The lignification of strawberry fruit: molecular basis and effects on fruit quality
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | kostenfrei https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130121-1113252-0-9 Inhaltsverzeichnis |
| Beschreibung: | XIV, 165 S. Ill., graph. Darst. |
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CONTENTS
TABLE OF CONTENTS
ABBREVIATIONS V
ZUSAMMENFASSUNG VIII
SUMMARY XII
I. INTRODUCTION.... 1
1. STRAWBERRY 1
1.1 BACKGROUND 1
1.2 FRUIT GROWTH AND FIRMNESS 2
1.3 APPLICATION O F GENETIC BIOTECHNOLOGIES FOR IMPROVING FRUIT TEXTURE
3
2. BIOSYNTHESIS PATHWAYS AND CORE GENES 5
2.1 BIOSYNTHESIS PATHWAYS 5
2.2 REGULATORY CORE GENES FOR MONOLIGNOL BIOSYNTHESIS 10
3. LIGNIN AND LIGNIFICATION 15
3.1 LIGNIN 15
3.2 LIGNIFICATION 17
4. CHANGES IN THE LIGNIN CONTENT AND COMPOSITION 19
4.1. STRESSES 19
4.2. TRANSGENIC AND MUTAGENIC APPROACHES 21
5. RNA SILENCING IN PLANTS 24
6. INTENTION OF THE STUDY 26
II. MATERIALS AND METHODS 27
1. MATERIALS 27
1.1 PLANT MATERIALS 27
1.2 CHEMICALS 27
1.3 BACTERIAL AND YEAST STRAINS 28
1.4 ENZYMES, MARKERS, AND OTHER 28
1.5 VECTORS 29
1.6 VECTORS CONTAINING ANTIBIOTICS FOR BACTERIAL SELECTION AND PRIMERS
FOR SEQUENCING. 30 1.7 ANTIBIOTICS 30
1.8 PRIMERS 30
1.9 MEDIA, BUFFERS, AND SOLUTIONS 33
1.10 COMMERCIAL KITS 36
2. APPARATUS 36
2.1 FPLC (FAST PROTEIN LIQUID CHROMATOGRAPHY) 36
2.2 LIQUID CHROMATOGRAPHY ULTRAVIOLET ELECTRO-SPRAY IONIZATION MASS
SPECTROMETRY (LC- UV-ESI-MS ) 37
2.3 GAS CHROMATOGRAPHY-MASS SPECTROPHOTOMETER (GC-MS) 40
2.4 OTHER EQUIPMENT 4 0
2.5 SOFTWARE AND INTERNET SOURCES 41
HTTP://D-NB.INFO/1032162309
IMAGE 2
CONTENTS II
3. METHODS 42
3.1 MOLECULAR BIOLOGY EXPERIMENTS 42
3.1.1 EXTRACTION O F GENOMIC DNA 42
3.1.2 EXTRACTION O F TOTAL RNA 42
3.1.3 PCR REACTION 43
3.1.4 AGAROSE GEL ELECTROPHORESIS 44
3.1.5 PURIFICATION O F DNA FRAGMENTS FROM AGAROSE GELS 44
3.1.6 RESTRICTION ENDONUCLEASE REACTION 44
3.1.7 LIGATION REACTION 44
3.1.8 PREPARATION O F CHEMICALLY COMPETENT CELLS 45
3.1.9 TRANSFORMATION 45
3.1.10 ISOLATION O F PLASMID DNA AND GLYCEROL STOCKS 46
3.1.11 REMOVAL O F GENOMIC DNA FROM TOTAL RNA 46
3.1.12 FIRST-STRAND SYNTHESIS O F CDNAS 47
3.1.13 QUANTITATIVE REAL-TIME PCR (QRT-PCR) ANALYSIS 47
3.1.14 BRADFORD ASSAY 49
3.1.15 SDS-PAGE 49
3.2 PRIMER DESIGN, CLONING, AND SEQUENCE ANALYSIS 50
3.3 CONSTRUCTION O F IHPRNA AND OVEREXPRESSION O F INDIVIDUAL FACCR,
FACAD, AND FAPOD 50
3.3.1 IHPRNA CONSTRUCTS 50
3.3.2 OVEREXPRESSION CONSTRUCTS 52
3.4 RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION 52
3.4.1 CONSTRUCTION O F EXPRESSION PLASMID 52
3.4.2 EXPRESSION AND PURIFICATION O F GST-FUSED PROTEINS IN E. COLI BL21
(DE3) PLYSS 53
3.4.3 EXPRESSION AND PURIFICATION O F HIS-FUSED PROTEINS IN E. COLI BL21
(DE3) PLYSS 54
3.4.4 CONSTRUCTION AND EXPRESSION O F THE RECOMBINANT PROTEIN IN YEAST
(SACCHAROMYCES CEREVISAE INVSC. 1) 55
3.5 FACCR ACTIVITY ASSAY 56
3.5.1 PREPARATION O F HYDROXYCINNAMOYL-COA AS FACCR SUBSTRATES 56
3.5.2 DETERMINATION O F PH AND TEMPERATURE OPTIMA 57
3.5.3 DETERMINATION O F KINETICS 58
3.5.4 FACCR ACTIVITY CALCULATION 58
3.5.5 IDENTIFICATION O F REACTION PRODUCTS BY LC-UV-ESI-MS 58
3.6 FACAD ACTIVITY ASSAYS AND ANALYSIS O F STANDARD CHEMICALS VIA
LC-UV-ESI-MS . 59 3.7 FAPOD ACTIVITY ASSAYS 61
3.8 STRAWBERRY FRUIT 61
3.8.1 GENE EXPRESSION STUDIES 61
3.8.2 WOUNDING TREATMENT 61
3.8.3 PATHOGEN TREATMENT 62
IMAGE 3
CONTENTS III
3.8.4 DOWN-REGULATION AND UP-REGULATION O F LIGNIN BIOSYNTHETIC GENES (
FACCR, FACAD, AND FAPOD) IN FRUIT 62
3.8.4.1 PREPARATION O F THE AGMBACTERIUM SUSPENSION AND INFILTRATION O F
FRUITS 63 3.8.4.2 FRUIT FIRMNESS 63
3.8.4.3 GENE EXPRESSION LEVEL ASSAY 63
3.8.4.4 ENZYME EXTRACTION AND ASSAYS 63
3.8.4.5 EXTRACTION AND METABOLITE ANALYSIS VIA LC-UV-ESI-MS 64
3.8.4.6 LIGNIN HISTOCHEMICAL STAINING 65
3.8.4.7 LIGNIN CONTENT 66
3.8.4.7.1 CELL WALL PREPARATION 66
3.8.4.7.2 HYDROLYSIS 67
3.8.4.7.3 THIOGLYCOLIC-ACID ASSAY 67
3.8.4.8 LIGNIN COMPOSITION 67
3.8.4.8.1 PREPARATION O F PLANT MATERIAL 67
3.8.4.8.2 ISOLATION O F LIGNIN 68
3.8.4.8.3 THIOACIDOLYSIS 68
3.8.4.9 BOX PLOTS AND STATISTICAL ANALYSIS 69
III. RESULTS 70
1. SEQUENCE ANALYSIS 70
1.1 CLONING AND CHARACTERIZATION O F FACCR, FACAD , AND FAPOD 70
1.2 A POSSIBLE CCR, CAD, AND POD GENE FAMILY FROM FRAGARIA VESCA 71
1.3 PHYLOGENETIC TREE O F PARTIAL GENOME AND GENE SEQUENCES 75
1.4 PHYLOGENETIC ANALYSIS O F PROTEINS 79
2. BIOCHEMICAL CHARACTERISTICS O F THE RECOMBINANT FACCR 84
2.1 HETEROLOGOUS EXPRESSION O F FACCR IN E. COLI 84
2.2 DETERMINATION O F OPTIMAL PH AND TEMPERATURE FOR GST-FACCR ACTIVITY
85 2.3 SUBSTRATE SPECIFICITY O F GST-FACCR 87
2.4 IDENTIFICATION O F THE GST-FACCR REACTION PRODUCTS 88
3. HETEROLOGOUS EXPRESSION O F FACAD IN E. COLI AND YEAST CELLS 89
4. HETEROLOGOUS EXPRESSION O F FAPOD AND FAPOD27: TWO PEROXIDASE
ISOFORMS. 92 5. LIGNIN BIOSYNTHETIC GENES IN STRAWBERRY FRUIT 94
5.1 EXPRESSION O F LIGNIN BIOSYNTHETIC GENES IN VEGETATIVE TISSUES,
FLOWERS, AND FRUIT DEVELOPMENTAL STAGES 94
5.2 STRAWBERRY FRUIT IN RESPONSE TO STRESS TOLERANCE 95
5.2.1 EFFECT O F WOUNDING AND PATHOGEN ON GENE EXPRESSION LEVELS IN
FRUIT 95 5.2.2 EFFECT O F WOUNDING AND PATHOGEN INFECTION ON FRUIT
FIRMNESS 99
5.2.3 EFFECT O F AGROBACTERIUM- INFECTION ON FRUIT FIRMNESS AND LIGNIN
CONTENT 100 5.3 DOWN- AND UP-REGULATION O F LIGNIN BIOSYNTHETIC GENES IN
F. X ANANASSA CV. ELSANTA FRUIT 101
5.3.1 IHPRNA AND OVEREXPRESSION CONSTRUCTS USED FOR SILENCING AND
OVEREXPRESSION O F LIGNIN BIOSYNTHETIC GENES IN FRUIT 102
5.3.2 PHENOTYPE AND LIGNIN STAINING 102
5.3.3 FRUIT TEXTURE AND LIGNIN CONTENT 104
5.3.4 GENE EXPRESSION LEVELS 104
5.3.5 ACTIVITY ASSAY 107
5.3.6 METABOLITE LEVELS 109
IMAGE 4
CONTENTS IV
5.3.7 THIOACIDOLYSIS REACTION AND LIGNIN COMPOSITION 112
5.3.8 DETECTION O F FERULIC ACID IN ALL TREATED FRUITS FOLLOWING
THIOACIDOLYSIS 112 5.4 DOWN- AND UP-REGULATION O F LIGNIN BIOSYNTHETIC
GENES IN TRANSGENIC ANTISENSE CHS CALYPSO LINES 115
5.4.1 PHENOTYPE 115
5.4.2 LIGNIN TEXTURE AND LIGNIN CONTENT 116
5.4.3 GENE EXPRESSION LEVELS 117
5.4.4 METABOLITE LEVELS 121
IV. DISCUSSION 123
1. NUCLEOTIDE SEQUENCES 123
2. AMINO ACID SEQUENCES AND BIOCHEMICAL CHARACTERIZATION 123
2.1 FACCR 123
2.2 FACAD 125
2.3 FAPOD 126
3. EXPRESSION LEVELS OF MONOLIGNOL GENES IN DIFFERENT PARTS O F A
STRAWBERRY PLANT... 127 4. FRUITS IN RESPONSE TO WOUNDING AND
AGROBACTERIUM ATTACK 128
5. INDIVIDUAL DOWN-AND UP-REGULATION OF MONOLIGNOL GENES IN FRUITS 130
6. DOWN- AND UP-REGULATION O F MONOLIGNOL GENES IN CHS FRUITS 136
V. CONCLUSION 137
VI. REFERENCES 138
VII. APPENDIX 151
A. DEGENERATE PRIMERS DESIGNED 151
B. THE MAIN CODING SEQUENCE USED FOR CONSTRUCTS 152
C. THE PARTIAL CODING SEQUENCE USED FOR IHPRNA CONSTRUCTS 154
D. STANDARD CURVES 155
E. STATISTICAL ANALYSIS 156
F. PARTIAL GENOME SEQUENCES 163
G SDS-PAGE 166
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| spelling | Yeh, Su-Ying Verfasser aut The lignification of strawberry fruit molecular basis and effects on fruit quality Su-Ying Yeh 2012 XIV, 165 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2012 Erdbeere (DE-588)4136965-8 gnd rswk-swf Lagerfähigkeit (DE-588)4434453-3 gnd rswk-swf Genexpression (DE-588)4020136-3 gnd rswk-swf Lignifizierung (DE-588)4482853-6 gnd rswk-swf Peroxidase (DE-588)4564742-2 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Erdbeere (DE-588)4136965-8 s Lignifizierung (DE-588)4482853-6 s Lagerfähigkeit (DE-588)4434453-3 s Peroxidase (DE-588)4564742-2 s Genexpression (DE-588)4020136-3 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20130121-1113252-0-9 http://mediatum.ub.tum.de/node?id=1113252 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130121-1113252-0-9 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025741524&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Yeh, Su-Ying The lignification of strawberry fruit molecular basis and effects on fruit quality Erdbeere (DE-588)4136965-8 gnd Lagerfähigkeit (DE-588)4434453-3 gnd Genexpression (DE-588)4020136-3 gnd Lignifizierung (DE-588)4482853-6 gnd Peroxidase (DE-588)4564742-2 gnd |
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| title | The lignification of strawberry fruit molecular basis and effects on fruit quality |
| title_auth | The lignification of strawberry fruit molecular basis and effects on fruit quality |
| title_exact_search | The lignification of strawberry fruit molecular basis and effects on fruit quality |
| title_full | The lignification of strawberry fruit molecular basis and effects on fruit quality Su-Ying Yeh |
| title_fullStr | The lignification of strawberry fruit molecular basis and effects on fruit quality Su-Ying Yeh |
| title_full_unstemmed | The lignification of strawberry fruit molecular basis and effects on fruit quality Su-Ying Yeh |
| title_short | The lignification of strawberry fruit |
| title_sort | the lignification of strawberry fruit molecular basis and effects on fruit quality |
| title_sub | molecular basis and effects on fruit quality |
| topic | Erdbeere (DE-588)4136965-8 gnd Lagerfähigkeit (DE-588)4434453-3 gnd Genexpression (DE-588)4020136-3 gnd Lignifizierung (DE-588)4482853-6 gnd Peroxidase (DE-588)4564742-2 gnd |
| topic_facet | Erdbeere Lagerfähigkeit Genexpression Lignifizierung Peroxidase Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1113252 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130121-1113252-0-9 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025741524&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT yehsuying thelignificationofstrawberryfruitmolecularbasisandeffectsonfruitquality |