The role of thylakoid ATP synthase subunit gamma and attempts to transform the organelles of A. thaliana [Elektronische Ressource] / vorgelegt von Cristina Dal Bosco

Cristina Dal Bosco

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

The role of thylakoid ATP synthase subunit gamma
and attempts to transform the organelles of
A. thaliana





Dissertation der Fakultät für Biologie
der Ludwig-Maximilians-Universität München




vorgelegt von
Cristina Dal Bosco
aus Italien



2006




























1. Gutachter: Prof. Dr. Reinhold G. Herrmann
2. Gutachter: Prof. Dr. Hugo Scheer

Tag der mündlichen Prüfung: März, den 31, 2006

2 Content
CONTENT
ABBREVIATIONS............................................................................................................................................ 6
1 INTRODUCTION..... 8
1.1 THYLAKOID MEMBRANE PHOTOSYNTHETIC COMPLEXES IN HIGHER PLANTS................ 8
1.2 ATP SYNTHASE: STRUCTURE, FUNCTION AND REGULATION............................................... 9
1.3 ATP SYNTHASE γ SUBUNIT ......................................................................................................... 11
1.3.1 REDOX REGULATION........................................................................................................... 11
1.3.2 ATPC1 VERSUS ATPC2............................................................................................................ 12
1.4 PHOTOCHEMISTRY ..................................................................................................................... 13
1.4.1 NON-PHOTOCHEMICAL QUENCHING - NPQ.................................................................... 13
1.4.2 THE CENTRAL ROLE OF PSBS FOR qE .............................................................................. 14
1.5 TOWARDS ORGANELLE TRANSFORMATION IN ARABIDOPSIS THALIANA ......................... 14
1.5.1 ARABIDOPSIS THALIANA: A MODEL FOR HIGHER PLANTS .......................................... 15
1.5.2 PLASTID TRANSFORMATION ............................................................................................. 16
1.6 GOALS OF THE PROJECT ........................................................................................................... 19
2 MATERIALS ......................................................................................................................................... 20
2.1 CHEMICALS, RADIACTIVE SUBSTANCES, WORKING MATERIAL ......................................... 20
2.2 PLANT MATERIAL ........................................................................................................................ 20
2.3 BACTERIAL STRAINS ................................................................................................................... 21
2.4 PLASMIDS...... 21
2.4.1 CLONING VECTORS .............................................................................................................. 21
2.4.2 EXPRESSION VECTORS ........................................................................................................ 21
2.4.3 BINARY VECTORS................................................................................................................. 21
2.5 OLIGONUCLEOTIDES 22
2.6 MEDIA............................................................................................................................................ 24
2.6.1 MEDIA FOR BACTERIA......................................................................................................... 24
2.6.2 SOLUTIONS AND MEDIA FOR PROTOPLAST AND TISSUE CULTURE ....................... 24
2.7 ENZYMES........ 26
2.8 ANTIBODIES.. 27
3 METHODS.............. 28
3.1 BACTERIA GROWTH AND MANIPULATION.............................................................................. 28
3.1.1 CULTIVATION OF BACTERIA ............................................................................................. 28
3.1.2 PREPARATION OF COMPETENT CELLS............................................................................ 28
3.1.3 TRANSFORMATION OF BACTERIA (HEAT SHOCK) ....................................................... 29
3.2 PLANT GROWTH AND MANIPULATION.................................................................................... 29
3.2.1 SEED STERILIZATION, PLANT GROWTH AND MUTANT SELECTION ....................... 29
3.2.2 PROTOPLAST ISOLATION.................................................................................................... 30
3.2.3 PEG TREATMENT OF PROTOPLAST................................................................................... 30
3.2.4 DNA TRANSFER BY THE BIOLISTIC METHOD................................................................ 31
3
3.2.5 AGROBACTERIA MEDIATED NUCLEAR TRANSFORMATION OF ARABIDOPSIS........ 31
3.2.6 GUS ASSAY ............................................................................................................................. 32
3.3 NUCLEIC ACID MANIPULATION ............................................................................................... 32
3.3.1 RAPID ISOLATION OF GENOMIC DNA FOR PCR USE .................................................... 32
3.3.2 ISOLATION OF TOTAL RNA................................................................................................. 33
3.3.3 ISOLATION OF DNA FOR PCR USE FROM YEAST........................................................... 33
3.3.4 PLASMID ISOLATION FROM E. COLI................................................................................. 33
3.3.5 ENZYMATIC ASSAYS............................................................................................................ 34
3.3.6 NORTHERN ANALYSIS......................................................................................................... 35
3.3.7 HYBRIDIZATION OF NUCLEIC ACIDS............................................................................... 35
3.3.8 REVERSE TRANSCRIPTION (RT)-PCR................................................................................ 36
3.3.9 QUANTITATIVE REAL-TIME RT-PCR 36
3.4 PROTEIN AND PIGMENT ANALYSES ......................................................................................... 37
3.4.1 EXTRACTION OF THYLAKOID MEMBRANE PROTEINS................................................ 37
3.4.2 EXTRACTION OF TOTAL PROTEINS.................................................................................. 38
3.4.3 ISOLATION OF MAJOR THYLAKOID MEMBRANE COMPLEXES OF ARABIDOPSIS.39
3.4.4 ISOELECTRO FOCUSING OF THYLAKOID MEMBRANE PROTEINS............................ 40
3.4.5 IN VIVO LABELLING OF LEAF PROTEINS......................................................................... 40
3.4.6 MEASURMENT OF PROTEIN AND CHLOROPHYLL CONCENTRATION ..................... 41
3.4.7 SODIUM DODECYL SULFATE POLYACRILAMIDE GEL ELECTROPHORESIS (SDS-
PAGE) 41
3.4.8 PROTEIN DETECTION ........................................................................................................... 41
3.5 CHLOROPHYLL A FLUORESCENCE ANALYSIS........................................................................ 43
3.6 GENETIC METHODS.................................................................................................................... 44
3.6.1 MAPPING OF THE DPA1 MUTATION.................................................................................. 44
3.6.2 GENERATION OF THE PSBSxDPA1 DOUBLE MUTANT .................................................. 44
3.7 ELECTRON MICROSCOPY .......................................................................................................... 45
3.8 PHOTOPHOSPHORYLATION ...................................................................................................... 45
4 RESULTS ............................................................................................................................................... 46
4.1 KNOCK-OUT MUTANT OF THE ATP SYNTHASE γ SUBUNIT, DPA1 (DEFICIENCY OF
PLASTID ATP SYNTHASE 1)....................................................................................................................... 46
4.1.1 ISOLATION AND PHENOTYPE OF THE DPA1 MUTANT................................................. 46
4.1.2 INACTIVATION OF THE ATPC1 GENE................................................................................ 47
4.1.3 ACCUMULATION OF THE MAJOR PHOTOSYNTHETIC COMPLEXES IN DPA1 ......... 49
4.1.4 PLASTID ATP SYNTHASE ACTIVITY IN DPA1 MUTANT ............................................... 50
4.1.5 PHOTOSYNTHETIC ACTIVITY IN DPA1 ............................................................................ 51
4.1.6 LIGHT INDUCED THYLAKOID SWELLING IN DPA1....................................................... 55
4.1.7 PSBSXDPA1 DOUBLE MUTANT........................................................................................... 55
4.1.8 EXPRESSION OF NUCLEAR-ENCODED GENES IN DPA1 58
4.2 DPA1 COMPLEMENTED WITH A MUTATED FORM OF ATPC1 ......................................