Analisis proteomico

Proteomic analysis of the biological
control fungus Trichoderma

Jasmine Grinyer
Bachelor of Technology (Biotechnology), Macquarie University
Bachelor of Technology (Biotechnology) Hons,Macquarie University

A thesis presented to Macquarie University in partial fulfillment for the
degree of Doctor of Philosophy.

Proteome Systems Ltd.,
The Department of BiologicalSciences, Macquarie University and
Department of Chemistry and Biomolecular Sciences, Macquarie
University,
Sydney, NSW, Australia
August 2006 Table of contents
Abstract 4
Declaration 6Publications 7
Abbreviations 9
Acknowledgements 10
1. Introduction 12
1.1 Proteomics and two-dimensional electrophoresis 13
1.1.1 Recent advances in the preparation ofproteomic samples 15
1.1.2 Simplification to ultimately resolve more of the proteome 20
1.1.3 Visualisation of the proteome 24
1.1.4 Protein identification by massspectrometry and alternative
techniques
27
1.1.5 Differential gel expression (DIGE) and image analysis 33
1.2 A proteomic approach to study the filamentous fungus - Trichoderma35
1.2.1 Overview of biological control mediated by Trichoderma spp. 36
1.2.2 Cell wall degrading enzymes of Trichoderma 38
1.2.3 Synergism of fungalbiocontrol agents and enzymes 51
1.2.4 Enhancing the biocontrol capabilities of Trichoderma spp. 52
1.3 Aims of the thesis 55
2. Materials and Methods 56
3. Results and Discussion57
3.1 Method development for the display and identification of fungal
proteins by 2DE and mass spectrometry
57
3.1.1 Improved 2-DE of microorganisms after acidic extraction(Publication I)
58
3.1.2 Fungal proteomics: initial mapping of biological control strain
Trichoderma harzianum (Publication II)
70
3.1.3 Fungal proteomics: mapping the...