Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2016 The function and regulation of CIA5/CCM1 in Chlamydomonas reinhardtii Bo Chen Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Genetics Commons Recommended Citation Chen, Bo, "The function and regulation of CIA5/CCM1 in Chlamydomonas reinhardtii" (2016). Graduate Theses and Dissertations. 15101. https://lib.dr.iastate.edu/etd/15101 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The function and regulation of CIA5/CCM1 in Chlamydomonas reinhardtii by Bo Chen A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Program of Study Committee: Martin H. Spalding, Major Professor Steven R. Rodermel Gururaj A. Rao Bing Yang Yanhai Yin Iowa State University Ames, Iowa 2016 Copyright © Bo Chen, 2016. All rights reserved. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS...................................................................................................... v ABBREVIATIONS ................................................................................................................ vi CHAPTER 1. GENERAL INTRODUCTION AND LITERATURE REVIEW ............................. 1 1. General Introduction ..................................................................................................... 1 2. Literature Review .......................................................................................................... 2 2.1 The model organism Chlamydomonas reinhardtii ................................................ 2 2.2 CO2 concentrating mechanism (CCM) ................................................................. 3 2.3 Important components of CCMs .......................................................................... 5 2.4 The schematic model for Chlamydomonas CCM ............................................... 12 2.5 Chlamydomonas CIA5 ....................................................................................... 13 3. Dissertation Organization ............................................................................................ 15 References ..................................................................................................................... 17 Figure Legend ................................................................................................................ 24 CHAPTER 2. CIA5 ACTIVATION DOMAIN ......................................................................... 26 Abstract .......................................................................................................................... 27 1. Introduction ................................................................................................................. 27 2. Material and Methods ................................................................................................. 30 2.1 Cell strains and culture conditions...................................................................... 30 2.2 Modified yeast two-hybrid and LacZ assay ........................................................ 30 2.3 Chlamydomonas dTALE construction and colorimetric assay ............................ 30 2.4 Gene expression analysis .................................................................................. 31 2.5 Western immunoblot analysis ............................................................................ 32 2.6 Expression of protein fragments in E. coli .......................................................... 32 3. Results ....................................................................................................................... 32 3.1 CIA5/CCM1 auto-activation domain identification in yeast ................................. 32 3.2 Activation domain confirmation in Chlamydomonas ........................................... 33 3.3 Region responsible for abnormal migration of CIA5 during SDS-PAGE ............. 36 3.4 Region indispensable for complementation of cia5 mutant ................................ 38 4. Conclusion and Discussion ......................................................................................... 39 References ..................................................................................................................... 41 iii Figure and Table Legends .............................................................................................. 44 Supplemental Information ............................................................................................... 54 CHAPTER 3. CIA5 DNA BINDING DOMAIN ....................................................................... 61 Abstract .......................................................................................................................... 61 1. Introduction ................................................................................................................. 62 2. Materials and Methods ............................................................................................... 64 2.1 Cell strains and culture conditions...................................................................... 64 2.2 Preparation of competent E.coli and transformation ........................................... 65 2.3 CIA5 constructs for overexpression in E. coli ..................................................... 66 2.4 Random binding site selection ........................................................................... 67 2.5 Gel mobility shift assay ...................................................................................... 68 2.6 Constructs for ad1 complementation .................................................................. 69 2.7 Gene expression analysis .................................................................................. 69 2.8 Western immunoblot analysis ............................................................................ 70 3. Results ....................................................................................................................... 70 3.1 CIA5/CCM1 protein 3-D structure prediction ...................................................... 70 3.2 CIA5 overexpression in E. coli ........................................................................... 71 3.3 Random binding site selection (RBSS) .............................................................. 72 3.4 Gel mobility shift assay ...................................................................................... 73 3.5 The effect of replaced promoter on complementation with ad1 mutant .............. 74 4. Conclusions and Discussion ....................................................................................... 75 References ..................................................................................................................... 77 Figure and Table Legends .............................................................................................. 80 Supplemental Information ............................................................................................... 89 CHAPTER 4. MINI RECOMBINANT CIA5 ......................................................................... 100 Abstract ........................................................................................................................ 100 1. Introduction ............................................................................................................... 101 2. Material and Methods ............................................................................................... 103 2.1 Cell strains, culture conditions and growth ....................................................... 103 2.3 Selection for suppressors and growth spot-tests .............................................. 104 2.4 Gene expression analysis ................................................................................ 105 iv 2.5 Western immunoblot analysis .......................................................................... 105 2.6 Chlorophyll measurement ................................................................................ 106 3. Results ..................................................................................................................... 106 3.1 Recombinant mini-CIA5 construct can complement cia5 ................................. 106 3.2 Downstream gene expression in mini-CIA5 transformants ............................... 107 3.3 Liquid growth experiments ............................................................................... 108 3.4 Screening/selection for cia5 suppressors ......................................................... 109 4. Conclusions and Discussion ..................................................................................... 110 References ................................................................................................................... 113 Figure and Table Legends ............................................................................................ 116 CHAPTER 5. GENERAL SUMMARY ...............................................................................