| dc.contributor.advisor | Karlovsky, Petr Prof. Dr. | de |
| dc.contributor.author | Utermark, Jan | de |
| dc.date.accessioned | 2013-01-22T15:48:32Z | de |
| dc.date.available | 2013-01-30T23:50:59Z | de |
| dc.date.issued | 2008-05-28 | de |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-000D-F16B-6 | de |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-3473 | |
| dc.description.abstract | Zearalenon ist ein makrozyklisches Lacton der Resorcinolsäure, welches von phytopathogenen Pilzen Fusarium graminearum und Fusarium culmorum gebildet wird. Wegen seiner östrogenen Wirkung auf Säugetiere wird Zearalenon als Mykotoxin betrachtet. Infolge der von Fusarien hervorgerufenen Pflanzenkrankheiten (Ährenfusariose von Getreide und Kolbenfäule von Mais) können Lebensmittel und Futtermittel mit gesundheitsgefährdenden Mengen von Zearalenon kontaminiert kommen. Neben ihrer östrogenen Aktivität wirken Zearalenon und seine Derivate auch anabolisch, sie besitzen jedoch nur eine geringe akute Toxizität. Viele Mikroorganismen reduzieren Zearalenon zu α- oder β-Zearalenol. Diese Transformation stellt keine Detoxifizierung dar, da die beiden Produkte weiterhin eine östrogene Wirkung aufweisen. Der Mykoparasit Gliocladium roseum hydrolysiert Zearalenon mithilfe einer spezifischen Lactonase, die vom Gen zes2 codiert wird. Nach einer spontanen Decarboxylierung wird das hydrolysierte Zearalenon in 1-(3,5-Dihydroxyphenyl)-10’-hydroxy-1’E-undecene-6’-on transformiert, welches keine östrogene Wirkung mehr besitzt.
Die biologische Funktion der zearalenonspezifischen Lactonase in G. roseum war lange unbekannt. In unserem Labor wurde das Lactonase-Gen zes2 mithilfe einer Agrobacterium tumefaciens vermittelten Transformation inaktiviert. Durch die Insertion eines Markergens sind die zes2-Mutanten nicht mehr in der Lage Zearalenon zu detoxifizieren. Diese Ergebnisse zeigten, dass die zearalenon-abbauende Lactonase den Mykoparasiten G. roseum vor der fungiziden Wirkung des Zearalenons schützt.
Um die Untersuchung der Regulation der Expression von Zearalenon-Lactonase in G. roseum zu vereinfachen, wurden der Promotorbereich von zes2 mit zwei Reportergenen fusioniert, dem Gen für Green Fluorescent Protein (GFP) und dem Luciferase-Gen aus Gaussia princeps verwendet. Durch Agrobacterium tumefaciens-vermittelten Transfer der Genkonstrukte in G. roseum wurde die codierende Sequenz von zes2 durch die Reportergene ersetzt. Die Aktivierung des Promotors von zes2 führt in diesen Stämmen zur Fluoreszenz (GFP) bzw. Lichemission (Luciferase), die empfindlich und mit hohem Probendurchsatz gemessen werden kann.
Zearalenon induzierte die Expression der Reportergene in einer konzentrations- und zeitabhängigen Art und Weise. Alpha-Zearalenol, Alpha- und Beta-Zearalanol sowie Zearalanon wirkten ebenfalls induzierend. Keine Reaktion zeigte der GFP-Indikatorstamm jedoch nach Inkubation mit Beta-Zearalenol. Auch das Phytoöstrogen Genestein und das Steroid Beta-Estradiol induzierten die Expression der Reporterfusionen nicht. | de |
| dc.format.mimetype | application/pdf | de |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/ | de |
| dc.title | Ecological role of mycotoxin zearalenone in interactions among fungi and its enzymatic detoxification | de |
| dc.type | doctoralThesis | de |
| dc.title.translated | Biologische Funktion des Mykotoxins Zearalenon und seine enzymatische Detoxifizierung | de |
| dc.contributor.referee | Tiedemann, Andreas von Prof. Dr. | de |
| dc.date.examination | 2008-05-22 | de |
| dc.subject.dnb | 570 Biowissenschaften, Biologie | de |
| dc.subject.gok | WF 200 | de |
| dc.description.abstracteng | Fungal secondary metabolites can not only
cause toxic effects in animals and humans, but also exhibit toxic
properties to microorganisms and plants. The mycotoxin zearalenone
is a resorcylic lactone derivative produced by plant pathogenic
Fusarium species, which exerts estrogenic and anabolic effects on
mammals. The biological role of zearalenone in fungal biology has
been unknown. We established that zearalenone and its derivatives
inhibit the growth of filamentous fungi. Only the mycoparasitic
ascomycete Gliocladium roseum, which is known to hydrolyse
zearalenone to a far less estrogenic product by the activity of a
zearalenone-specific lactonase, was not affected by zearalenone.
The zearalenone lactonase encoding gene zes2 in G. roseum was
inactivated using Agrobacterium tumefaciens-mediated genetic
transformation. The mutants were unable to hydrolyse the lactone
bond of zearalenone and displayed an increased susceptibility to
zearalenone. Thus it appears that zearalenone acts as an agent of
interference competition (active protection of substrate from
colonization by competitors) and that zearalenone lactonase
protects G. roseum from its fungitoxic effect.
To assess the effect of zearalenone on plants a sensitive in vitro
seedling growth assay was used to investigate the phytotoxic effect
of zearalenone on Arabidopsis thaliana wild type and transformants
expressing the gene zes2 from G. roseum. While wild type plants
after exposure to zearalenone exhibited dwarfism. The transgenic
plants were more tolerant to zearalenone showing almost no
symptoms. These data indicate that zearalenone lactonase protects
transgenic Arabidopsis plants from the toxic effects of this
mycotoxin.
Genetic and methodical requirements for the A. tumefaciens-mediated
transformation of G. roseum and the identification of mutants
deficient in zearalenone lactonase production were developed. We
further applied the protocol to transform different species of
filamentous fungi (Leptosphaeria maculans, Verticillium
longisporum, Fusarium verticillioides and Trichoderma harzianum).
In order to maximize the number of transformants, the ratio of A.
tumefaciens cells to fungal spores and the duration of the
co-cultivation was optimized. A practical method for suitable
saturationmutagenesis is described that can be used to generate
hundreds of independent transformation events in one
experiment.
Specific knowledge of concentrations which actually occur in the
infected tissue would facilitate the understanding of the
phytotoxic effects caused by zearalenone. We harnessed the
zearalenone-sensing ability of G. roseum to develop a novel kind of
biosensor for zearalenone. Genetically engineered fusions of the
zearalenone-sensing promoter-element and reporter genes encoding
Green Fluorescent Protein (GFP) and Gaussia princeps lucifease were
constructed. G. roseum strains harbouring the fusion constructs act
as specific and highly sensible systems in a bioassay which exhibit
fluorescence (GFP) or emit light (luciferase) upon exposure to
zearalenone. The strains were used for real-time macroscopic
imaging of zearalenone biodistribution on Fusarium infected maize
tissue. Based on these imaging properties, the place of origin of
native zearalenone synthesis in target tissue was discovered.
Fluorescent proteins like the green fluorescent protein (GFP) have
become enormously popular as tools for monitoring gene expression
in filamentous fungi. Biochemical assays using GFP reporter gene
technique are usually performed in 96-well microtiter plates and
the fluorescence is determined with a fluorometer. A new method was
developed which uses a real time thermal cycler to measure GFP
expression. In this way, fluorometers which are not commonly
encountered in molecular biology laboratories, can be replaced by
widely accessible thermal cyclers. | de |
| dc.subject.topic | Agricultural Sciences | de |
| dc.subject.ger | Zearalenon | de |
| dc.subject.ger | Gliocladium roseum | de |
| dc.subject.ger | Agrobacterium tumefaciens | de |
| dc.subject.ger | Biosensor | de |
| dc.subject.ger | Biologische Funktion | de |
| dc.subject.eng | Zearalenone | de |
| dc.subject.eng | Gliocladium roseum | de |
| dc.subject.eng | Agrobacterium tumefaciens | de |
| dc.subject.eng | biosensor | de |
| dc.subject.eng | biological function | de |
| dc.subject.bk | 42.13 | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-webdoc-1799-5 | de |
| dc.identifier.purl | webdoc-1799/ | de |
| dc.identifier.ppn | 588951773 | de |