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Arabidopsis Thaliana Und Solanum Tuberosum Hydroxyzimtsäureamide als Abwehrstoffe gegen Phytophthora infestans in Arabidopsis thaliana und Solanum tuberosum Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg vorgelegt von Frau Diplom-Biochemikerin Melanie Dobritzsch geboren am 04.02.1983 in Leisnig Gutachter: 1. Prof. Dr. Dierk Scheel (Martin-Luther-Universität Halle-Wittenberg) 2. Prof. Dr. Ingo Heilmann (Martin-Luther-Universität Halle-Wittenberg 3. Prof. Dr. Felix Mauch (Universität Fribourg; Schweiz) Datum der Verteidigung: 24.05.2016 Inhaltsverzeichnis i. Inhaltsverzeichnis i. Inhaltsverzeichnis .............................................................................................................................................. i ii. Abkürzungsverzeichnis ................................................................................................................................. vi 1. Einleitung .................................................................................................................................................... 1 1.1. Solanum tuberosum ............................................................................................................................. 1 1.2. Phytophthora infestans ......................................................................................................................... 2 1.3. Pflanzliche Immunantwort ........................................................................................................... 4 Nichtwirts-Resistenz ............................................................................................................ 8 1.4. Immunabwehr an der Zellperipherie in Pflanzen ................................................................... 11 Chemische Abwehr in S. tuberosum gegen P. infestans .................................................... 16 Hydroxyzimtsäureamide .................................................................................................... 17 1.5. Acyltransferasen ............................................................................................................................ 23 BAHD-Acyltransferasen ................................................................................................... 23 GCN5-Acyltransferasen .................................................................................................... 24 1.6. Zielstellung der Arbeit ................................................................................................................. 26 Pflanzen ............................................................................................................................... 27 Pathogen .............................................................................................................................. 27 Bakterien .............................................................................................................................. 28 Chemikalien ......................................................................................................................... 28 Medien ................................................................................................................................. 28 Nukleinsäuren-Isolierungen .............................................................................................. 30 Southern-Blot für Southernhybridisierung ..................................................................... 31 Northern-Blot für Northernhybridisierung ................................................................... 32 Herstellung radioaktiv markierter Sonden und Hybridisierung .................................. 32 Waschen der Nylonmembran und Storage Phospo-Screen – Analyse ........................ 32 Polymerase-Ketten-Reaktion (PCR) ................................................................................ 32 Restriktionsverdau .............................................................................................................. 34 Kreuzung der Arabidopsis thaliana-Mutanten pen2 und mate1 ....................................... 34 Genotypisierung von Arabidopsis thaliana-Mutanten ..................................................... 35 Klonierungen ...................................................................................................................... 35 Transformation von kompetenten A. tumefaciens AGL-0-Zellen ................................ 39 i i. Inhaltsverzeichnis Plasmid-DNA-Isolierung aus A. tumefaciens ................................................................... 39 Stabile Transformation von Kartoffelpflanzen mit A. tumefaciens .............................. 40 Transiente Transformation von Tabakpflanzen mit A. tumefaciens ............................. 41 Transformation von Arabidopsis-Protoplasten ............................................................. 41 Konfokale Laserscan-Mikroskopie .................................................................................. 41 Zoosporengewinnung von P. infestans ............................................................................. 41 Gewinnung von P.-infestans-Sporangien .......................................................................... 42 Kulturführung der Kartoffel-Meristem-Sterilkultur ..................................................... 42 Probennahme für LC/MS/MS-Messungen von A. thaliana ........................................ 42 Probennahme für LC/MS/MS-Messungen von S. tuberosum ...................................... 43 LC/MS/MS-Messungen ................................................................................................... 43 Identifizierung von Metaboliten ...................................................................................... 45 Relative Quantifizierung von P.-infestans-Biomasse ....................................................... 47 In vitro-Inhibierungsassay von p-Cumaroylagmatin auf P.-infestans- Myzelwachstum ... 47 P.-infestans-Zoosporen Inhibierungs-Assay ..................................................................... 48 2.3. Statistische Signifikanzberechnungen von Messwerten ......................................................... 48 3. Ergebnisse ................................................................................................................................................ 50 3.1. Expression von AtACT1 und AtMATE in A. thaliana ......................................................... 50 3.2. Subzelluläre Lokalisation von AtACT1 in A. thaliana ............................................................ 51 Transiente AtACT1-YFP Expression in Nicotiana benthamiana ................................... 51 Transiente AtACT1-YFP Expression in Zellkulturprotoplasten ............................... 52 3.3. Charakterisierung von AtACT1- und AtMATE-knock out-Linien ...................................... 53 p-Cumaroylagmatingehalte in Blättern von A.–thaliana-knock-out-Linien ................... 54 p-Cumaroylagmatingehalte in Tropfen von A.-thaliana-knock-out-Linien ................... 56 3.4. Die Rolle von p-Cumaroylagmatin in der Abwehr gegen P. infestans .................................. 60 In-vitro-Inhibierungstest von p-Cumaroylagmatin auf das Myzelwachstum von P. infestans 60 In-vitro-Inhibierungstest von p-Cumaroylagmatin auf die Zoosporenkeimung von P. infestans 61 In-vitro-Inhibierungstest von Inokulierungstropfen auf das Myzelwachstum von P. infestans 62 In-vitro-Inhibierungstest von Inokulierungstropfen auf die Zoosporenkeimung von P. infestans ............................................................................................................................................ 64 ii i. Inhaltsverzeichnis 3.5. Vergleich p-Cumaroylagmatingehalte in S. tuberosum (Wildtyp) und A. thaliana (Wildtyp) 65 3.6. Transgene Expression von AtACT1 in Kartoffel .................................................................. 66 Überprüfung auf T-DNA-Einbau von AtACT1 exprimierenden transgenen Kartoffellinien .................................................................................................................................... 66 Nachweis der Expression von AtACT1-Transkript in transgenen Kartoffellinien . 67 Nachweis von AtACT1 in transgenen Kartoffellinien per qRT-PCR ....................... 68 3.7. Veränderte HCAA-Gehalte in transgenen AtACT1 exprimierenden Kartoffellinien ... 69 3.8. Gehalt an sezerniertem p-Cumaroylagmatin in transgenen AtACT1 exprimierenden Kartoffellinien ............................................................................................................................................... 72 p-Cumaroylagmatin in Blättern von transgenen AtACT1-exprimierenden Kartoffellinien .................................................................................................................................... 72 p-Cumaroylagmatin in Tropfen auf transgenen AtACT1 exprimierenden Kartoffellinien .................................................................................................................................... 73 3.9. Transgene Expression von AtACT1-AtMATE in Kartoffel ............................................... 75 Expression von AtACT1-AtMATE in transgenen Kartoffellinien ........................... 76 3.10. Morphologischer Phänotyp der transgenen p35S-AtACT1-p35S-AtMATE exprimierenden Kartoffellinien .................................................................................................................
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