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(Vitis Vinifera L.) Cv. `Spätburgunder

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(Vitis Vinifera L.) Cv. `Spätburgunder Aus dem Institut für Pflanzenbau und Pflanzenzüchtung I der Justus-Liebig-Universität Gießen Lehrstuhl für Pflanzenzüchtung Leiter: Prof. Dr. Dr. h.c. Wolfgang Friedt Genexpressionsanalyse zur Ausprägung der Lockerbeerigkeit der Weinrebe ( Vitis vinifera L.) cv. `Spätburgunder´ Dem Fachbereich Agrarwissenschaften, Ökotrophologie und Umweltmanagement der Justus – Liebig – Universität Gießen Als Dissertation zur Erlangung des akademischen Grades eines Doktors der Agrarwissenschaften (Dr. agr.) vorgelegt von M. Sc. agr. Achim Schmitt aus Leinach Gießen, im Juli 2009 Gutachter: Prof. Wolfgang Friedt Gutachter: Prof. Reinhard Töpfer Tag der Disputation: 28.09.2009 2 Inhaltsverzeichnis Inhaltsverzeichnis Inhaltsverzeichnis............................................................................................................ I Verzeichnis der Tabellen................................................................................................ V Verzeichnis der Abbildungen........................................................................................ VI Abkürzungsverzeichnis............................................................................................... VIII 1 Einleitung ..................................................................................................................... 1 1.1 Bedeutung des Weinbaus in Deutschland................................................................... 1 1.2 `Blauer Spätburgunder´............................................................................................... 1 1.3 Entwicklung der Weinrebe .......................................................................................... 2 1.4 Grauschimmelfäule und Lockerbeerigkeit als physikalischer Resistenzfaktor ............. 4 1.4.1 Grauschimmelfäule .................................................................................................. 4 1.4.2 Lockerbeerigkeit als physikalischer Resistenzfaktor................................................. 7 1.5 Literatur basierende Kandidatengene ......................................................................... 9 1.5.1 Sprossverzweigung.................................................................................................. 9 1.5.2 Infloreszenzmorphologie .........................................................................................10 1.6 Zielsetzung der Arbeit ................................................................................................11 2 Literatur.......................................................................................................................13 2.1 Microarray Hybridisierungen ......................................................................................13 2.1.1 Nachweis von mRNA – klassische Methoden .........................................................13 2.1.2 DNA – Chip Technologie / Microarrays ...................................................................14 2.1.3 Microarray Typen....................................................................................................15 2.1.4 Herstellung..............................................................................................................17 2.1.4.1 Trägersubstanzen.....................................................................................17 2.1.4.2 Photolithografisches Verfahren / in situ Technik .......................................18 2.1.4.3 Mechanisches Microspotting ....................................................................19 2.1.4.4 Gendatenbanken......................................................................................21 2.1.5 Validierungsmethoden für Microarray Experimente.................................................21 2.2 Real Time PCR..........................................................................................................22 2.2.1 Prinzip und Technik ................................................................................................22 2.2.2 SYBR Green I .........................................................................................................23 2.2.3 Quantitative Real Time PCR ...................................................................................24 2.2.3.1 Absolute Quantifizierung...........................................................................24 2.2.3.2 Relative Quantifizierung............................................................................24 2.2.4 PCR – Effizienz.......................................................................................................25 2.2.5 Auswertung.............................................................................................................26 3 Material & Methoden...................................................................................................29 I Inhaltsverzeichnis 3.1 Material......................................................................................................................29 3.1.1 Chemikalien, Reagenzien & Enzyme ......................................................................29 3.1.2 Primer .....................................................................................................................30 3.1.2.1 Primer ”Housekeeping Genes” für quantitative Real Time PCR................30 3.1.2.2 Primer für die Kandidatengene aus der Microarray Analyse .....................31 3.1.2.3 Abgeleitete Primer für die Kandidatengene aus der Literatur....................33 3.1.2.4 Sequenzierprimer (inklusive der M13 – Universalsequenz) ......................34 3.1.3 Plasmid...................................................................................................................34 3.1.4 Bakterienstämme....................................................................................................34 3.1.5 Geräte, Kits & Zubehör ...........................................................................................35 3.1.6 Software..................................................................................................................37 3.1.7 Größenstandards....................................................................................................38 3.1.8 Pflanzenmaterial für die Genexpressionsstudien.....................................................38 3.1.8.1 Auswahl des Pflanzenmaterials 2006 .......................................................39 3.1.8.2 Phänotypische Beobachtung 2006 ...........................................................40 3.1.8.3 Probennahme 2006..................................................................................40 3.1.8.4 Auswahl des Pflanzenmaterials 2007 und 2008 .......................................41 3.1.8.5 Phänotypische Beobachtung 2007 und 2008............................................42 3.1.8.6 Probennahme 2007 und 2008 ..................................................................42 3.1.9 Pflanzenmaterial für Arbeiten mit genomischer DNA...............................................42 3.2 Methoden...................................................................................................................44 3.2.1 Arbeiten mit RNA aus Pflanzenmaterial ..................................................................44 3.2.1.1 Allgemeine Arbeitsschritte ........................................................................44 3.2.1.2 Präparation der Gesamt – RNA aus der Weinrebe ...................................45 3.2.1.3 DNase – Behandlung und anschließend Aufreinigung..............................46 3.2.1.4 Reverse Transkription ..............................................................................48 3.2.2 DNA Extraktion aus der Weinrebe...........................................................................48 3.2.3 Gelelektrophorese...................................................................................................50 3.2.3.1 Agarose – Gelelektrophorese ...................................................................50 3.2.3.2 DNA – Agarose – Gelelektrophorese........................................................50 3.2.3.3 RNA – Agarose – Gelelektrophorese........................................................51 3.2.4 Bestimmung der Konzentration von Nukleinsäuren.................................................51 3.2.4.1 Spektralphotometrische Konzentrationsbestimmung ................................51 3.2.4.2 Elektrophoretische Konzentrationsbestimmung ........................................52 3.2.5 Polymerase – Ketten – Reaktion (PCR) ..................................................................52 3.2.5.1 Reverse Transkriptase – PCR ..................................................................53 3.2.5.2 Gradienten – PCR ....................................................................................54 II Inhaltsverzeichnis 3.2.5.3 Touchdown – PCR ...................................................................................55 3.2.5.4 Kolonie – PCR..........................................................................................55 3.2.6 Microarray Analyse .................................................................................................56 3.2.6.1 Experimentelles Design............................................................................56 3.2.6.2 Genexpressionsanalyse ...........................................................................56 3.2.6.3 Bioinformatische Auswertung ...................................................................57 3.2.6.4 Einteilung nach molekularer
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