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Molecular Genetic Studies of Senescence in Anthurium

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Molecular Genetic Studies of Senescence in Anthurium MOLECULAR GENETIC STUDIES OF SENESCENCE IN ANTHURIUM A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOSCIENCES AND BIOENGINEERING AUGUST 2012 By Pierriden Azucena Perez Dissertation Committee: David Christopher, Chairperson Anne Alvarez Richard Criley John Hu Gernot Presting Keywords: Anthurium senescence, Agrobacterium-mediated transformation ABSTRACT Senescence is a complex physiological process and has become an attractive area of research in plant molecular biology. The autoregulated production of cytokinin in plants transformed with the PrSAG12-IPT gene construct significantly delayed leaf senescence, and created plants that lived longer, produced more flowers with improved vase life, and an overall increased productivity. The promoter region of an arabidopsis cysteine protease served as the senescence-activated switch for the cytokinin gene IPT, and the discovery of a homolog in anthurium (ANTH17) made possible the cloning and isolation of its promoter. The sequence contained motifs and cis-elements characteristic of senescence response, and transformation of arabidopsis with PrANTH17-IPT showed similar traits with those transformed with PrSAG12-IPT. Agrobacterium-mediated transformation of anthurium with the senescence-activated gene constructs proved challenging, and stable transformation of plants was confirmed by screening for the reporter gene GFP using molecular methods. An effort to establish a protoplast transient expression system in anthurium was initiated in order to study protein subcellular signaling and localization, and is still in the process of optimization. Transcriptomic analysis of senescing leaf and spathe identified proteins involved in tissue-specific development, and provided an enormous collection of over 17,000 gene sequences that are differentially expressed. An examination of the major anthurium seed development proteins provided initial results in understanding the connection between senescence and embryo development, two very similar molecular processes in plants. ii TABLE OF CONTENTS ABSTRACT ...................................................................................... ii LIST OF TABLES ........................................................................... vii LIST OF FIGURES ......................................................................... viii CHAPTER I. INTRODUCTION AND LITERATURE REVIEW...................................................................................... 1 Biochemical changes during senescence ................................... 2 Changes in gene expression associated with senescence ........................................................................... 5 Role of hormones and plant growth regulators .......................... 6 Cytokinin & isopentenyl transferase ......................................... 8 Transgenic expression of cytokinin in plants ............................. 9 A system to regulate cytokinin production in transgenic plants ................................................................. 10 Anthurium andreanum ......................................................... 12 Anthurium breeding and genetic transformation ...................... 13 Green Fluorescent Protein as a useful reporter gene .................................................................................. 14 Seed development and senescence ........................................ 15 CHAPTER II. HYPOTHESES, SIGNIFICANCE OF RESEARCH AND OBJECTIVES ............................................ 17 Hypotheses ........................................................................ 17 Significance of Research ....................................................... 18 Objectives .......................................................................... 21 iii CHAPTER III. PLANT TRANSFORMATION USING SENESCENCE REGULATED IPT CONSTRUCTS ..................... 23 Introduction ..................................................................... 23 Materials and Methods ..................................................... 24 Isolation of the promoter region of anth17 .................... 24 Generation of IPT constructs ........................................ 26 Anthurium plants, culture and transformation ................ 29 Arabidopsis transformation .......................................... 31 Screening of transformants by Western blot .................. 32 Results ............................................................................. 33 Isolation of the anth17 promoter region ........................ 33 Anthurium transformation ........................................... 39 Arabidopsis transformation .......................................... 43 Discussion ........................................................................ 50 Isolation of the promoter region ................................... 50 Anthurium transformation ........................................... 55 Arabidopsis transformation .......................................... 59 Conclusion ........................................................................ 60 Future studies .................................................................. 61 CHAPTER IV. EXPRESSION OF GFP IN ANTHURIUM PROTOPLASTS .................................................................. 62 Introduction ..................................................................... 62 Materials and Methods ..................................................... 63 Isolation of protoplasts ............................................... 63 iv Protoplast transfection and GFP expression ................... 64 Results ............................................................................. 65 Isolation of protoplasts and transfection ........................ 66 Discussion ........................................................................ 68 Conclusion ........................................................................ 69 Future research ................................................................ 70 CHAPTER V. CHARACTERIZATION OF SENESCENCE RELATED GENE TRANSCRIPTS IN ANTHURIUM SPATHE AND LEAVES ........................................................ 71 Introduction ..................................................................... 71 Materials and Methods ..................................................... 73 Spathe and leaf RNA extraction, transcriptome sequencing and annotation ..................... 73 Sequence selection, primer design and transcript expression levels ......................................... 75 Results ............................................................................. 76 RNA isolation from leaf and spathe ............................... 76 Transcriptome sequencing and annotation ..................... 77 Sequence selection, primer design and transcript expression levels ......................................... 78 Discussion ........................................................................ 88 Transcriptome sequencing, annotation and sequence selection ..................................................... 88 Transcript expression levels ......................................... 91 Conclusion ........................................................................ 93 Future studies .................................................................. 93 v CHAPTER VI. ANTHURIUM SEED DEVELOPMENT .......................... 95 Introduction ..................................................................... 95 Materials and Methods ..................................................... 96 Pollination of flowers, seed development and harvesting ................................................................. 96 Protein extraction, analysis and mass mapping ................................................................... 97 Results ............................................................................. 99 Pollination, seed development & harvesting ................... 99 Total protein from seeds ........................................... 100 Protein types based on solubility ................................ 101 Peptide sequencing results ........................................ 103 Discussion ...................................................................... 104 Pollination of flowers, seed development and harvesting ............................................................... 104 SDS-PAGE analysis of seed proteins ........................... 105 Protein extraction, analysis and mass mapping ................................................................. 107 Conclusion ...................................................................... 108 Future studies ................................................................ 109 Appendix A – PlantCARE Database search results ............................. 110 Appendix B – RT-PCR primers designed for the selected sequences ........................................................................ 119 CHAPTER VII. LITERATURE CITED ............................................. 120 vi LIST OF TABLES TABLE PAGE 3.1 Media composition used for in vitro culture of anthurium .......................................................................... 29 3.2 A search of the PlantCARE database using the PrANTH17 and PrSAG12 sequences revealed the presence of regions involved in transcription regulation common in both ................................................... 38 5.1 Illumina
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