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Untersuchung Der Benzoxazinoidbiosynthese in Den Dikotyledonen Consolida Orientalis Und Lamium Galeobdolon

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Untersuchung Der Benzoxazinoidbiosynthese in Den Dikotyledonen Consolida Orientalis Und Lamium Galeobdolon TECHNISCHE UNIVERSITÄT MÜNCHEN Untersuchung der Benzoxazinoidbiosynthese in den Dikotyledonen Consolida orientalis und Lamium galeobdolon Dissertation Laura Hannemann TECHNISCHE UNIVERSITÄT MÜNCHEN Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl für Pflanzenzüchtung Untersuchung der Benzoxazinoidbiosynthese in den Dikotyledonen Consolida orientalis und Lamium galeobdolon Laura Hannemann 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. Erwin Grill Prüfende der Dissertation: 1. Prof. Dr. Alfons Gierl 2. Prof. Dr. Wilfried Schwab Die Dissertation wurde am 06.05.2019 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 22.08.2019 angenommen. Für Mama Inhaltsverzeichnis Inhaltsverzeichnis Abbildungsverzeichnis .......................................................................................................... IV Tabellenverzeichnis .............................................................................................................. VI Abkürzungsverzeichnis ........................................................................................................ VII 1 Einleitung ....................................................................................................................... 1 1.1 Sekundärmetabolite und ihre Funktion .................................................................... 1 1.2 Benzoxazinoide ....................................................................................................... 2 1.2.1 Struktur und Eigenschaften .............................................................................. 2 1.2.2 Vorkommen der Benzoxazinoide ...................................................................... 3 1.2.3 Biosynthese der Benzoxazinoide...................................................................... 6 1.3 Die Enzyme der BX Biosynthese ............................................................................. 7 1.3.1 Oxygenasen als Modifikationsenzyme ............................................................. 8 1.3.2 Detoxifizierende Glukosyltransferasen ............................................................. 9 1.3.3 Β-Glukosidasen in der Bioaktivierung ..............................................................11 1.4 Ziel der Arbeit .........................................................................................................12 2 Material und Methoden ..................................................................................................14 2.1 Material ..................................................................................................................14 2.1.1 Pflanzenmaterial und Anzucht .........................................................................14 2.1.2 Bakterienstämme ............................................................................................15 2.1.3 Hefestämme ....................................................................................................15 2.1.4 Plasmide .........................................................................................................15 2.1.5 Oligonukleotide ...............................................................................................16 2.1.6 Chemikalien und Reagenzien ..........................................................................18 2.2 Molekularbiologische Methoden .............................................................................19 2.2.1 DNA-Isolierung ................................................................................................19 2.2.2 RNA-Isolierung und cDNA-Synthese ...............................................................19 2.2.3 Allgemeine DNA-Klonierungstechniken ...........................................................19 2.2.4 PCR-Verfahren ................................................................................................20 2.2.5 Transkriptionelle Analyse nach Verwundungsstress ........................................21 2.2.6 Codonoptimierung von P450 Genen auf das Hefeexpressionssystem ............21 2.2.7 DNA-Sequenzierung .......................................................................................21 2.2.8 Sequenzierung des L. galeobdolon Transkriptoms ..........................................21 2.2.9 Sequenzierung von genomischer L. galeobdolon DNA ....................................22 2.3 Hefe- und Gewebekulturen .....................................................................................23 2.3.1 Anzucht und Induktion von Hefe ......................................................................23 I Inhaltsverzeichnis 2.3.2 Transformation von S. cerevisiae ....................................................................23 2.3.3 Transiente Proteinexpression in N. benthamiana Agroinfiltration .....................24 2.3.4 Erzeugung und Analyse von transgenen A. thaliana-Pflanzen ........................25 2.4 Proteinbiochemische Methoden .............................................................................25 2.4.1 Proteinrohextraktion aus C. orientalis und L. galeobdolon ...............................25 2.4.2 SDS-Polyacrylamid-Elektrophorese (SDS-PAGE) ...........................................26 2.4.3 Proteinkonzentrationsbestimmung nach Bradford ...........................................26 2.4.4 Proteinextraktion aus N. benthamiana .............................................................26 2.4.5 Heterologe Proteinexpression in E. coli und Reinigung über Nickel- Affinitätschromatographie bzw. Glutathion Agarose Säule ............................................26 2.4.6 Mikrosomenisolierung aus S. cerevisiae ..........................................................27 2.4.7 Mikrosomenisolierung aus C. orientalis und Z. mays .......................................27 2.5 Isolierung von Naturstoffen .....................................................................................28 2.5.1 Isolierung von DIBOA aus L. galeobdolon bzw. DIMBOA aus Z. mays ............28 2.5.2 Isolierung von GDIBOA aus L. galeobdolon ....................................................28 2.5.3 Isolierung von GDIMBOA aus Z. mays ............................................................28 2.6 Analyse von Naturstoffen .......................................................................................29 2.6.1 Hochleistungsflüssigkeitschromatographie (HPLC) .........................................29 2.7 Enzymtests ............................................................................................................31 2.7.1 Test der Zimtsäure-4-Hydroxylase-Aktivität .....................................................31 2.7.2 In vitro Enzymtests mit Mikrosomen ................................................................31 2.7.3 Tests auf Glukosyltransferase-Aktivität............................................................31 2.7.4 Tests auf β-Glukosidase-Aktivität ....................................................................31 2.8 Identifikation von POR Sequenzen aus dem C. orientalis Transkriptom .................33 2.9 Phylogenetische Analysen .....................................................................................34 3 Ergebnisse ....................................................................................................................35 3.1 Untersuchung von C. orientalis P450-Enzymen .....................................................35 3.1.1 Hydroxylierung von Intermediaten der DIBOA-Synthese durch Consolida orientalis Mikrosomen ...................................................................................................35 3.1.2 Identifikation der P450 Kandidaten aus dem Transkriptom ..............................36 3.1.3 Expression der P450-Enzyme in Hefe .............................................................38 3.2 Analysen zur Identifizierung von Bx-Genen in L. galeobdolon ................................42 3.2.1 Verifzierung von Gen-Assemblierungen des Transkriptoms ............................42 3.2.2 Enzyme der Detoxifizierung und Bioaktivierung der Benzoxazinoide in L. galeobdolon ...............................................................................................................45 3.2.3 Untersuchung von L. galeobdolon UDP-Glukosyltransferasen ........................45 3.2.4 Charakterisierung der L. galeobdolon BGLUs .................................................48 II Inhaltsverzeichnis 3.3 Phylogenetische Untersuchung der L. galeobdolon Enzyme ..................................64 3.3.1 Phylogenie der β-Glukosidasen .......................................................................65 3.3.2 Phylogenie der P450-Enzyme .........................................................................67 3.3.3 Phylogenie der UGTs ......................................................................................69 4 Diskussion .....................................................................................................................72 4.1 Zwei-Komponenten-Systeme als Strategie der Pflanzenabwehr. ...........................72 4.2 Analyse von C. orientalis P450-Enzymen ...............................................................74 4.3 Analyse der BX-Biosynthese in L. galeobdolon
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