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Biology and Function of the Osalmt1 Gene in Rice

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Biology and Function of the Osalmt1 Gene in Rice Biology and function of the OsALMT1 gene in rice (Oryza sativa L.) by Jie Liu Submitted in fulfilment of the requirement for the Degree of Doctor of Philosophy University of Tasmania December 2015 DECLARATION DECLARATION The thesis contains no material, which has been accepted for the award of any other degree or diploma in any tertiary institution, and to the best of my knowledge, contains no material previously published or written by any other person, except where due reference is made in the text of this thesis. Jie Liu This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. Jie Liu University of Tasmania I ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Four year is not a long term for one’s whole life, but the last four years will be the most precious time along my life. Looking back, I still remember the first day when I arrived at this special place, but the completion of my thesis and subsequent Ph.D. has been a long journey. I could not have succeeded without the invaluable support of my supervisors, colleagues, friends and family. Without these supporters, especially the select few I’m about to mention, I may not have gotten to where I am today, at least not sanely. First and foremost I would like to express my special appreciation and thanks to my supervisors Associate Professor Meixue Zhou, Dr. Peter Ryan, Dr. Emmanual Delhaize and Professor Sergey Shabala, for giving me the chance to study with them and supporting me during these past four years. I would like to thank all of you for encouraging my research and for helping on my life. Your advices on both research as well as on my career have been priceless. I would especially like to thank Dr. Peter Ryan who entirely guide my research throughout my Ph.D. with his wide knowledge and deep intuitions. I appreciate all his contributions of time, patience, ideas and funding to make my Ph.D. experience productive and stimulating. Without his guidance and constant feedback this Ph.D. would not have been achievable. I offer my sincerest gratitude to people who provided help and advises during my experiment. Many thanks to Professor Jianfeng Ma who gave the advises on plant inner manganese measurement, to Dr. Sergio Moroni who shared the knowledge on physiological leaf spots, to Greg Cawthray who helped on the organic acids measurement by HPLC, to Miguel Pineros who attempted to measure OsALMT1 malate efflux by using oocytes experiments, to Richard James who helped on osmotic potential calculation of salt and mannitol, to Gonzalo Estavillo who performed the photosynthesis measurements, to Rosemary White who helped a lot with microscopy and to Tina Rathjen, Kumara Weligama and David Lewis who assisted with technique support. I gratefully acknowledge the living allowance funding received towards my PhD from the China Scholarship Council (CSC). I am also grateful to the funding received through the University of Tasmania for offering me with the tuition fee scholarship. My deep II ACKNOWLEDGEMENTS appreciation goes out to Professor Youliang Zheng, Professor Yuming Wei and Associate Professor Yaxi Liu for their supporting and encouraging to apply for the CSC scholarship and restart my Ph.D. at the University of Tasmania. The years spent in Australia would not have been as wonderful without my Chinese friends, including Dr. Qing Liu, Dr. Zhongyi Li, Dr. Ming Luo, Yao Zhi, Lijun Tian, Gaofeng Zhou, Muyun Xu, Ting Tang and Feiyi Wang. Also thanks to my friends outside Australia, including Shihang Liu, Zehou Liu, Ma Yu and Junyan Feng for their valuable friendship these years and forever. Last, but certainly not least, I must acknowledge with tremendous and deep thanks to my family who support me throughout my study overseas. I appreciate both my parents and my parents-in-law always who always believing in me and encouraging me to follow my dreams. And finally to Liang Zhang, who has been by my side throughout this Ph.D., accompanying every single minute of it, and without whom, I would not have had the courage to embark on this journey in the first place. Jie Liu December, 2015 III PUBLICATIONS PUBLICATIONS Liu J, Delhaize E, Zhou M, Shabala S, Ryan PR (2014) Functional Characterisation of OsALMT1 in Rice. ComBio2014, 28 September - 02 October Liu J, Delhaize E, Zhou M, Shabala S, Ryan PR (2015) Characterisation of the malate- permeable anion channel in rice, OsALMT1. The 9th Symposium of the International Society of Root Research (ISRR9), 06-09 October Liu J, Zhou M, Delhaize E, Shabala S, Ryan PR (2016) OsALMT1 encodes a malate permeable anion channel in rice (Oryza sativa L.) which is required for normal growth in conditions of low light and chloride deficiency. Plant Physiology (in preparation) Liu J, Zhou M, Delhaize E, Shabala S, Ryan PR (2016) Over-expression of the anion channel OsALMT1 in transgenic rice (Oryza sativa L.) increases sensitivity to manganese and enhances tolerance to boron toxicity. New Phytologist (in preparation) V Table of Contents Table of Contents DECLARATION ............................................................................................................. I ACKNOWLEDGEMENTS ............................................................................................ II PUBLICATIONS............................................................................................................V Table of Contents ............................................................................................................V List of Figures .............................................................................................................. XII List of Tables ............................................................................................................. XVI List of Abbreviations ................................................................................................ XVII Abstract ................................................................................................................... XIXX CHAPTER 1 General Introduction .................................................................................. 1 1.1 The ALMT gene family ......................................................................................... 1 1.2 Functions of the ALMT family .............................................................................. 2 1.2.1 The first member of the ALMT gene family was identified in wheat ............... 2 1.2.2 ALMT genes contributing to Al resistance ....................................................... 4 1.2.3 ALMT genes involved in guard cell function ................................................. 11 1.2.4 ALMT genes involved with mineral nutrition ................................................ 14 1.2.5 ALMT genes contributing to fruit and berry acidity ....................................... 14 1.3 Structural analysis and evolution of the ALMT family ........................................ 16 1.3.1 Phylogeny of the ALMT family .................................................................... 16 1.3.2 Secondary structure ...................................................................................... 19 1.3.3 The structure-function relationship of ALMT proteins .................................. 20 1.4 Objectives ........................................................................................................... 26 1.5 References ........................................................................................................... 28 CHAPTER 2 General materials and methods ............................................................... 35 2.1 Plant material and growth conditions ................................................................... 35 V Table of Contents 2.1.1 Plant material and seed germination .............................................................. 35 2.1.2 Plant growth conditions ................................................................................ 35 2.2 Online sequence resources ................................................................................... 35 2.3 Primer design ...................................................................................................... 36 2.4 DNA and RNA extractions .................................................................................. 36 2.4.1 Standard genomic DNA (gDNA) extraction .................................................. 36 2.4.2 High throughput DNA isolation for genotype test ......................................... 37 2.4.3 RNA extraction and 1st strand cDNA synthesis ............................................. 42 2.5 PCR reactions ...................................................................................................... 43 2.5.1 Standard PCR ............................................................................................... 43 2.5.2 Proof-reading PCR ....................................................................................... 43 2.5.3 Quantitative reverse transcription PCR (qRT-PCR) ...................................... 44 2.5.4 PCR products and gel purification ................................................................ 44 2.5.5 Fusion PCR .................................................................................................. 45 2.6 Cloning ............................................................................................................... 46 2.6.1 Preparation of E. coli cells for electroporation .............................................. 46 2.6.2
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