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Biogenesis of Lipid Bodies in Lobosphaera Incisa

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Biogenesis of Lipid Bodies in Lobosphaera Incisa Biogenesis of Lipid Bodies in Lobosphaera incisa Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program GGNB Microbiology and Biochemistry of the Georg-August University School of Science (GAUSS) submitted by Heike Siegler from Münster Göttingen 2016 Members of the Thesis Committee Prof. Dr. Ivo Feußner Department for Plant Biochemistry, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen Prof. Dr. Volker Lipka Department of Plant Cell Biology, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen Prof. Dr. Thomas Friedl Department of Experimental Phycology and Culture Collection of Algae at the University of Göttingen, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen Members of the Examination Board Prof. Dr. Ivo Feußner (Referee) Department for Plant Biochemistry, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen Prof. Dr. Volker Lipka (2nd Referee) Department of Plant Cell Biology, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen Prof. Dr. Thomas Friedl Department of Experimental Phycology and Culture Collection of Algae at the University of Göttingen, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen Prof. Dr. Andrea Polle Department of Forest Botany and Tree Physiology, Büsgen Institute, University of Göttingen PD Dr. Thomas Teichmann Department of Plant Cell Biology, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen Dr. Martin Fulda Department for Plant Biochemistry, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen Date of oral examination: 30.05.2016 Affidavit I hereby declare that I wrote the present dissertation on my own and with no other sources and aids than quoted. Heike Siegler Göttingen, February 2017 Index 1 Introduction .................................................................................................................................... 1 1.1 TAG biosynthesis in algae .................................................................................................... 1 1.2 TAG degradation in algae ..................................................................................................... 3 1.3 Diversity of lipid bodies ........................................................................................................ 4 1.4 Structural features of LB proteins ........................................................................................ 8 1.5 The oleaginous microalga Lobosphaera incisa ................................................................. 9 1.6 Aims of this study ................................................................................................................ 12 2 Methods ......................................................................................................................................... 13 2.1 Cultivation of Lobosphaera incisa ................................................................................... 13 2.2 Isolation of LBs .................................................................................................................... 13 2.3 Identification of LB proteins .............................................................................................. 14 2.3.1 Protein isolation ........................................................................................................... 14 2.3.2 Protein identification ................................................................................................... 14 2.3.3 Enrichment analysis of identified proteins ............................................................... 16 2.3.4 In silico analyses of putative LB proteins ............................................................... 17 2.4 Gene expression analysis ..................................................................................................... 17 2.4.1 L. incisa growth under conditions of nitrogen starvation and recovery ............ 17 2.4.2 Quantitative real-time PCR (qRT-PCR) ................................................................... 19 2.5 Isolation and modification of nucleic acids ...................................................................... 20 2.5.1 Isolation of RNA from L. incisa ............................................................................. 20 2.5.2 Isolation of RNA from Arabidopsis thaliana seeds ............................................ 20 2.5.3 cDNA synthesis ........................................................................................................... 21 2.5.4 Polymerase chain reaction (PCR) .............................................................................. 21 2.5.5 Agarose gel electrophoresis ........................................................................................ 21 2.5.6 Subcloning ..................................................................................................................... 22 2.5.7 Restriction cloning ....................................................................................................... 23 2.5.8 Gateway cloning ........................................................................................................... 23 2.5.9 Generation of chemically competent bacteria ......................................................... 24 2.6 Transient gene expression in Nicotiana tabacum pollen tubes ................................... 24 2.7 Microscopy ............................................................................................................................ 25 2.7.1 Detection of neutral lipids in L. incisa cells and isolated LBs ............................. 25 2.7.2 Localization studies in N. tabacum pollen tubes ................................................... 26 2.7.3 Selection of Arabidopsis thaliana seeds expressing the mCherry gene ........... 26 2.8 Complementation of Arabidopsis thaliana mutants .................................................... 27 2.8.1 Arabidopsis thaliana growth conditions ............................................................... 27 2.8.2 Transformation of Agrobacteria ................................................................................ 27 2.8.3 Agrobacterium-mediated transformation of Arabidopsis thaliana ................... 28 2.8.4 Selection of transgenic plants ..................................................................................... 28 2.8.5 Hypocotyl growth assay .............................................................................................. 29 2.8.6 Lipase activity assay ..................................................................................................... 30 2.9 Analysis of fatty acids .......................................................................................................... 31 2.9.1 Analysis of fatty acids in Arabidopsis thaliana seeds and seedlings ................. 31 2.9.2 Analysis of fatty acids in TAG and total extracts of L. incisa ............................. 32 3 Materials ......................................................................................................................................... 33 3.1 Equipment ............................................................................................................................. 33 3.2 Software ................................................................................................................................. 34 3.3 Consumables ......................................................................................................................... 35 3.4 Chemicals............................................................................................................................... 35 3.5 Media and buffers................................................................................................................. 36 3.5.1 Media ............................................................................................................................. 36 3.5.2 Buffers for cell disruption ........................................................................................... 38 3.5.3 Buffers for SDS-PAGE .............................................................................................. 39 3.5.4 Buffers for nucleic acid extraction ............................................................................ 40 3.5.5 Buffers for agarose gel electrophoresis ..................................................................... 41 3.5.6 TFP buffer for generation of competent E. coli.................................................... 41 3.5.7 Tris EDTA buffer (TE buffer) for Gateway cloning ............................................. 42 3.5.8 Solutions for lipid extraction ...................................................................................... 42 3.5.9 Solutions for lipase activity assays ............................................................................. 42 3.6 Organisms ............................................................................................................................. 43 3.6.1 Algae and plant lines .................................................................................................... 43 3.6.2 Bacterial strains ............................................................................................................. 43 3.7 Oligonucleotides ..................................................................................................................
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