Oat Taxonomy and Genome ............................................................................................ 6 2.3 Nutritional content of Oat ................................................................................................ 8 2.4 1,3;1,4-β-D-glucan (β-glucan) components in oat ........................................................... 9 2.5 Genetic of β-glucan ........................................................................................................ 11 2.6 Genomic Approaches to Understand gene function ..................................................... 14 2.6.1 RNA interference ..................................................................................................... 14 2.6.2 RNAi as Functional Genomic Resource ................................................................... 17 2.7 Plant Transposable Elements ..................................................................................... 18 2.7.1 Ac/Ds transposons ................................................................................................... 23 2.7.2 Transposon as functional genomic tools ................................................................. 25 CONNECTING STATEMENT BETWEEN CHAPTER II & III ........................................................... 27 CHAPTER III : Functional Characterization of Genes Associated with β-glucan in Oat ........... 28 3.1 Abstract .......................................................................................................................... 28 3.2 Introduction .................................................................................................................... 28 II 3.3 Materials and Method ................................................................................................... 30 3.3.1 In-silico Identification of Oat TLP8 .......................................................................... 30 3.3.2 Generation of RNAi Construct ................................................................................. 30 3.3.3 Transformation to E. Coli ......................................................................................... 31 3.3.4 Plasmid Isolation...................................................................................................... 31 3.3.5 Plant Materials and In Vitro Culture ........................................................................ 32 3.3.6 Biolistic Transformation .......................................................................................... 33 3.3.7 Selection of Putative Transgenic Calli ..................................................................... 33 3.3.8 Regenerating Plants ................................................................................................. 33 3.3.9 Basta Leaf Painting .................................................................................................. 34 3.3.10 Genomic DNA Extraction ....................................................................................... 34 3.3.11 Molecular characterization ................................................................................... 34 3.3.12 RNA isolation and cDNA synthesis ........................................................................ 35 3.3.13 RT-PCR analysis of TLP8 ......................................................................................... 35 3.3.14 qRT-PCR analysis of TLP8 ....................................................................................... 35 3.3.15 Stastitical Data Analysis ......................................................................................... 36 3.3.16 Examination of Seeds Under Selected Regeneration Medium ............................. 36 3.3.17 Comparison of Germination Stages Between Transgenic and Non-transgenic Plants ................................................................................................................................ 36 3.4 Results ............................................................................................................................ 36 3.4.1 The Ortholog TLP8 Gene Retrieval .......................................................................... 36 3.4.2 The AsTLP8.pMCG161 binary vector construction.................................................. 37 3.4.3 Sequencing of AsTLP8.RNAi construct .................................................................... 39 3.4.4 Selection of Putative Transgenic Calli ..................................................................... 40 3.4.5 Basta Leaf Painting .................................................................................................. 41 3.4.6 Molecular Characterization in T0 Plants ................................................................. 41 3.4.7 Molecular Characterization in T1 Plants ................................................................. 42 3.4.8 Examination of T2 Transformants during Germination and Vegetative Stages ..... 43 3.4.9 TLP8 Expression in T2 Transgenic seeds .................................................................. 44 3.4.10 Comparison of Germination Growth Stages Between Transgenic and Non- transgenic Plants .............................................................................................................. 45 3.5 Discussion ....................................................................................................................... 46 III 3.6 Conclusion ...................................................................................................................... 49 CONNECTING STATEMENT BETWEEN CHAPTER III & IV .......................................................... 50 CHAPTER IV: Standardizing the Tissue Culture and Transformation System and Developing Transposon Functional Genomic Resources in Diploid Oat ..................................................... 51 4.1 Abstract .......................................................................................................................... 51 4.2 Introduction .................................................................................................................... 51 4.3 Materials and Method ................................................................................................... 53 4.3.1 Sample Preparation and Tissue Culture Induction .................................................. 53 4.3.2 Biolistic Transformation .......................................................................................... 54 4.3.3 Ac/Ds Plasmid Construct ......................................................................................... 54 4.3.4 Transient Gus Expression ........................................................................................ 56 4.3.5 Selection of Putative Transgenic Calli ..................................................................... 57 4.3.6 Regeneration of Plants ...........................................................................................
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