Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | kostenfrei Inhaltsverzeichnis |
| Beschreibung: | Nebentitel: Identifizierung und Charakterisierung von Regulatoren der 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |
| Beschreibung: | IX, 143 Bl. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |c by Wei Guo |
| 246 | 1 | 3 | |a Identifizierung und Charakterisierung von Regulatoren der 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |
| 264 | 1 | |c 2013 | |
| 300 | |a IX, 143 Bl. |b Ill., graph. Darst. | ||
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| 500 | |a Nebentitel: Identifizierung und Charakterisierung von Regulatoren der 2-Cys-Peroxiredoxin A in Arabidopsis thaliana | ||
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Datensatz im Suchindex
| _version_ | 1804151493111054336 |
|---|---|
| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
TABLE OF CONTENTS I
SUMMARY V
LIST OF ABBREVIATIONS VII
1 INTRODUCTION 1
1.1 COMMUNICATION BETWEEN NUCLEUS AND ORGANELLES 1
1.1.1 FROM ENDOSYMBIONTS TO ORGANELLES 1
1.1.2 COMMUNICATION BETWEEN PLASTIDS AND NUCLEUS 1
1.2 RETROGRADE SIGNALING FROM CHLOROPLAST TO NUCLEUS 2
1.2.1 REACTIVE OXYGEN SPECIES (ROS)
4
1.2.2 REDOX STATE OF THE COMPONENTS IN THE PHOTOSYNTHETIC ELECTRON
TRANSPORT CHAIN 7
1.2.3 TETRAPYRROLE BIOSYTHESIS
9
1.2.4 PHOSPHOADENOSINE PHOSPHATE (PAP)
11
1.2.5 PLASTID GENE EXPRESSION
12
1.2.6 PROTEINS MOVING BETWEEN PLASTIDS AND NUCLEUS
14
1.2.7 METHYLERYTHRITOLCYCLODIPHOSPHATE (MECPP)
14
1.3 ANTIOXIDANT SYSTEM IN CHLOROPLAST 15
1.3.1 NON-ENZYMATIC ANTIOXIDANTS
15
1.3.2 ENZYMATIC ANTIOXIDANTS IN CHLOROPLASTS
16
1.4 TRANSCRIPTIONAL REGULATION OF THE
CHLOROPLAST ANTIOXIDANT ENZYMES 19
1.4.1 DIFFERENT RESPONSES OF DIFFERENT CHLOROPLAST ANTIOXIDANT ENZYMES
TO ABIOTIC
STRESSES
20
1.4.2 TRANSCRIPTION FACTOR OF SAPX IN ARABIDOPSIS
20
1.4.3 REGULATION OF GPX1 AND GPX7 EXPRESSION IN ARABIDOPSIS
21
1.4.4 REGULATION OF 2-CYS-PRXA (2CPA)
21
1.5 AIM OF THIS STUDY 24
2 MATERIAL AND METHODS 25
2.1 PLANT MATERIALS 25
2.2 GROWTH CONDITIONS 25
2.2.1 STERILIZATION AND VERNALIZATION OF
ARABIDOPSIS THALIANA
SEEDS
25
2.2.2 GROWTH OF
ARABIDOPSIS THALIARIA
SEEDLINGS ON STERILE MS MEDIUM
26
2.2.3 GROWTH OF
ARABIDOPSIS THALIANA
PLANTS ON SOIL
26
2.2.4 SHORT-DAY AND LONG-DAY GROWING CONDITION
27
2.3 CROSSING OF ARABIDOPSIS THALIANA PLANTS 27
2.4 CO-SEGREGATION ANALYSIS 27
2.5 PHENOTYPE ANALYSIS OF THE PLANTS 28
2.6 DETERMINATION OF LUCIFERASE ACTIVITY IN VIVO 28
I
HTTP://D-NB.INFO/1045331023
TABLE OF CONTENTS
2.6.1 MEASUREMENT WITH FLUOSKAN ASCENT FL LUMINOMETER
28
2.6.2 MEASUREMENT WITH NIGHTSHADE LB 985 IN VIVO PLANT IMAGING SYSTEM
28
2.7 AGAROSE GEL ELECTROPHORESIS 29
2.7.1 DNA GELELECTROPHORESIS
29
2.7.2 RNA GELELECTROPHORESIS
29
2.8 DNA ISOLATION 30
2.8.1 ISOLATION OF GENOMIC DNA
30
2.8.2 ISOLATION OF NUCLEI DNA FOR LLLUMINA SEQUENCING
31
2.8.3 TESTING THE QUALITY OF ISOLATED DNA
32
2.9 RNA ISOLATION 33
2.9.1 ISOLATION OF RNA FROM THE PLANT MATERIAL
33
2.9.2 TESTING THE QUALITY OF ISOLATED RNA
33
2.10 POLYMERASE CHAIN REACTION (PCR) 33
2.10.1 STANDARD PCR
34
2.10.2 PCR WITH OPTITAQ
34
2.11 SEQUENCING OF PCR FRAGMENTS AND PLASMIDS 35
2.11.1 SEQUENCING OF PCR FRAGMENTS
35
2.11.2 SEQUENCING OF PLASMIDS
35
2.11.3 SEQUENCE ANALYSIS
36
2.12 SEQUENCING WITH ILLUMINA SYSTEM 36
2.13 GENE EXPRESSION ANALYSIS 37
2.14 FIRST STRAND CDNA SYNTHESIS 37
2.14.1 EXPRESSION ANALYSIS
37
2.15 GENETIC MAPPING OF MUTANT LOCI WITH SINGLE SEQUENCE LENGTH
POLYMORPHISMS
(SSLP) 40
2.15.1 DETERMINATION OF INDIVIDUALS WITH MUTANT ALLELES FROM THE
SEGREGATING
F2 POPULATION
40
2.15.2 SSLPS MARKERS DESIGN USING CEREON DATABASE
40
2.15.3 MAPPING ANALYSIS OF THE MUTANT LOCI WITH SSLPS MARKERS
41
2.16 GENETIC MAPPING OF MUTANT LOCI WITH NEXT GENERATION SEQUENCING 41
2.16.1 ANALYSIS OF THE SEQUENCING RESULTS OF LLLUMINA SEQUENCING
SHOREMAP
42
2.16.2 CONFIRMATION OF THE FINAL CANDIDATE LOCUS
42
2.17 GENOTYPING AND ISOLATION OF HOMOZYGOUS T-DNA INSERTION
LINES BY PCR 42
2.18 ESCHERICHIA COLI MANIPULATIONS 43
2.18.1 GENERATION OF CHEMICALLY COMPETENT E. COLI
43
2.18.2 PLASMID TRANSFORMATION INTO E.COLI
44
2.19 PLASMID MANIPULATION 44
2.19.1 PURIFICATION OF PCR PRODUCTS FROM AGAROSE GEL
44
2.19.2 CLONING INTO ENTRY VECTOR (PCR8/GW/TOPO)
44
2.19.3 CONFIRMATION OF THE INSERTION WITH COLONY PCR
45
2.19.4 ISOLATION OF PLASMIDS USING MINIPREP KITS
46
2.19.5 LR REACTION
46
2.20 AGROBACTERIUM TUMEFACIENS MANIPULATIONS 48
II
TABLE OF CONTENTS
2.20.1 PRODUCTION OF COMPETENT A. TUMEFACIENS CELLS
48
2.20.2 TRANSFORMATION OF A. TUMEFACIENS
49
2.21 COMPARISON AND ANALYSIS OF PROMOTERS IN PLANTS 49
2.21.1 ANALYSIS OF THE PROMOTER SEQUENCES IN COL-0 AND LER
49
2.21.2 TRANSIENT GENE EXPRESSION
49
2.22 METAL-CONTENT ANALYSIS 51
2.23 COEXPRESSION ANALYSIS AND FUNCTION PREDICTION 51
2.24 ANALYSIS OF AMINO ACID SEQUENCES AND PROTEIN STRUCTURES 52
3 RESULTS 53
3.1 PHENOTYPIC CHARACTERIZATION OF RIMB3 AND RIMB6 MUTANTS 53
3.1.1 GROWTH HABITS OF THE MUTANTS
53
3.1.2 ANALYSIS OF REPORTER GENE (LUCIFERASE) EXPRESSION
58
3.2 IDENTIFICATION AND CHARACTERIZATION OF MUTANT RIMB6 61
3.2.1 THE CONTENTS OF MINERAL ELEMENTS IN RIMB6 AND T19-2
61
3.2.2 GENETIC MAPPING OF RIMB6 WITH NEXT GENERATION SEQUENCING
62
3.2.3 CONFIRMATION OF THE FINAL CANDIDATE LOCUS OF RIMB6 BY
TRANSCRIPTION ANALYSIS USING
T-DNA KNOCK-OUT LINE
67
3.2.4 AT4G12560, THE FINAL CANDIDATE LOCUS FOR RIMB6
67
3.2.5 RIMB6 MIGHT BE CORRELATED WITH PLASMA METAL TRANSPORTERS ATLRT 1
AND ATPDR8
68
3.3 AN EQTL
OF 2CPA REGULATOR BETWEEN COL-0
AND LER
WAS IDENTIFIED ON TOP ARM
OF CHROMOSOME III 69
3.3.1 GENETIC MAPPING OF THE RIMB3 MUTATION WITH SSLP MARKERS
69
3.3.2 AN EQTL OF 2CPA WAS IDENTIFIED BASED ON LER GENETIC BACKGROUND
70
3.3.3 CONFIRMATION OF THE EQTL
71
3.3.4 GENETIC MAPPING OF THE EQTL WITH SSLP 72
3.3.5 TRANSCRIPTION ANALYSIS OF THE CANDIDATE GENES OF THE EQTL USING
T-DNA
KNOCK-OUT LINE 75
3.3.6 NATURAL VARIATION OF AT3G21660 BETWEEN COL-0 AND LER
76
3.3.7 PREDICTION OF PROTEIN AT3G21660 WITH CONSERVED DOMAIN DATABASE OF
NCBI
78
3.3.8 AT3G21660 POSITIVELY REGULATES THE 2CPA AND 2CPB
79
3.3.9 TEST FOR THE POLYMORPHISM IN PROMOTER OF COL-0 AND LER
80
3.3.10 PREDICTION OF THE FUNCTION NETWORK OF AT3G21660
81
3.3.11 COEXPRESSION ANALYSIS OF UBOX PROTEIN WITH CDC48, PUX2 AND 2CPA
82
3.3.12 THE EQTL AND RIMB6 MIGHT BE CORRELATED VIA ASK9 AND ASK16
86
3.4 IDENTIFICATION AND CHARACTERIZATION OF MUTANT RIMB3 90
3.4.1 CO-SEGREGATION ANALYSIS OF LOW LUCIFERASE AND CHLOROSIS PHENOTYPE
90
3.4.2 GENETIC MAPPING OF RIMB3 WITH NEXT GENERATION SEQUENCING
91
3.4.3 ONE STEP SEQUENCING WITH LLLUMINA G2 ANALYZER
92
3.4.4 TRANSCRIPT ABUNDANCE ANALYSIS OF RIMB3 AND 2CPA
96
3.4.5 COEXPRESSION ANALYSIS OF RIMB3 AND RIMB1 (RCD1)
97
3.4.6 COEXPRESSION ANALYSIS OF RIMB3 AND ATCDC48
101
4 DISCUSSION 102
4.1 GENETIC MAPPING OF LOCI RIMB3, RIMB6ANDTHE
EQTLOF2CPA 102
III
TABLE OF CONTENTS
4.2 RIMB6 103
4.2.1 RIMB6 (CPR30) IS A NEGATIVE REGULATOR OF PLANT DEFENSE RESPONSE
SYSTEM
104
4.2.2 RIMB6 IS INVOLVED IN CROSS-TALK BETWEEN REDOX SIGNALING AND PLANT
DEFENSE
RESPONSE.
106
4.2.3 RIMB6 IS INVOLVED IN THE METAL HOMEOSTASIS SYSTEM IN PLANT CELL
106
4.2.4 ATPDR8 MAY BE REPRESSED IN RIMB6
107
4.2.5 ATLRT
1 AND CNG CHANNELS MIGHT BE DISTURBED IN RIMB6
109
4.2.6 POSSIBLE REGULATION NETWORK OF RIMB6 WITH EQTL
110
4.3 THE EQTL
IN 2CPA REGULATION 111
4.3.1 NATURAL VARIATION OF AT3G21660
112
4.3.2 THE EQTL POSITIVELY REGULATES 2-CYS-PEROXIREDOXIN EXPRESSION
112
4.3.3 THE EQTL BELONGS TO UBX CONTAINING PROTEIN FAMILY THAT PHYSICALLY
INTERACTS WITH
CDC48
113
4.3.4 PUX2 IS A POSITIVE REGULATOR OF CDC48 AND NEGATIVELY CORRELATES
WITH THE EQTL...
114
4.3.5 THE EQTL IS COEXPRESSED WITH ASK9 AND ASK16
114
4.3.6 HYPOTHESIS OF TWO POSSIBLE MODELS OF EQTL REGULATION NETWORK
115
4.3.7 AN ALTERNATIVE APPROACH TO DISCOVER THE NEW REGULATOR USING THE
NATURAL VARIATION
RECOURSES OF
ARABIDOPSIS THALIANA 116
4.4 RIMB3 118
4.4.1 THE PREDICTED STRUCTURE AND FUNCTIONS OF RIMB3
118
4.4.2 RIMB3 COEXPRESSES WITH ATCDC48
119
4.4.3 RIMB3 POSITIVELY CORRELATES WITH RIMB1 AND FUNCTIONS UPSTREAM OF
RIMB1
119
4.4.4 POSSIBLE REGULATION NETWORK OF RIMB3
120
4.5 CONCLUSION 121
5 REFERENCES 123
APPENDIX 138
IV
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| spelling | Guo, Wei Verfasser (DE-588)104360832X aut Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana by Wei Guo Identifizierung und Charakterisierung von Regulatoren der 2-Cys-Peroxiredoxin A in Arabidopsis thaliana 2013 IX, 143 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Nebentitel: Identifizierung und Charakterisierung von Regulatoren der 2-Cys-Peroxiredoxin A in Arabidopsis thaliana Berlin, Freie Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:kobv:188-fudissthesis000000095396-7 http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000095396 kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026840088&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Guo, Wei Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |
| subject_GND | (DE-588)4113937-9 |
| title | Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |
| title_alt | Identifizierung und Charakterisierung von Regulatoren der 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |
| title_auth | Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |
| title_exact_search | Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |
| title_full | Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana by Wei Guo |
| title_fullStr | Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana by Wei Guo |
| title_full_unstemmed | Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana by Wei Guo |
| title_short | Identification and characterization of regulators of 2-Cys-Peroxiredoxin A in Arabidopsis thaliana |
| title_sort | identification and characterization of regulators of 2 cys peroxiredoxin a in arabidopsis thaliana |
| topic_facet | Hochschulschrift |
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