Interaktion Camalexin-Spezifischer Enzyme Und Etablierung Von Eutrema Salsugineum Als Modellsystem

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Interaktion Camalexin-Spezifischer Enzyme Und Etablierung Von Eutrema Salsugineum Als Modellsystem Dissertation 2017 Phytoalexinbiosynthese in Brassicaceen : Interaktion Camalexin-spezifischer Enzyme und Etablierung von Eutrema salsugineum als Modellsystem Stefanie Mucha Technische Universität München Technische Universität München Lehrstuhl für Botanik Phytoalexinbiosynthese in Brassicaceen : Interaktion Camalexin-spezifischer Enzyme und Etablierung von Eutrema salsugineum als Modellsystem Stefanie Mucha Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Prof. Dr. Ralph Hückelhoven Prüfer der Dissertation: 1. apl. Prof. Dr. Erich Glawischnig 2. Prof. Dr. Brigitte Poppenberger-Sieberer Die Dissertation wurde am 09.11.2017 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 19.01.2018 angenommen. für meinen Opa Manfred († 2014) Inhaltsverzeichnis Inhaltsverzeichnis Inhaltsverzeichnis .................................................................................................................. 1 Abbildungsverzeichnis ........................................................................................................... 5 Tabellenverzeichnis ............................................................................................................... 8 Abkürzungsverzeichnis .........................................................................................................10 Zusammenfassung ...............................................................................................................14 Summary ..............................................................................................................................16 1. Einleitung ......................................................................................................................18 1.1. Sekundäre Pflanzenstoffe in Pflanzen ....................................................................18 1.1.1. Phenolische Sekundärmetabolite ....................................................................20 1.1.2. Isoprenoide Sekundärmetaboliten - Terpene ...................................................21 1.1.3. Stickstoff- und schwefelhaltige Sekundärmetabolite ........................................22 1.2. Phytoalexine als Bestandteil der pflanzlichen Abwehr ............................................25 1.3. Cytochrom P450-Monooxygenasen .......................................................................29 1.4. Arabidopsis thaliana und Eutrema salsugineum als Modellsysteme der Phytoalexinbiosynthese ....................................................................................................34 1.4.1. Camalexin und die Rolle von Glutathiontransferasen in A. thaliana .................34 1.4.2. Eutrema salsugineum und Wasalexin als deren typisches Phytoalexin ...........41 2. Zielsetzung der Arbeit ...................................................................................................44 3. Material und Methoden .................................................................................................45 3.1. Chemikalien und Lösungsmittel ..............................................................................45 3.2. Medien ...................................................................................................................45 3.3. Antikörper ...............................................................................................................46 3.4. Organismen und Anzuchtbedingungen...................................................................47 3.4.1. Arabidopsis thaliana ........................................................................................47 3.4.2. Eutrema salsugineum ......................................................................................48 3.4.3. Nicotiana benthamiana ....................................................................................48 3.4.4. Escherichia coli ...............................................................................................48 1 Inhaltsverzeichnis 3.4.5. Agrobacterium tumefaciens .............................................................................49 3.4.6. Saccharomyces cerevisiae ..............................................................................49 3.5. Methoden ...............................................................................................................50 3.5.1. Isolierung von Nukleinsäuren ..........................................................................50 3.5.2. cDNA-Synthese ...............................................................................................50 3.5.3. Agarosegelelektrophorese...............................................................................51 3.5.4. Präparation von DNA aus Agarosegelen .........................................................51 3.5.5. Restriktion von DNA ........................................................................................52 3.5.6. Dephosophorylierung von DNA .......................................................................52 3.5.7. Sequenzierungsreaktion ..................................................................................52 3.5.8. Polymerasekettenreaktion ...............................................................................53 3.5.9. Klonierungsstrategien ......................................................................................55 3.5.10. Transformation ............................................................................................57 3.5.11. Yeast-two-Hybrid .........................................................................................61 3.5.12. Samensterilisation .......................................................................................61 3.5.13. Selektion auf Resistenzmarker (BASTA, Hyg) .............................................61 3.5.14. Induktion der Phytoalexinbiosynthese ..........................................................62 3.5.15. Metabolitextraktion .......................................................................................62 3.5.16. Infiltration von N. benthamiana ....................................................................64 3.5.17. Mikrosomenpräparation ...............................................................................64 3.5.18. Bradford-Assay ............................................................................................66 3.5.19. Aktivitätsbestimmung von S. cerevisiae -Mikrosomen ...................................66 3.5.20. HPLC-Analyse .............................................................................................66 3.5.21. Co-Immunopräzipitation ...............................................................................67 3.5.22. Polyacrylamidgelelektrophorese und Western Blot ......................................68 3.5.23. Konfokale Mikroskopie .................................................................................68 3.6. Internetressourcen und Datenbanken.....................................................................69 4. Ergebnisse ...................................................................................................................70 4.1. Metabolonbildung von Enzymen der Camalexinbiosynthese in A. thaliana .............70 2 Inhaltsverzeichnis 4.1.1. Lokalisationsstudien in N. benthamiana ..........................................................70 4.1.2. Saccharomyces cerevisiae als in vivo System zum Nachweis von Protein-Protein Interaktionen .................................................................................................................75 4.1.3. Etablierung von FRET-Mikroskopie als Mittel für Interaktionsstudien ..............80 4.1.4. Identifizierung potentieller Interaktionspartner mittels FLIM-FRET ...................84 4.1.5. Nachweis von Protein-Protein Interaktionen mittels Co-Immunpräzipitation ....88 4.1.6. Analyse der Substrataffinitäten von in Interaktion stehenden Camalexin- spezifischen Enzymen ..................................................................................................90 4.2. Beteiligung von Arabidopsis-GSTs an der Camalexinbiosynthese ..........................92 4.2.1. Etablierung und Optimierung des Expressionssystems S. cerevisiae für die Expression von AtGSTs und P450-Enzymen ................................................................92 4.2.2. Einfluss von AtGSTs auf die GS-IAN-Bildung durch CYP71B20, CYP71B15 und CYP71A13 in Hefe........................................................................................................99 4.2.3. Die Rolle von AtGSTU2 und AtGSTU4 bei der Camalexinbiosynthese.......... 101 4.2.4. Weitere AtGSTs und ihre Bedeutung während der Synthese von Camalexin nach Botrytis cinerea -Infektion in A. thaliana ....................................................................... 105 4.3. Eutrema salsugineum als alternatives Modellsystem zur Charakterisierung von Phytoalexinen ................................................................................................................. 108 4.3.1. Induktion von Wasalexin A und B als Abwehrreaktion auf biotische und abiotische Stressoren in E. salsugineum Ökotyp Shandong und Yukon ..................... 108 4.3.2. Expressionsanalyse von Genen koexprimierter Biosynthesewege ................ 111 4.3.3.
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