Development of tools for inducible gene expression in chloroplasts:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | VIII, 110 Bl. Ill., graph. Darst. |
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| 100 | 1 | |a Emadpour, Masoumeh |e Verfasser |0 (DE-588)1070538671 |4 aut | |
| 245 | 1 | 0 | |a Development of tools for inducible gene expression in chloroplasts |c von Masoumeh Emadpour |
| 264 | 1 | |c 2014 | |
| 300 | |a VIII, 110 Bl. |b Ill., graph. Darst. | ||
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| 502 | |a Potsdam, Univ., Diss., 2014 | ||
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Datensatz im Suchindex
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| adam_text | INDEX
INDEX I
ABBREVIATIONS VI
1. INTRODUCTION 1
1.1. PLASTID GENE EXPRESSION 2
1.1.1. T RANSCRIPTION 2
1.1.2. POST-TRANSCRIPTIONAL REGULATION OF PLASTID GENE EXPRESSION 3
1.1.3. TRANSLATION AND POST-TRANSLATIONAL REGULATION 4
1.2. TRANSPLASTOMIC PLANTS 6
1.2.1. CHLOROPLASTS AS BIOREACTORS FOR MOLECULAR FARMING 6
1.2.2. INDUCIBLE EXPRESSION OF FOREIGN PROTEINS FROM THE PLASTID GENOME
8
1.2.2.1. RIBOSWITCHES AS NEW TOOLS TO INDUCE TRANSGENE EXPRESSION 9
1.3. ACETYL-COENZYME A CARBOXYLASE (ACCASE) AND DE NOVO FATTY ACID
BIOSYNTHESIS
IN PLASTIDS 12
1.4. ACCD: THE ONLY PLASTID-ENCODED COMPONENT OF PLANT LIPID METABOLISM
13
1.5. LIPID BIOSYNTHESIS IN PLANTS 14
1.5.1. LIPID COMPOSITION IN PLANT CELLS 14
1.5.2. PROKARYOTIC AND EUKARYOTIC PATHWAYS OF LIPID BIOSYNTHESIS IN
PLANT CELLS 15
1.6. MUTANTS OF FATTY ACID SYNTHESIS 16
1.7. INDUCIBLE REPRESSION OF ENDOGENOUS GENES IN CYANOBACTERIA AND
PLASTIDS 17
1.8. AIMS AND STRATEGIES OF THE PROJECTS IN THIS STUDY 19
1.8.1. THEOPHYLLINE-RESPONSIVE RIBOSWITCH AS A NEW TOOL TO REGULATE THE
TRANSCRIPTION OF FOREIGN GENES BY INDUCIBLE TRANSLATION OF T7 RNA
POLYMERASE 19
1.8.2. A THEOPHYLLINE-RESPONSIVE RIBOSWITCH AS A NOVEL TOOL FOR
CONDITIONAL
REPRESSION OF THE ESSENTIAL PLASTID GENE ACCD 20
2. MATERIAL AND METHODS 22
2.1. CHEMICALS AND CONSUMABLES 22
2.1.1. SUPPLIERS FOR COMMON CHEMICALS AND CONSUMABLES 22
2.1.2. SPECIAL CHEMICALS 22
2.1.3. CONSUMABLES 23
2.2. EQUIPMENTS 23
2.3. ENZYMES AND REACTION KITS 24
2.3.1. RESTRICTION ENZYMES 24
HTTP://D-NB.INFO/1070689157
2.3.2. ENZYMES 24
2.3.3. REACTION KITS 24
2.4. MOLECULAR WEIGHT MARKERS 25
2.5. VECTORS 25
2.6. OLIGONUCLEOTIDES 27
2.7. CULTIVATION OF ESCHERICHIA COLI 28
2.7.1. BACTERIAL MEDIA 28
2.8. GENOTYPE OF ESCHERICHIA COLI STRAIN 28
2.9. CULTIVATION OF PLANTS 29
2.9.1. CULTIVATION OF NICOTIANA TABACUM CV. PETIT HAVANA : 29
2.9.1.1. STERILIZATION OF NICOTIANA TABACUM SEEDS 29
2.9.1.2. CULTIVATION / REGENERATION OF NICOTIANA TABACUM UNDER STERILE
CONDITIONS. 29
2.9.1.2.1. CULTURE MEDIUM 29
2.9.1.2.2. MEDIA USED FOR TISSUE CULTURE AND SHOOT REGENERATION 30
2.9.1.3. CULTIVATION OF NICOTIANA TABACUM IN SOIL 30
2.9.1.4. INDUCTION OF GENE EXPRESSION BY THEOPHYLLINE TREATMENT 30
2.10. MOLECULAR BIOLOGICAL AND BIOCHEMICAL METHODS 31
2.10.1. CLONING PROCEDURES 31
2.10.1.1. DIGESTION OF PLASMID DNA OR PCR PRODUCTS WITH RESTRICTION
ENZYMES 31
2.10.1.2. LIGATION OF DNA FRAGMENTS 31
2.10.1.3. PREPARATION OF ESCHERICHIA COLI HEAT-SHOCK-COMPETENT CELLS 31
2.10.1.4. HEAT-SHOCK TRANSFORMATION OF ESCHERICHIA COLI 32
2.10.2. PLASTID TRANSFORMATION OF NICOTIANA TABACUM 32
2.10.3. EXTRACTION OF NUCLEIC ACIDS 32
2.10.3.1. ISOLATION OF PLASMID DNA 32
2.10.3.2. ISOLATION OF PLANT GENOMIC DNA 33
2.10.3.3. ISOLATION OF TOTAL PLANT RNA 33
2.10.4. SPECTROPHOTOMETRIC QUANTIFICATION OF NUCLEIC ACIDS 33
2.10.5. PURIFICATION OF DNA FRAGMENTS FROM SOLUTIONS OR AGAROSE GELS 33
2.10.6. POLYMERASE CHAIN REACTION (PCR) 34
2.10.7. SEPARATION OF DNA BY GEL ELECTROPHORESIS 35
2.10.8. SEPARATION OF RNA BY DENATURING AGAROSE GEL ELECTROPHORESIS 35
2.10.9. NUCLEIC ACID DETECTION BY SOUTHERN AND NORTHERN BLOT ANALYSIS 36
2.10.9.1. DNA DETECTION BY SOUTHERN BLOT 36
2.10.9.2. RNA DETECTION BY NORTHERN BLOT 36
2.10.9.3. RADIOACTIVE LABELLING OF DNA PROBES 36
2.10.9.4. HYBRIDIZATION OF IMMOBILIZED NUCLEIC ACIDS WITH RADIOACTIVE
PROBES 37
2.10.10. ANALYSIS OF PROTEINS 37
2.10.10.1. PHENOLIC EXTRACTION OF TOTAL PROTEINS 37
2.10.10.2. SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS 38
2.10.10.3. SENSITIVE COOMASSIE STAINING OF PROTEINS IN POLYACRYLAMIDE
GELS 39
2.10.10.4. ELECTROBLOT OF PROTEINS 39
2.10.10.5. IMMUNOLABELLING AND DETECTION OF PROTEINS ON PVDF MEMBRANES
39
2.11. LIPID ANALYSIS 39
2.11.1. LIPID EXTRACTION 39
2.11.2. PEAK EXTRACTION AND QUANTIFICATION 40
2.12. MICROSCOPY 40
2.12.1. DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPY OF LEAF
MESOPHYLL
CELLS 40
2.12.2. CONFOCAL MICROSCOPY OF CHLOROPLASTS 41
2.12.3. FLUORESCENCE MICROSCOPY 41
2.12.4. MICROSCOPIC ANALYSIS OF SEED CAPSULES 41
2.12.5. MICROSCOPY OF LEAF CROSS SECTIONS 41
2.12.6. TRANSMISSION ELECTRON MICROSCOPY (TEM) OF CHLOROPLASTS 42
3. RESULTS 43
3.1. THEOPHYLLINE-RESPONSIVE RIBOSWITCH AS A NEW TOOL TO REGULATE THE
TRANSCRIPTION
OF FOREIGN GENES BY INDUCIBLE TRANSLATION OF THE T7 RNA POLYMERASE 43
3.1.1. GENERATION OF A VECTOR DESIGNED TO REGULATE THE EXPRESSION OF GFP
UNDER THE
CONTROL OF THE RNA AMPLIFICATION-ENHANCED RIBOSWITCH (RAMPER) 43
3.1.2. PLASTID TRANSFORMATION AND SELECTION OF TRANSPLASTOMIC LINES 44
3.1.2.1. RFLP ANALYSIS AND INHERITANCE ASSAY 45
3.1.3. THEOPHYLLINE-DEPENDENT GFP EXPRESSION IN RAMPER TRANSPLASTOMIC
TOBACCO
PLANTS: IN VITRO CULTURE OF SEEDLINGS 45
3.1.4. OPTIMIZATION OF GFP INDUCTION IN RAMPER PLANTS BY WATERING WITH
THEOPHYLLINE: SOIL-GROWN PLANTS 48
3.1.5. T7 RNA POLYMERASE AND GFP MRNA ACCUMULATION IN LEAVES OF
GREENHOUSE-
GROWN RAMPER LINES 50
3.1.6. INDUCTION OF GFP ACCUMULATION IN TRANSPLASTOMIC PLANTS BY
WATERING WITH
THEOPHYLLINE 51
3.1.6.1. GFP EXPRESSION IN TRANSPLASTOMIC LINE EXPRESSING GFP UNDER
DIRECT
RIBOSWITCH CONTROL 51
3.1.7. PHENOTYPIC CHARACTERIZATION OF TRANSPLASTOMIC LINES 52
3.1.8. GENERATION OF TRANSPLASTOMIC PLANTS EXPRESSING THE HIV-1 ANTIGEN
NEF UNDER
THE CONTROL OF THE RAMPER SYSTEM 53
3.1.9. PLASTID TRANSFORMATION AND SELECTION OF TRANSPLASTOMIC LINES 55
3.1.10. 77 RNA POLYMERASE AND NEF MRNA ACCUMULATION IN LEAVES OF
TRANSPLASTOMIC PLANTS 55
3.1.11. INDUCTION OF NEF EXPRESSION IN TRANSPLASTOMIC PLANTS 56
3.1.12. PHENOTYPIC CHARACTERIZATION OF NEF-EXPRESSING TRANSPLASTOMIC
MUTANTS 57
3.2. A THEOPHYLLINE-RESPONSIVE RIBOSWITCH AS A NOVEL TOOL FOR
CONDITIONAL REPRESSION
OF THE ESSENTIAL PLASTID GENE ACCD 59
3.2.1. GENERATION OF A PLASTID TRANSFORMATION VECTOR DESIGNED FOR
INDUCIBLE
REPRESSION OF ACCD EXPRESSION 59
3.2.2. PLASTID TRANSFORMATION OF TOBACCO PLANTS AND SELECTION OF
PUTATIVE
TRANSFORMANTS 61
3.2.3. RFLP ANALYSIS OF TRANSPLASTOMIC LINES 61
3.2.4. PHENOTYPIC CHARACTERIZATION OF RS-ACCD TRANSFORMANTS 66
3.2.5. INHERITANCE ASSAY 68
3.2.6. SEED PHENOTYPE OF RS-ACCD TRANSPLASTOMIC PLANTS 69
3.2.7. MESOPHYLL CELL MORPHOLOGY AND CHLOROPLAST STRUCTURE IN RS-ACCD
PLANTS 70
3.2.8. CHLOROPLASTS IN EPIDERMAL AND MESOPHYLL CELLS OF RS-ACCD PLANTS
72
3.2.9. THEOPHYLLINE-DEPENDENT PHENOTYPE RECOVERY IN TRANSPLASTOMIC
PLANTS 73
3.2.10. ACCD EXPRESSION ANALYSIS IN LEAVES BEFORE AND AFTER INDUCTION
WITH
THEOPHYLLINE 75
3.2.11. ANALYSIS OF POLAR LIPIDS IN LEAVES OF RS-ACCD TRANSPLASTOMIC
PLANTS BEFORE
AND AFTER INDUCTION WITH THEOPHYLLINE 76
4. DISCUSSION 79
4.1. DESIGN OF THE RAMPER SYSTEM: IMPROVING RIBOSWITCH-MEDIATED
INDUCIBLE GENE
EXPRESSION BY RNA AMPLIFICATION 79
4.1.1. RAMPER-DRIVEN REPORTER GENE EXPRESSION FROM THE PLASTID GENOME 80
4.1.2. RAMPER FACILITATES EXPRESSION OF A CYTOTOXIC VACCINE PROTEIN, THE
HIV-1
ANTIGEN NEF 80
4.1.3. PHENOTYPIC CHARACTERIZATION OF NEF-EXPRESSING TRANSPLASTOMIC
PLANTS 81
4.1.4. OUTLOOK 82
4.2. A THEOPHYLLINE-BINDING RIBOSWITCH AS A NEW SYSTEM FOR INDUCIBLE
REPRESSION OF
AN ESSENTIAL PLASTID GENE IN HIGHER PLANTS 83
4.2.1. ACCD HAS AN ESSENTIAL ROLE IN TOBACCO 84
4.2.2. DIFFICULTIES IN REPLACING THE NATIVE 5 UTR OF THE ACCD GENE WITH
RIBOSWITCH,
INDICATE A HIGH RATE OF GENE CONVERSION 84
4.2.3. REPRESSION OF ACCD GENE EXPRESSION BY THE THEOPHYLLINE-RESPONSIVE
RIBOSWITCH CAUSES CHIMERIC LEAVES, WHICH LOSE PART OF THE LEAF LAMINA 87
4.2.4. HOMOPLASMY FOR RS-ACCD IN THE L2 LEAF LAYER CAN EXPLAIN THE SEED
ABORTION .. 88
4.2.5. CHLOROPLAST MORPHOLOGY IN RS-ACCD TRANSFORMANTS IN THE ABSENCE OF
THEOPHYLLINE 90
4.2.6. EXPRESSION ANALYSIS IN ACCD DOWN-REGULATION MUTANTS 91
4.2.7. SUPRESSED ACCD EXPRESSION RESULTS IN ALTERED GLYCEROLIPID LEVELS,
WHICH
RECOVER TO THE WILD-TYPE LEVEL AFTER THEOPHYLLINE APPLICATION 91
4.2.8. OUTLOOK 92
5. SUMMARY 93
5.1. THEOPHYLLINE-RESPONSIVE RIBOSWITCH AS A NEW TOOL TO REGULATE THE
TRANSCRIPTION
OF FOREIGN GENES BY INDUCIBLE TRANSLATION OF A T7 RNA POLYMERASE FROM
THE PLASTID
GENOME 93
5.2. A THEOPHYLLINE-RESPONSIVE RIBOSWITCH AS A NOVEL TOOL FOR
CONDITIONAL REPRESSION
OF ESSENTIAL PLASTID GENES IN HIGHER PLANTS 94
6. LITERATURE 95
7. APPENDIX 104
7.1. FIGURES 104
7.2. TABLES 106
8. PUBLICATION AND CONFERENCE ATTENDANCE 107
8.1. PUBLICATIONS 107
8.2. CONFERENCE ATTENDANCE 107
9. EIDESTATTLICHE ERKLARUNG 108
10. ACKNOWLEDGEMENTS 109
|
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| spelling | Emadpour, Masoumeh Verfasser (DE-588)1070538671 aut Development of tools for inducible gene expression in chloroplasts von Masoumeh Emadpour 2014 VIII, 110 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Potsdam, Univ., Diss., 2014 Chloroplast (DE-588)4147821-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Chloroplast (DE-588)4147821-6 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027991795&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Emadpour, Masoumeh Development of tools for inducible gene expression in chloroplasts Chloroplast (DE-588)4147821-6 gnd |
| subject_GND | (DE-588)4147821-6 (DE-588)4113937-9 |
| title | Development of tools for inducible gene expression in chloroplasts |
| title_auth | Development of tools for inducible gene expression in chloroplasts |
| title_exact_search | Development of tools for inducible gene expression in chloroplasts |
| title_full | Development of tools for inducible gene expression in chloroplasts von Masoumeh Emadpour |
| title_fullStr | Development of tools for inducible gene expression in chloroplasts von Masoumeh Emadpour |
| title_full_unstemmed | Development of tools for inducible gene expression in chloroplasts von Masoumeh Emadpour |
| title_short | Development of tools for inducible gene expression in chloroplasts |
| title_sort | development of tools for inducible gene expression in chloroplasts |
| topic | Chloroplast (DE-588)4147821-6 gnd |
| topic_facet | Chloroplast Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027991795&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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