Molecular biological and biochemical studies of proteolytic enzymes of the cereal pathogen fusarium graminearum [Elektronische Ressource] / vorgelegt von Matthias Hellweg

Matthias Hellweg

Sujets

• Biologie
• Garten

Informations

• Publié par: westfalische_wilhelms-universitat_munster
• Publié le: 01 janvier 2003
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Botanik
Molecular biological and biochemical studies of
proteolytic enzymes of the cereal pathogen
Fusarium graminearum
Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften im Fachbereich Biologie
der Mathematisch-Naturwissenschaftlichen Fakultät
der Westfälischen Wilhelms-Universität Münster
vorgelegt von
Matthias Hellweg
aus Bremen
- 2003 -
Dekan: Prof. Dr. A. Steinbüchel
Erster Gutachter: Prof. Dr. B. Moerschbacher
Zweiter Gutachter: Prof. Dr. W. Stöcker
Tag der mündlichen Prüfung: 22.7.2003
Tag der Promotion: groovy, groovy, funky fungi
- anonymous 2003 -Contents I
Table of Contents
Table of contents................................................................................................I
Abbreviations....................................................................................................V
1 INTRODUCTION...........................................................................1
1.1 Introduction to plant disease resistance mechanisms..............2
1.2 The structure of plant cell walls................................................................4
1.3 Cell wall degrading enzymes.....................................................................8
1.4 General classification of proteases..........................................................9
1.5 Fusarium graminearum............................................................................10
1.6 Objectives of the present work...............................................................13
2 MATERIALS & METHODS..................................................................14
2.1 Materials.....................................................................................................14
2.1.1 Chemicals and materials.....................................................................14
2.1.2 Organisms – growth conditions and strain maintenance.....................14
2.1.2.1 Fungal isolates.......................................................................................14
2.1.2.2 Plant cultivars.........................................................................................15
2.1.2.3 Bacteria..................................................................................................15
2.2 Methods......................................................................................................15
2.2.1 Cultivation of Fusarium graminearum in submerged culture...............15
2.2.1.1 CM-medium, minimal medium and protease induction medium.15
2.2.1.2 Preculture...............................................................................................16
2.2.1.3 Induction of conidia................................................................................16
2.2.1.4 Induction of protease secretion..............................................................18
2.2.1.5 Growth rate and kinetics of protease secretion......................................18
2.2.1.6 Kinetics of initial protease secretion – Mass inoculation .....................18
2.2.1.7 Variations in protein derived nitrogen levels...........................................19
2.2.1.8 Variations in carbohydrate levels...........................................................20
2.2.1.9 Isolated wheat cell wall material as substrate........................................20Contents II
2.2.2 Cultivation of Fusarium graminearum on agar plates..........................20
2.2.3 In planta cultivation – Detached leaf petri dish assay..........................21
2.2.4 Protein biochemical methods..............................................................22
2.2.4.1 Photometric assay for protease activity..................................................22
2.2.4.2 pH-profile and temperature optimum.....................................................23
2.2.4.3 Inhibitor studies......................................................................................23
2.2.4.4 Cation exchange perfusion chromatography..........................................24
2.2.4.5 SDS polyacrylamide gel elecrophoresis (SDS-PAGE)...........................25
2.2.4.6 'Slow' Coomassie staining of acrylamide gels........................................27
2.2.4.7 Silver staining of acrylamide gels...........................................................27
2.2.4.8 Standard zymography............................................................................28
2.2.4.9 Blotting zymography...............................................................................28
2.2.5 Molecular biology................................................................................30
2.2.5.1 Isolation of fungal DNA..........................................................................30
2.2.5.2 Standard PCR.......................................................................................31
2.2.5.3 Touch-down PCR..................................................................................32
2.2.5.4 Inverse PCR..........................................................................................32
2.2.5.5 TAIL PCR..............................................................................................34
2.2.5.6 Standard agarose gel electrophoresis...................................................35
2.2.5.7 Purification of PCR fragments...............................................................36
2.2.5.8 Cloning of PCR fragments and colony PCR..........................................36
2.2.5.9 Plasmid purification and sequencing of cloned PCR fragments............37
2.2.5.10 Isolation of fungal RNA..........................................................................38
2.2.5.11 cDNA production and RT-PCR..............................................................40
2.2.5.12 Quantitative expression analysis...........................................................40
2.3 Computer aided methods.........................................................................41
2.3.1 Documentation of polyacrylamide gels, agarose gels, macroscopic
and microscopic images......................................................................41
2.3.2 Analysis of DNA and amino acid sequences.......................................41
2.3.3 Databases...........................................................................................42
2.3.4 Sequence alignment............................................................................42
2.3.5 Prediction of protein characteristics based on amino acid
sequences...........................................................................................42Contents III
3 RESULTS......................................................................................43
3.1 General characterization of fungal growth.............................................43
3.1.1 Growth on CM agar.............................................................................43
3.1.2 Submerged culture in CM-medium......................................................44
3.1.3 Formation of conidia............................................................................45
3.2 Optimization of the photometric protease assay...................................46
3.3 Development of protease induction medium.........................................47
3.4 Characterization of growth in protease induction medium..................49
3.4.1 Growth on PI-agar compared to CM- and minimal-agar......................49
3.4.2 Submerged culture in PI-medium compared to CM- and
minimal medium..................................................................................51
3.5 Kinetics of protease secretion.................................................................53
3.5.1 Overview of protease secretion – daily samples.................................53
3.5.2 Basic characterization of the protease activity induced in
PI-medium...........................................................................................54
3.5.2.1 pH-optimum............................................................................................54
3.5.2.2 Temperature optimum............................................................................54
3.5.2.3 Inhibitor studies......................................................................................55
3.5.3 Kinetics of initial protease secretion – Mass inoculation......................56
3.5.4 Variations in nitrogen and carbohydrate levels – Mass inoculation.....58
3.5.5 Growth in CM- and PI-medium – Mass inoculation.............................61
3.5.6 pH of the medium during cultivation in PI- and pPI-medium with
variations in carbon levels – Mass inoculation....................................62
3.5.7 Protease activity and pH of the medium during growth in