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Genetic Compartmentalization in the Complex Plastid of Amphidinium Carterae

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Genetic Compartmentalization in the Complex Plastid of Amphidinium Carterae Genetic compartmentalization in the complex plastid of Amphidinium carterae and The endomembrane system (ES) in Phaeodactylum tricornutum Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Vorgelegt dem Fachbereich Biologie der Philipps-Universität Marburg FB 17 – Biologie, Zellbiologie von Xiaojuan Liu aus Fujian, China Marburg/Lahn 2015 Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation angenommen am: 2015 Erstgutachter: Prof. Dr. Uwe-G. Maier Zweitgutachterin: Prof. Dr. Ralf Jacob Prof. Dr. Andrea Maisner Prof. Dr. Susanne Önel Tag der Disputation am: 2015 Publications X. liu, F. Hempel, S. Stork, K. Bolte, D. Moog, T. Heimerl, UG. Maier, S. Zauner (2015). Addressing various compartments via sub-cellular marker proteins of the diatom model organism Phaeodactylum tricornutum. Algal Research. In preparation. In preparation: X. liu, C. Grosche, UG. Maier, S. Zauner (2015). Isolation of individual minicircles via a novel transposon-insertion based approach in Amphidinium carterae. Forthcoming. Contents Summary ................................................................................................................................................. 1 Zusammenfassung ................................................................................................................................... 2 Abbreviations .......................................................................................................................................... 3 Figures and Tables ................................................................................................................................... 4 1 Introduction .......................................................................................................................................... 6 1.1 The evolution of complex plastids ............................................................................................. 6 1.1.1 The primary endosymbiosis ........................................................................................... 6 1.1.2 The secondary endosymbiosis........................................................................................ 7 1.1.3 Plastid genome and gene transfer ............................................................................... 10 1.3 Endomembrane system ........................................................................................................... 11 1.3.1 The endomembrane system ......................................................................................... 11 1.3.2 Vacuolar protein transport within endomembrane system ........................................ 12 1.3.2.1 Vacuolar sorting determinants .......................................................................... 13 1.3.2.2 Vacuolar protein transport via vesicles ............................................................. 14 1.3.3 The biosynthesis of N-Glycoproteins on endomembrane system ............................... 17 1.3.4 Tonoplast intrinsic proteins (Tips) ................................................................................ 18 1.3.5 Vacuolar-type H+-ATPases (V-ATPase) ......................................................................... 19 2. Aim..................................................................................................................................................... 21 3 Results ................................................................................................................................................ 22 3.1 Genetic compartmentalization in the complex plastid of Amphidinium carterae .................. 22 3.1.1 The enrichment and isolation of minicircles ................................................................ 22 3.1.2 Analysis of individual minicircles .................................................................................. 23 3.1.2.1 The overview of all individual minicircles .......................................................... 24 3.1.2.2 The core regions of minicircles in A. carterae CCAM0512 ................................ 27 3.1.2.3 Transcription and RNA editing analyses of individual minicircles ..................... 28 3.1.3 Evolution analyses of four A. carterae strains’ minicircles........................................... 29 3.1.3.1 Overall genome characteristics and open reading frames................................ 29 3.1.3.2 Phylogenetic analysis of psbA genes for 15 minicircles of dinoflagellates........ 31 3.1.3.3 Alignment analysis of core regions from four A. carterae strains ..................... 32 3.1.3.4 Phylogenetic analysis of partial LSU/SSU rDNA ................................................. 34 3.2 The endomembrane system (ES) in Phaeodactylum tricornutum .......................................... 37 3.2.1 Identification of marker proteins ................................................................................. 37 3.2.2 Tonoplast intrinsic proteins (Tips) ................................................................................ 37 3.2.2.1 In vivo-localization of Tip1 ................................................................................. 38 3.2.2.2 In vivo-localization of Tip2 ................................................................................. 39 3.2.2.3 In vivo-localization of Tip3 and Tip5 .................................................................. 40 3.2.2.4 In vivo-localization of Tip4 ................................................................................. 42 3.2.3 In vivo localization of Golgi proteins ............................................................................ 44 3.2.4 In vivo localization of retromer complex ...................................................................... 45 3.2.5 In vivo localization of vacuolar H+-ATPase proteins ..................................................... 47 4 Discussion ........................................................................................................................................... 50 4.1 Genetic compartmentalization of peridinin-containing dinoflagellates ................................. 50 4.1.1 Minicircles of the peridinin-containing dinoflagellate Amphidinium carterae CCAM0512 ............................................................................................................................. 50 4.1.1.1 Minicircles with coding genes ........................................................................... 51 4.1.1.2 Empty minicircles .............................................................................................. 54 4.1.2 The evolutional relationship of minicircles .................................................................. 55 4.2 “The endomembrane system (ES) in Phaeodactylum tricornutum” ....................................... 57 4.2.1 Identification of tonoplast intrinsic proteins (Tips) ...................................................... 57 4.2.2 Identification of Golgi-marker proteins ........................................................................ 61 4.2.3 Identification of retromer complex proteins................................................................ 62 4.2.4 Identification of vacuolar type H+-ATPases .................................................................. 64 5 Materials and Methods ...................................................................................................................... 65 5.1 Materials .................................................................................................................................. 65 5.1.1 Instruments .................................................................................................................. 65 5.1.2 Membranes and filters ................................................................................................. 66 5.1.3 Antibodies..................................................................................................................... 66 5.1.4 Chemicals ...................................................................................................................... 66 5.1.5 Enzymes ........................................................................................................................ 66 5.1.6 Software and bioinformatic applications ..................................................................... 67 5.1.7 DNA and protein markers ............................................................................................. 67 5.1.8 Oligonucleotide primers ............................................................................................... 67 5.1.9 Vectors .......................................................................................................................... 68 5.1.10 organisms ................................................................................................................... 68 5.2 Methods .................................................................................................................................. 68 5.2.1 Culture of E.coli TOP10 ................................................................................................. 68 5.2.2 Culture of Phaeodactylum tricornutum CCAP 1055/1.................................................. 69 5.2.3 Culture of Amphidinium carterae CCAM0512 .............................................................. 69 5.2.4 Nucleic acid analytics ...................................................................................................
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