A proteomic approach to investigate agronomic traits in barley using the genetic variation of mapping populations [Elektronische Ressource] / von Katja Witzel

Lachmann

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

A proteomic approach to investigate agronomic
traits in barley using the genetic variation of
mapping populations


Dissertation

Zur Erlangung des akademischen Grades
Doctor rerum naturalium (Dr. rer. nat.)

vorgelegt der

Naturwissenschaftlichen Fakultät I
Biowissenschaften
der Martin-Luther-Universität Halle-Wittenberg

von

Frau Katja Witzel
Geboren am 07.04.1978 in Nordhausen

Gutachter/in
1. Prof. Dr. Klaus Humbeck
2. Priv. Doz. Dr. Hans-Peter Mock
3. Assoc. Prof. Dr. Christine Finnie

Halle/Saale, den 11.09.2008


urn:nbn:de:gbv:3-000014437
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000014437] i
1. Abstract ........................................................................................................................... 1
2. Introduction .................................................................................................................... 4
2.1. Approaches for crop improvement using mapping populations....................................... 4
2.2. Maturation and germination of barley grains ................................................................... 7
2.3. Adaptation to salt stress and characteristics of salinity tolerance .................................... 8
2.3.1. Physiology of salt stress ........................................................................................ 9
2.3.2. Plant strategies for adaptation to salt stress......................................................... 11
2.3.3. Targets for improving salt tolerance in plants..................................................... 11
Control of salt uptake and transport .................................................................... 11
Protection against osmotic stress ........................................................................ 12
Promotion of plant growth in saline soil ............................................................. 13
2.4. Proteomics for the dissection of stress responses........................................................... 13
2.5. Scientific aims of the work............................................................................................. 15
3. Materials and Methods ................................................................................................ 17
3.1. Plant material.................................................................................................................. 17
3.1.1. Barley genotypes and mapping populations........................................................ 17
3.1.2. Plant growth in hydroponic culture and salinity treatments................................ 17
3.1.3. Growth measurements......................................................................................... 18
3.2. Protein extraction methods and concentration measurements........................................ 18
3.2.1. Extraction of water-soluble protein fraction from mature grains........................ 18
3.2.2. Extraction of storage proteins from mature grains.............................................. 18
3.2.3. Protein extraction from roots............................................................................... 19
3.2.4. Enrichment and extraction of plasma membrane proteins from roots ................ 19 ent of plasma membranes by two-phase partitioning method............... 19
Enrichment of hydrophobic proteins by batch reversed-phase chromatography 20
3.2.5. Determination of protein concentration in crude mixtures ................................. 21
3.3. Protein separation methods............................................................................................. 21
3.3.1. SDS-PAGE.......................................................................................................... 21
3.3.2. Two-dimensional gel electrophoresis.................................................................. 21
3.4. Visualization of proteins and image acquisition............................................................. 22
3.4.1. Colloidal Coomassie Brilliant Blue staining....................................................... 22
3.4.2. Ruthenium staining 22
3.4.3. Silver staining...................................................................................................... 23
3.5. Relative quantitation of proteins and peptides ............................................................... 23
3.5.1. Image analysis of 2-dimensional gel patterns ..................................................... 23
3.5.2. Label-free quantitation of tryptic peptides.......................................................... 23
Protein digest preparation.................................................................................... 23
Liquid chromatography and mass spectrometry configuration........................... 24
Data processing and protein identification 24
3.5.3. Western blotting.................................................................................................. 24
3.6. Protein identification...................................................................................................... 25
3.6.1. Peptide mass fingerprinting by MALDI-TOF MS.............................................. 25
3.6.2. De novo sequencing of peptides by tandem MS/MS .......................................... 25

ii
3.7. Molecular cloning techniques......................................................................................... 26
3.7.1. Bacterial strains, vectors and oligonucleotides ................................................... 26
3.7.2. RNA preparation and Northern blot analysis...................................................... 27
3.7.3. DNA preparation and Southern blot analysis 28
3.8. Plant transformation....................................................................................................... 28
3.9. Statistical analysis.... 28
3.9.1. Cluster analysis of protein patterns ..................................................................... 28
3.9.2. Quantitative trait loci analysis for protein expression......................................... 29
3.9.3. Principle component analysis of peptide profiles derived from LC-based mass
spectrometry........................................................................................................ 29
4. Results............................................................................................................................ 30
4.1. Grain protein profiling of the Brenda x HS213 mapping population............................. 30
4.1.1. Construction of a protein reference map from barley cv. Brenda mature grains 30
4.1.2. Expression profiling of proteins in mature grains............................................... 32
Analysis of the water-soluble protein fraction of barley grains from the first
experiment............................................................................................ 33 the second
experiment 37
Calculation of QTL for protein expression ......................................................... 40
Analysis of alcohol-soluble grain storage proteins ............................................. 45
4.2. Grain proteome analysis of accessions from the Oregon Wolfe Barley mapping
population differing in salt stress response .................................................................... 48
4.2.1. Comparative proteome profiling ......................................................................... 48
4.2.2. Identification of candidate proteins by mass spectrometry................................. 51
4.2.3. Cloning and overexpression of candidate proteins ............................................. 53
4.3. Proteome analysis of accessions from the Steptoe Morex mapping population with
contrasting response towards salt stress at different developmental stages ................... 57
4.3.1. Comparative proteome profiling of mature grains.............................................. 57 oteins by mass spectrometry................................. 60
Cloning and overexpression of candidate proteins ............................................. 61
4.3.2. Assessment of salt tolerance at the seedling stage of the Steptoe and Morex
parental lines ....................................................................................................... 62
4.3.3. Cultivar-specific and salt stress-affected protein expression in roots of Steptoe
and Morex ........................................................................................................... 64
4.3.4. Identification of progeny lines showing a similar response towards salt stress at
the seedling stage as the parent lines................................................................... 75
4.3.5. Plasma membrane protein expression in roots of Steptoe and Morex subjected to
salt stress ............................................................................................................. 76
Enrichment of plasma membranes using two-phase partitioning method .......... 77
Identification of plasma membrane proteins by LC-based mass spectrometry .. 78
Comparative analysis of the plasma membrane proteome of Steptoe and Morex
roots under salt stress conditions ......................................................... 83
5. Discussi