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Untersuchung Der Transkriptionellen Regulation Von Kandidatengenen Der Pathogenabwehr Gegen Plasmopara Viticola in Der Weinrebe

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Untersuchung Der Transkriptionellen Regulation Von Kandidatengenen Der Pathogenabwehr Gegen Plasmopara Viticola in Der Weinrebe Tina Moser Institut für Rebenzüchtung Untersuchung der transkriptionellen Regulation von Kandidatengenen der Pathogenabwehr gegen Plasmopara viticola in der Weinrebe Dissertationen aus dem Julius Kühn-Institut Julius Kühn-Institut Bundesforschungsinstitut für Kulturpfl anzen Kontakt/Contact: Tina Moser Arndtstraße 6 67434 Neustadt Die Schriftenreihe ,,Dissertationen aus dem Julius Kühn-lnstitut" veröffentlicht Doktorarbeiten, die in enger Zusammenarbeit mit Universitäten an lnstituten des Julius Kühn-lnstituts entstanden sind The publication series „Dissertationen aus dem Julius Kühn-lnstitut" publishes doctoral dissertations originating from research doctorates completed at the Julius Kühn-Institut (JKI) either in close collaboration with universities or as an outstanding independent work in the JKI research fields. Der Vertrieb dieser Monographien erfolgt über den Buchhandel (Nachweis im Verzeichnis lieferbarer Bücher - VLB) und OPEN ACCESS im lnternetangebot www.jki.bund.de Bereich Veröffentlichungen. The monographs are distributed through the book trade (listed in German Books in Print - VLB) and OPEN ACCESS through the JKI website www.jki.bund.de (see Publications) Wir unterstützen den offenen Zugang zu wissenschaftlichem Wissen. Die Dissertationen aus dem Julius Kühn-lnstitut erscheinen daher OPEN ACCESS. Alle Ausgaben stehen kostenfrei im lnternet zur Verfügung: http://www.jki.bund.de Bereich Veröffentlichungen We advocate open access to scientific knowledge. Dissertations from the Julius Kühn-lnstitut are therefore published open access. All issues are available free of charge under http://www.jki.bund.de (see Publications). Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation In der Deutschen Nationalbibliografie: detaillierte bibliografische Daten sind im lnternet über http://dnb.d-nb.de abrufbar. Bibliographic information published by the Deutsche Nationalbibliothek (German National Library) The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.dnb.de. ISBN 978-3-95547-019-7 DOI 10.5073/dissjki.2015.004 Herausgeber l Editor Julius Kühn-lnstitut, Bundesforschungsinstitut für Kulturpflanzen, Quedlinburg, Deutschland Julius Kühn-lnstitut, Federal Research Centre for Cultivated Plants, Quedlinburg, Germany © Julius Kühn-lnstitut, Bundesforschungsinstitut für Kulturpflanzen sowie der genannten Universität, 2015. Das Werk ist urheberrechtlich geschützt. Die dadurch begründeten Rechte, insbesondere die der Übersendung, des Nachdrucks, des Vortrages, der Entnahme von Abbildungen, der Funksendung, der Wiedergabe auf fotomechanischem oder ähnlichem Wege und der Speicherung in Datenverarbeitungsanlagen, bleiben, auch bei nur auszugsweiser Verwertung, vorbehalten. Untersuchung der transkriptionellen Regulation von Kandidatengenen der Pathogenabwehr gegen Plasmopara viticola in der Weinrebe Zur Erlangung des akademischen Grades eines DOKTORS DER NATURWISSENSCHAFTEN (Dr. rer. nat.) Fakultät für Chemie und Biowissenschaften Karlsruher Institut für Technologie (KIT) - Universitätsbereich genehmigte DISSERTATION von Dipl.-Biol. Tina Moser aus Görlitz Dekan: Prof. Dr. Peter Roesky Referent: apl. Prof. Dr. Eva Zyprian Korreferent: Prof. Dr. Holger Puchta Tag der mündlichen Prüfung: 17.04.2014 Inhaltsverzeichnis Inhaltsverzeichnis ABBILDUNGSVERZEICHNIS ......................................................................................................III TABELLENVERZEICHNIS .......................................................................................................... VI ABKÜRZUNGSVERZEICHNIS ................................................................................................... VII ZUSAMMENFASSUNG .................................................................................................................. 1 1 EINLEITUNG ....................................................................................................................... 3 1.1 FALSCHER MEHLTAU IN DER REBE ............................................................................... 4 1.2 MODELL FÜR DIE EVOLUTION DER PATHOGENABWEHR BEI PFLANZEN ........................ 7 1.3 RESISTENZGENE ........................................................................................................... 8 1.3.1 Ethylen-Response Transkriptionsfaktoren (ERFs) ............................................... 9 1.3.2 PR-Proteine ........................................................................................................ 10 1.3.3 NBS-LRR Gene ................................................................................................... 11 1.4 PHYTOHORMONE BEI DER PATHOGENABWEHR ........................................................... 12 1.4.1 Salicylsäure ........................................................................................................ 12 1.4.2 Ethylen ............................................................................................................... 13 1.4.3 Methyljasmonat .................................................................................................. 14 1.5 ZIELSETZUNG ............................................................................................................. 15 2 MATERIAL UND METHODEN ............................................................................................ 16 2.1 MATERIAL .................................................................................................................. 16 2.1.1 Geräte ................................................................................................................. 16 2.1.2 Software ............................................................................................................. 16 2.1.3 Chemikalien ....................................................................................................... 17 2.1.5 Pflanzenmaterial ................................................................................................. 19 2.1.6 Plasmopara viticola – Gewinnung von Sporangienmaterial ............................. 20 2.2 METHODEN ................................................................................................................. 20 2.2.1 DNA-Extraktion ................................................................................................. 20 2.2.2 Primerdesign ....................................................................................................... 21 2.2.3 Polymerasekettenreaktion (PCR) ....................................................................... 21 2.2.4 Klonierung der Promotoren ausgewählter Kandidatengene ............................... 23 2.2.5 Bioinformatische Analyse .................................................................................. 23 2.2.6 Luciferaseassay (in Kooperation mit dem DLR Neustadt, AG Bogs) ............... 24 2.2.7 Mutation von WRKY-Bindestellen im PR10.1-Promotor ................................. 26 2.2.8 SNP Genotypisierung ......................................................................................... 27 2.2.9 Verrechnung der SNP-Daten mit Boniturdaten ................................................. 30 I Inhaltsverzeichnis 2.2.10 Probenahme für Genexpressionsanalysen .......................................................... 30 2.2.11 RNA-Extraktion und cDNA-Synthese ............................................................... 31 2.2.12 Genexpressionsanalysen ..................................................................................... 32 3 ERGEBNISSE ..................................................................................................................... 36 3.1 VERGLEICHENDE PROMOTORANALYSEN .................................................................... 36 3.2 UNTERSUCHUNG DES TRANSKRIPTIONSFAKTORS ERF5 DER WEINREBE .................... 42 3.3 SNP-GENOTYPISIERUNG DER CORE-KOLLEKTION VON RESISTENTEN WEINREBEN .... 48 3.4 UNTERSUCHUNG DER HORMONELLEN REGULATION VON GENEN DER PATHOGENABWEHR ............................................................................................................... 51 3.5 GENEXPRESSIONSANALYSEN VON EINZELLOCI-TRAGENDEN GENOTYPEN .................. 57 4 DISKUSSION ...................................................................................................................... 66 4.1 IN SILICO ANALYSE VON PROMOTORBEREICHEN ......................................................... 66 4.2 TRANSKRIPTIONELLE REGULATION VON PR10.1 ........................................................ 67 4.3 ANALYSE DES TRANSKRIPTIONSFAKTORS ERF5 ........................................................ 67 4.4 ASSOZIATIONSANALYSEN FÜR SNP-DATEN DER CORE-KOLLEKTION VON RESISTENTEN WEINREBEN ..................................................................................................... 68 4.5 HORMONELLE REGULATION VON RESISTENZGENEN ................................................... 70 4.6 EXPRESSION VON RESISTENZGENEN BEI EINZELLOCI-TRAGENDEN GENOTYPEN ........ 75 4.7 SCHLUSSFOLGERUNG .................................................................................................. 77 LITERATURVERZEICHNIS ........................................................................................................ XI ANHANG ..................................................................................................................................
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