Michigan Technological University Digital Commons @ Michigan Tech Dissertations, Master's Theses and Master's Dissertations, Master's Theses and Master's Reports - Open Reports 2012 Functional analysis of rice bidirectional promoters Surendar R. Dhadi Michigan Technological University Follow this and additional works at: https://digitalcommons.mtu.edu/etds Part of the Biology Commons Copyright 2012 Surendar R. Dhadi Recommended Citation Dhadi, Surendar R., "Functional analysis of rice bidirectional promoters", Dissertation, Michigan Technological University, 2012. https://doi.org/10.37099/mtu.dc.etds/191 Follow this and additional works at: https://digitalcommons.mtu.edu/etds Part of the Biology Commons FUNCTIONAL ANALYSIS OF RICE BIDIRECTIONAL PROMOTERS By Surendar R Dhadi A DISSERTATION Submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Biological Sciences) MICHIGAN TECHNOLOGICAL UNIVERSITY 2012 Copyright © Surendar R Dhadi 2012 This dissertation, “Functional Analysis of Rice Bidirectional Promoters”, is hereby approved in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY IN BIOLOGICAL SCIENCES. Department of Biological Sciences Signatures: Dissertation Advisor ________________________________________ Ramakrishna Wusirika Department Chair ________________________________________ K. Michael Gibson Date ________________________________________ TABLE OF CONTENTS List of Figures........................................................................................................................... 6 List of Tables ............................................................................................................................ 8 Preface....................................................................................................................................... 9 Acknowledgements ................................................................................................................ 10 Abstract ................................................................................................................................... 11 Literature review .................................................................................................................... 12 CHAPTER 1: .......................................................................................................................... 17 GENOME-WIDE COMPARATIVE ANALYSIS OF PUTATIVE BIDIRECTIONAL PROMOTERS FROM RICE, ARABIDOPSIS AND POPULUS ....................................... 17 1.1 Abstract ....................................................................................................................... 18 1.2 Introduction ...................................................................................................................... 19 1.3 Methods ............................................................................................................................ 20 1.3.1 Promoter identification and selection ...................................................................... 20 1.3.2 Promoter sequence analysis ..................................................................................... 21 1.4 Results and Discussion .................................................................................................... 22 1.4.1 Computational identification of putative bidirectional promoters from rice, Arabidopsis, and Populus and their high GC content ...................................................... 22 1.4.2 Overrepresented and abundant motifs in putative bidirectional promoters in rice, Arabidopsis and Populus genomes represented in the PLACE database ....................... 23 1.4.3 Overrepresented motifs in putative bidirectional promoters in three plant genomes represented in PLANTCARE database ............................................................. 28 1.4.4 Representative bidirectional promoters and divergent genes ................................ 29 1.4.5 Bidirectional promoters: a new toolkit to manipulate plant genomes ................... 33 CHAPTER 2: .......................................................................................................................... 35 3 A NOVEL NON-WOUNDING TRANSIENT EXPRESSION ASSAY FOR CEREALS MEDIATED BY AGROBACTERIUM TUMEFACIENS .................................................... 35 2.1 Abstract ............................................................................................................................. 36 2.2 Introduction ...................................................................................................................... 37 2.3 Materials and Methods .................................................................................................... 37 2.3.1 Constructs Used in the Present Study ...................................................................... 37 2.3.2 Growth of Plants under Sterile Conditions ............................................................. 38 2.3.3 Growth of Young Cereal Plants and Arabidopsis in the Greenhouse ................... 38 2.3.4 Agrobacterium Co-cultivation and Stress Treatments ........................................... 38 2.3.5 Effect of Agrobacterium Co-cultivation and Plant Incubation Times in MS Salt Solution on Reporter Gene Expression............................................................................. 39 2.3.6 Downstream Process and Applications ................................................................... 39 2.3.7 Quantitative Expression Analysis of gus and egfp Reporter Genes ...................... 40 2.4 Results............................................................................................................................... 40 2.4.1 Utility of AmTEA as a Universal Method for Cereals ........................................... 40 2.4.2 Conserved Expression Patterns of Reporter Genes in Stable and Transiently Expressing Arabidopsis Plants .......................................................................................... 43 2.4.3 Agrobacterium Co-cultivation and Plant Incubation Times in MS Salt Solution Affect Reporter Gene Activity in Plants ........................................................................... 43 2.4.4 AmTEA - A Useful and Reliable Tool for Deciphering Promoter and Gene Function in Plants............................................................................................................... 44 2.5 Discussion......................................................................................................................... 47 2.8 Supplementary information ............................................................................................. 49 CHAPTER 3: .......................................................................................................................... 52 REGULATION OF BIDIRECTIONAL PROMOTER ACTIVITY IN RICE BY CIS- ELEMENTS IN 5’-UNTRANSLATED REGIONS............................................................ 52 3.1 Abstract ............................................................................................................................. 53 4 3.2 Introduction ...................................................................................................................... 54 3.3 Methods ............................................................................................................................ 55 3.3.1 Construction of a vector with two reporter genes for testing BDPs ...................... 55 3.3.2 Gateway cloning of BDP-reporter gene constructs ................................................ 55 3.3.3 Plant transient expression assay ............................................................................... 56 3.3.4 Deletion analysis ....................................................................................................... 56 3.3.5 Extraction of rice nuclear proteins ........................................................................... 56 3.3.6 DNA footprinting assay............................................................................................ 56 3.3.7 Motif analysis............................................................................................................ 57 3.3.8 Site-directed mutagenesis of the BDP constructs ................................................... 57 3.3.9 Qualitative and quantitative expression analysis of reporter genes ....................... 58 3.4 Results............................................................................................................................... 58 3.4.1 Structure of the divergent genes harboring the divergent promoters (BDP) chosen for functional evaluation .................................................................................................... 58 3.4.2 Deletion constructs identify bidirectional promoters ............................................. 59 3.4.3 Cis-regulatory motifs: identification, deletion and expression analysis................ 62 3.5 Discussion......................................................................................................................... 68 3.8 Supplementary information ............................................................................................. 72 CONCLUSION ...................................................................................................................... 76 FUTURE DIRECTIONS ......................................................................................................