Reduction of PSII accumulation through manipulating psbD translational initiation:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | VII, 130 Bl. Ill., graph. Darst. |
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| adam_text | IMAGE 1
TABLE O F CONTENTS
TABLE O F CONTENTS I
ABBREVIATIONS V
UNITS O F MEASURE AND SYMBOLS VII
1. INTRODUCTION 1
1.1. PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN 1
1.1.1. OVERVIEW 1
1.1.2. ADAPTATION O F PHOTOSYNTHETIC CAPACITY 4
1.2. ORGANIZATION O F THE PHOTOSYNTHETIC COMPONENTS IN THE THYLAKOID
MEMBRANE 6
1.2.1. THYLAKOID STRUCTURE 6
1.2.2. SEGREGATION O F THE PHOTOSYNTHETIC COMPONENTS 7
1.3. BIOGENESIS AND TURNOVER O F PHOTOSYSTEM II 9
1.3.1. ASSEMBLY AND PROTEIN SYNTHESIS O F PSII 9
1.3.2. DE NOVO SYNTHESIS O F THE D1 PROTEIN IN THE PSII REPAIR CYCLE 13
1.3.3. HETEROGENEITY O F PSII 13
1.4. KINETICS O F THE XANTHOPHYLL CYCLE IN THE ENERGY-DEPENDENT
NON-PHOTOCHEMICAL
QUENCHING 14
1.4.1. NON-PHOTOCHEMICAL QUENCHING 14
1.4.2. KINETICS O F THE XANTHOPHYLL CYCLE 15
1.5. OBJECTIVE 16
2. MATERIALS AND METHODS 18
2.1. MATERIALS 18
2.1.1. CHEMICALS AND CONSUMABLES 18
2.1.1.1. SUPPLIERS FOR COMMON CHEMICALS AND CONSUMABLES 18
2.1.1.2. SPECIAL CHEMICALS AND CONSUMABLES 18
2.1.2. ENZYMES AND REACTION KITS 21
2.1.3. MOLECULAR WEIGHT MARKERS 21
2.1.4. ANTIBODIES 21
2.1.5. ORGANISMS 22
2.1.5.1. BACTERIA 22
2.1.5.2. PLANT 22
2.1.6. MEDIA FOR BACTERIA AND PLANT TISSUE CULTURE 22
2.1.6.1. BACTERIAL MEDIA 22
2.1.6.2. MEDIA FOR PLANT TISSUE CULTURE 23
2.1.6.2.1. COMMON MINERAL SALTS FOR MEDIUM PREPARATION 23
2.1.6.2.2. MEDIA FOR PLANT MAINTENANCE AND SHOOT REGENERATION AFTER
NUCLEAR TRANSFORMATION 23
I
HTTP://D-NB.INFO/1031247572
IMAGE 2
2.1.6.2.3. MEDIA FOR PLANT MAINTENANCE (RM) AND SHOOT REGENERATION
(RMOP) AFTER
PLASTID TRANSFORMATION 23
2.1.6.3. ANTIBIOTICS 24
2.1.7. OLIGONUCLEOTIDES AND BASIC VECTORS 24
2.1.7.1. OLIGONUCLEOTIDES 24
2.1.7.2. BASIC VECTORS 26
2.1.8. EQUIPMENT 26
2.1.9. SOFTWARE AND DATABASES 27
2.2. METHODS 28
2.2.1. GROWTH CONDITIONS O F BACTERIA AND PLANTS 28
2.2.1.1. BACTERIAL GROWTH CONDITIONS 28
2.2.1.1.1. ESCHERICHIA COLI 28
2.2.1.1.2. AGROBACTERIUM TUMEFACIENS 28
2.2.1.2. PLANT GROWTH CONDITIONS IN TISSUE CULTURE AND SOIL 28
2.2.1.3. SEED STERILIZATION 29
2.2.2. MOLECULAR BIOLOGY METHODS 29
2.2.2.1. NUCLEIC ACID ANALYSIS 29
2.2.2.1.1. PLASMID DNA ISOLATION FROM ESCHERICHIA COLI 29
2.2.2.1.2. DNA ISOLATION FROM NICOTIANA TABACUM 30
2.2.2.1.3. POLYMERASE CHAIN REACTION (PCR) 30
2.2.2.1.4. DNA AGAROSE GEL ELECTROPHORESIS 31
2.2.2.1.5. PURIFICATION O F DNA FROM AGAROSE GELS 32
2.2.2.1.6. CLEAVAGE O F DNA WITH RESTRICTION ENZYMES AND RESTRICTION
FRAGMENT LENGTH POLYMORPHISM (RFLP) ANALYSIS 32
2.2.2.1.7. DEPHOSPHORYLATION O F LINEAR PLASMID DNA 32
2.2.2.1.8. LIGATION O F DNA FRAGMENTS 32
2.2.2.1.9. DNA SEQUENCING 33
2.2.2.1.10. ISOLATION OF TOTAL RNA 33
2.2.2.1.11. POLYSOME PROFILING ANALYSIS AND RNA PURIFICATION FROM
SUCROSE GRADIENTS 33
2.2.2.1.12. RNA DENATURING AGAROSE GEL ELECTROPHORESIS 35
2.2.2.1.13. DETERMINATION O F NUCLEIC ACID CONCENTRATIONS 35
2.2.2.1.14. IMMOBILIZATION O F NUCLEIC ACIDS 36
2.2.2.1.15. RADIOACTIVE LABELLING O F NUCLEIC ACIDS FOR HYBRIDIZATION 37
2.2.2.1.16. HYBRIDIZATION AND DETECTION O F IMMOBILIZED NUCLEIC ACIDS 37
2.2.2.1.17. REMOVAL O F HYBRIDIZED PROBES FROM THE MEMBRANE 38
2.2.2.2. PROTEIN ANALYSIS 38
2.2.2.2.1. SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) 38
2.2.2.2.2. PROTEIN IMMUNOBLOT 40
2.2.3. VECTOR CONSTRUCTION 41
2.2.3.1. CONSTRUCTION O F PLASTID TRANSFORMATION VECTORS WITH
SITE-DIRECTED MUTATIONS OF THE PSBD GENE 41
II
IMAGE 3
2.2.3.2. CONSTRUCTION O F A VDE RNAI VECTOR 43
2.2.4. BACTERIAL TRANSFORMATION 44
2.2.4.1. TRANSFORMATION O F ESCHERICHIA COLI 44
2.2.4.2. TRANSFORMATION O F AGROBACTERIUM TUMEFACIENS 44
2.2.5. TOBACCO TRANSFORMATION 44
2.2.5.1. PLASTID TRANSFORMATION 44
2.2.5.2. AGROBACTERIUM-MEDIATED NUCLEAR TRANSFORMATION 45
2.2.5.3. INHERITANCE TEST 46
2.2.6. PHYSIOLOGICAL MEASUREMENT METHODS 46
2.2.6.1. SPECTROSCOPIC AND PHOTOSYNTHESIS-RELATED MEASUREMENT 46
2.2.6.1.1. OXYGEN EXCHANGE MEASUREMENT 46
2.2.6.1.2. IN VIVO CHLOROPHYLL A FLUORESCENCE MEASUREMENT 46
2.2.6.1.3. REDUCTION KINETICS O F PC AND P700 47
2.2.6.1.4. ELECTROCHROMIC SHIFT (ECS) MEASUREMENT 48
2.2.6.1.5. CHLOROPHYLL DETERMINATION 48
2.2.6.1.6. LEAF ABSORPTANCE MEASUREMENT 49
2.2.6.1.7. ISOLATION O F PLANT THYLAKOIDS 49
2.2.6.1.8. LOW TEMPERATURE CHLOROPHYLL A FLUORESCENCE MEASUREMENT 50
2.2.6.1.9. PSI QUANTIFICATION IN ISOLATED THYLAKOIDS 50
2.2.6.1.10. QUANTIFICATION O F PSII AND THE CYT B ( F COMPLEX IN
ISOLATED THYLAKOIDS 51
2.2.6.1.11. SEPARATION O F PIGMENTED PHOTOSYNTHETIC COMPLEXES O F
THYLAKOIDS 52
2.2.6.2. ULTRASTRUCTURE O F THYLAKOID MEMBRANES BY TRANSMISSION ELECTRON
MICROSCOPY 52
2.2.6.3. QUANTIFICAITON O F XANTHOPHYLL CYCLE CAROTENOIDS BY
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 53
3. RESULTS 54
3.1. GENERATION O F TOBACCO PLASTID TRANSFORMANTS WITH THE MUTATED PSBD
GENE 54
3.1.1. CONSTRUCTION O F PLASTID TRANSFORMATION VECTORS AND SELECTION O F
HOMOPLASMIC TRANSPLASTOMIC LINES 54
3.1.2. CONFIRMATION O F HOMOPLASMY 54
3.2. PHENOTYPES O F THE TRANSPLASTOMIC PLANTS WITH THE MUTATED PSBD GENE
56
3.2.1. GROWTH PHENOTYPE O F THE TI TRANSPLASTOMIC PLANTS 56
3.2.2. GROWTH PHENOTYPE O F THE AS -PSBD PLANTS IN TISSUE CULTURE 56
3.2.3. CHLOROPHYLL CONTENT O F THE TRANSPLASTOMIC PLANTS 59
3.3. MOLECULAR ANALYSIS O F THE TRANSPLASTOMIC PLANTS WITH THE MUTATED
PSBD GENE.. 60
3.3.1. TRANSCRIPT EXPRESSION ANALYSIS 60
3.3.1.1. ANALYSIS O F PSBD AND PSBC MRNAS 60
3.3.1.2. ANALYSIS O F OTHER TRANSCRIPTS 64
3.3.2. POLYSOME PROFILING ANALYSIS 65
3.3.3. ANALYSIS O F PHOTOSYNTHETIC PROTEIN ABUNDANCE 67
III
IMAGE 4
3.4. CHLOROPLAST ULTRASTRUCTURE IN THE TRANSPLASTOMIC PLANTS WITH AADA
INSERTION
IN ANTISENSE ORIENTATION RELATIVE TO P S B D 71
3.5. FUNCTIONAL ANALYSIS O F THE TRANSPLASTOMIC PLANTS WITH THE MUTATED
PSBD GENE . 74
3.5.1. CHLOROPHYLL A TO B RATIO 74
3.5.2. CHLOROPHYLL A FLUORESCENCE ANALYSIS AND LOW TEMPERATURE
CHLOROPHYLL A FLUORESCENCE EMISSION SPECTRUM 75
3.5.3. MAXIMUM ELECTRON TRANSPORT RATES O F PSI AND PSII 81
3.5.4. REDUCTION KINETICS O F PC AND P700 83
3.5.5. SUCROSE GRADIENT ANALYSIS O F PIGMENTED PHOTOSYNTHETIC COMPLEXES
84
3.6. CHARACTERIZATION O F THE VDE RNAI TOBACCO PLANTS 85
3.6.1. CONSTRUCTION O F VDE RNAI VECTORS AND SELECTION O F RNAI LINES 85
3.6.2. EXPRESSION O F VDE TRANSCRIPTS, LIGHT-INDUCED ZEAXANTHIN
FORMATION, AND GROWTH PHENOTYPE IN THE VDE RNAI LINES 86
3.6.3. ANALYSIS O F NON-PHOTOCHEMICAL QUENCHING 87
3.6.4. ELECTROCHROMIC SHIFT (ECS) MEASUREMENT 88
4. DISCUSSION 90
4.1. EFFECTS O F THE AADA INSERTION AND MUTATIONS IN THE PSBD GENE 90
4.1.1. GENETIC APPROACH TO REDUCE PSII ACCUMULATION 90
4.1.2. MODULATION O F THE PSBD GENE EXPRESSION 91
4.1.3. INCREASE O F THE PSBA TRANSCRIPT ACCUMULATION IN THE AS -PSBD
LINES 92
4.1.4. FACTORS O F THE SOIL AND TISSUE CULTURE CONDITIONS WHICH AFFECT
THE PHENOTYPES O F THE TRANSPLASTOMIC PLANTS 93
4.1.5. STOICHIOMETRY O F THE PHOTOSYNTHETIC APPARATUS 94
4.1.6. LIGHT-HARVESTING, PSII PHOTOCHEMISTRY, AND LINEAR ELECTRON
TRANSPORT 95
4.1.7. ENERGY DISSIPATION AND MONOMERIZATION O F LHCII 99
4.1.8. CHLOROPLAST DEGRADATION 99
4.1.9. CONCLUDING REMARKS 100
4.2. OUTLOOK 100
5. SUMMARY 102
6. ZUSAMMENFASSUNG 104
7. APPENDIX 106
7.1. LIST O F FIGURES 106
7.2. LIST O F TABLES 108
8. ACKNOWLEDGEMENTS 109
9. CONFERENCE ATTENDANCE I L L
10. EIDESSTATTLICHE VERSICHERUNG 112
11. REFERENCES 113
IV
|
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| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.4623952 |
| dewey-search | 572.4623952 |
| dewey-sort | 3572.4623952 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | Fu, Han-Yi Verfasser aut Reduction of PSII accumulation through manipulating psbD translational initiation von Han-Yi Fu 2012 VII, 130 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Potsdam, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025404101&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Fu, Han-Yi Reduction of PSII accumulation through manipulating psbD translational initiation |
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| title | Reduction of PSII accumulation through manipulating psbD translational initiation |
| title_auth | Reduction of PSII accumulation through manipulating psbD translational initiation |
| title_exact_search | Reduction of PSII accumulation through manipulating psbD translational initiation |
| title_full | Reduction of PSII accumulation through manipulating psbD translational initiation von Han-Yi Fu |
| title_fullStr | Reduction of PSII accumulation through manipulating psbD translational initiation von Han-Yi Fu |
| title_full_unstemmed | Reduction of PSII accumulation through manipulating psbD translational initiation von Han-Yi Fu |
| title_short | Reduction of PSII accumulation through manipulating psbD translational initiation |
| title_sort | reduction of psii accumulation through manipulating psbd translational initiation |
| topic_facet | Hochschulschrift |
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