Fructan exohydrolases in corn: search for functions
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. Sprache |
| Beschreibung: | IV, 147 Bl. |
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| 245 | 1 | 0 | |a Fructan exohydrolases in corn |b search for functions |c [presented by Hongbo Zhao] |
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Datensatz im Suchindex
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| adam_text | FRACTAL! EXOHYDROLASES IN CORN - SEARCH FOR FUNCTIONS
REFEREES: PROFESSOR DR. THOMAS RAUSCH
PROFESSOR DR. RUDIGER HELL
HTTP://D-NB.INFO/1054939306
TABLE OF CONTENTS
TABLE OF CONTENTS
1. SUMMARY/ZUSAMMENFASSUNG 1
1.1 SUMMARY (ENGLISH) 1
1.2 ZUSAMMENFASSUNG (DEUTSCH) 3
2. INTRODUCTION 5
2.1 MAIZE AS A
MODEL..................................................................................................
5
2.2 SOURCE AND
SINK......................................................................................................
5
2.2.1 SOURCE-SINK RELATIONS 5
2.2.2 SOURCE-SINK REGULATION FACTORS 6
2 J
SUGAR......................................................................................................................
7
2.3.1 SUGAR SENSING AND SIGNALING 7
2.3.2 SUGAR TRANSPORT 8
2.3.3 SUCROSE TRANSPORT IN THE VACUOLE 9
2.4 SUCROSE SYNTHESIS AND
HYDROLYSIS........................................................................
9
2.4.1 SUCROSE SYNTHASE (SUSY) 10
2.4.2 INVERTASE 12
2.5 PHYSIOLOGICAL ROLES OF ACID
INVERTASES.................................................................
15
2.5.1 CWI IS ESSENTIAL FOR SUCROSE PARTITIONING, SEED AND FRUIT
DEVELOPMENT 15
2.5.2 VI AS A KEY MODULATOR FOR HEXOSE ACCUMULATION AND CELL EXPENSION
16
2.6 REGULATION OF
INVERTASES......................................................................................
17
2.6.1 ENVIRONMENTAL STIMULI 17
2.6.2HORMONES 18
2.6.3 SUGARS 18
2.6.4 WOUNDING AND PATHOGENS 18
2.7 PROTEINACEOUS INHIBITORS OF PLANT
INVERTASES.................................................... 19
2.7.1 STRUCTURAL FEATURES OF INVERTASE INHIBITOR PROTEINS 20
2.7.2 EXPRESSION AND PHYSIOLOGICAL ROLES OF INVERTASE INHIBITORS 21
2.7.3 REGULATION OF INVERTASE INHIBITORS 23
2.8FRUCTANS................................................................................................................
23
2.8.1 STRUCTURE AND OCCURRENCE OF FRUCTANS IN PLANTS AND BACTERIA 23
2.8.2 FUNCTIONS OF FRUCTANS IN PLANTS 25
2.8.3 APPLICATIONS OF FRUCTANS 25
2.9 FRUCTAN SYNTHESIS AND
HYDROLYSIS......................................................................
25
2.9.1 FRUCTAN SYNTHESIS 25
2.9.2 FRUCTAN HYDROLYSIS 27
2.10 FUNCTIONS OF FRUCTAN EXOHYDROLASES IN
PLANT................................................... 28
2.11 REGULATION OF FRUCTAN
EXOHYDROLASES................................................................
28
2.12 STRUCTURE AND EVOLUTION BETWEEN INVERTASES, FRUCTAN EXOHYDROLASES
AND
FRUCTAN BIOSYNTHETIC
ENZYMES........................................................................
29
3 RESEARCH AIMS
31
I
TABLE OF CONTENTS
4 RESULTS 32
4.1 MAKE IS NON FRUCTAN PLANT BUT WITH FEH
ENZYME........................................... 32
4.1.1 CARBOHYDRATE ANALYSIS IN SOURCE AND SINK PART OF MAIZE LEAF. 32
4.1.2 CARBOHYDRATE ANALYSIS IN OTHER TISSUES IN MAIZE 33
4.1.3 IN VITRO SYNTHESIS OF FRUCTAN TRISACCHARIDES 34
4.1.4 FEH ACTIVITIES IN DIFFERENT MAIZE TISSUES 35
4.2 MAIZE HAS FEHS BUT NO
FBES.............................................................................
35
4.2.1 CLONING, SEQUENCE ALIGNMENT AND PHYLOGENETIC ANALYSIS OF CWI/ FEHS
35
4.2.2 ENZYME CHARACTERIZATION 42
4.2.3 ECTOPIC EXPRESSION ENZYMES IN NICOTIANABENTHAMIANA. 49
4.2.4 SUBCELLULAR LOCALIZATION OF MAIZE FEHS 51
4.2.5 TRANSCRIPT LEVELS OF MAIZE FEHS IN DIFFERENT TISSUES 54
4.2.6 DROUGHT, ABA AND COLD REGULATE THE MAIZE FEHS IN TRANSCRIPT LEVEL
54
43 FEHS HYDROLYZE INVERTASE-MADE FRUCTANS
(KESTOSE)......................................... 56
4.4 IN CORN, 6&1-FEH EXPRESSION IS INDUCED BY
LEVAN........................................... 56
4.4.1 EXOGENOUS LEVAN AFFECT MAIZE FEHS EXPRESSION 56
4.4.2 ECTOPICALLY EXPRESSING ZM-6&1-FEH1 TRANSGENIC ARABIDOPSIS LINES DO
NOT
ALLEVIATE THE INFECTION OF PSEUDOMONAS SYRINGAE PV. TOMATO
DC3000. 57
4.5 NOVEL INVERTASE INHIBITORS IN
MAIZE...................................................................
59
4.5.1 CLONING CDNAS ENCODING THE INVERTASE INHIBITOR, SEQUENCE ALIGNMENT
AND
PHYLOGENETIC ANALYSIS 59
4.5.2 FUNCTIONAL ASSAY OF ZM-INVINHS 62
4.5.3 SUBCELLULAR LOCALIZATION OF ZM-INVINHS 66
4.5.4 TRANSCRIPT LEVEL OF ZM-INVINH4 UNDER DROUGHT, ABA AND
COLD STRESS 68
4.6 FEH ENZYMES ACTIVATE INVERTASES BY COMPETING FOR INVERTASE INHIBITOR
BINDING..................................................................................................................
69
4.6.1 FEHS PHYSICALLY INTERACT WITH INVERTASE INHIBITORS 69
4.6.2 MAIZE INVERTASE INHIBITORS AFFECT FEHS ACTIVITY 72
4.6.3 ZM-6&1-FEH2 TRANSGENIC ARABIDOPSIS LINES ALLEVIATE THE EFFECT OF
ABA
INDUCED BY AT-C/VIF
1 72
5 DISCUSSION 76
5.1 PROPERTIES AND LOCALIZATION OF PLANT
FEHS........................................................ 76
5.1.1 FEH ACTIVITIES IN MAIZE EXTRACTS 76
5.1.2 HETEROLOGOUS EXPRESSION, PURIFICATION AND IDENTIFICATION OF FEHS
IN N.
BENTHAMIANA LEAVES 76
5.1.3 HETEROLOGOUS EXPRESSION, PURIFICATION AND IDENTIFICATION OF FEHS
IN PICHIA
PAS TOR IS 77
5.2 PLANT FEHS ARE DERIVED FROM CELL WALL
INVERTASES........................................... 78
5J MAIZE IS A FRUCTOOLIGOSACCHARIDE-PLANT BUT NOT A FRUCTAN
PLANT................... 80
5.3.1 FRUCTOOLIGOSACCHARIDES IN MAIZE 80
II
TABLE OF CONTENTS
5.3.2 FUNCTIONS OF MAIZE FRUCTOOLIGOSACCHARIDES 81
5.3.3 MAIZE LACKS FRUCTAN BIOSYNTHESIS ENZYMES FOR FRUCTAN CHAIN
ELONGATION 82
5.4 REGULATION OF FEHS IN NON FRUCTAN PLANTS OR IN
MAIZE.................................... 82
5.4.1 REGULATION OF MAIZE FEHS EXPRESSION 82
5.4.2 MAIZE FEHS ARE REGULATED ON POST-TRANSCRIPTIONAL LEVEL? 84
5.4.3 MAIZE FEHS ARE REGULATED ON POST-TRANSLATIONAL LEVEL 85
5.5 FUNCTIONS OF FEHS IN
MAIZE................................................................................
88
5.5.1 SIGNALING? 88
5.5.2 PATHOGEN DEFENCE OR STABILIZING THE SYMBIOSIS 88
5.5.3 FEH INVOLVED IN INVERTASE-MEDIATED SUCROSE DEGRADATION AND
SIGNALING IN
SOURCE AND SINK 91
5.5.4 OTHER FUNCTIONS 93
6. MATERIAL AND METHODS 94
6.1 PLANT MATERIAL AND
CULTIVATION............................................................................
94
6.1.1 ZEA MAYS. 94
6.1.2 NICOTIANA BENTHAMICMA. 94
6.1.3 ALLIUM CEPA. 94
6.1.4 ARABIDOPSIS THALIANA. 94
6.2
MICROBIOLOGICAL
TECHNIQUES.................................................................................
94
6.2.1 BACTERIAL STRAINS 94
6.2.2 MEDIA AND ANTIBIOTICS 95
6.2.3 PREPARATION OF ELECTROCOMPETENT BACTERIAL AND YEAST CELLS 95
6.2.4 TRANSFORMATION OF BACTERIAL AND YEAST CELLS VIA ELECTROPORATION 96
6.2.5 PREPARATION OF GLYCEROL STOCKS 96
63
NUCLEIC ACID
TECHNIQUES.......................................................................................
96
6.3.1 AGAROSE GELS 96
6.3.2 POLYACRYLAMIDE GELS 96
6.3.3 EXTRACTION OF GENOMIC DNA FOR CLONING 97
6.3.4 RNA EXTRACTION 97
6.3.5 SEPARATION OF RNA BY AGAROSE GEL ELECTROPHORESIS 98
6.3.6 DETERMINATION OF NUCLEIC ACID CONCENTRATION 98
6.3.7 REVERSE TRANSCRIPTION 98
6.3.8 VECTORS 98
6.3.9 OLIGONUCLEOTIDES 99
6.3.10 POLYMERASE CHAIN REACTION (PCR) 101
6.3.11 QUANTITATIVE REAL TIME PCR 102
6.3.12 GEL EXTRACTION AND PCR PURIFICATION 103
6.3.13 ISOLATION OF PLASMID DNA FROM BACTERIAL CULTURE 103
6.3.14 DNA SEQUENCING 104
6.4 CLONING
TECHNIQUES............................................................................................
104
6.4.1 T/A
CLONING OF PCR PRODUCTS 104
6.4.2 CLONING USING RESTRICTION ENZYME DIGESTION 104
6.4.3 CLONING USING THE GATEWAY SYSTEM 104
HI
TABLE OF CONTENTS
6.5 PROTEIN
TECHNIQUES.............................................................................................
104
6.5.1 PROTEIN OVEREXPRESSION AND PURIFICATION IN E. CO/I. 104
6.5.2 PROTEIN OVEREXPRESSION AND PURIFICATION IN PICHIAPASTORIS. 105
6.5.3 EXTRACTION OF PROTEINS FROM NICOTIANA BENTHAMIANA LEAVES AND
ARABIDOPSIS
THALIANA. 106
6.5.4 EXTRACTION OF SOLUBLE AND CELL WALL PROTEINS FROM MAIZE 106
6.5.5 CARBOHYDRATE EXTRACTION FOR HPAEC-PAD 106
6.5.6 PME EXTRACTION 107
6.5.7 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS 107
6.5.8 COOMASIE STAINING 107
6.5.9 SILVER STAINING 107
6.5.10 IMMUNOBLOTTING 108
6.5.11 DIFFERENTIAL CENTRIFUGATION AND SALT ELUTION 108
6.5.12 IMMUNOPRECIPITATION 109
6.6 ENZYME ACTIVITY
ASSAYS......................................................................................
109
6.6.1 MEASUREMENT OF SOLUBLE AND CELL-WALL BOUND FEH OR INVERTASE
ACTIVITIES. 109
6.6.2 FUNCTIONAL ASSAY OF INVERTASE INHIBITOR RECOMBINANT PROTEINS 110
6.6.3 FUNCTIONAL ASSAY OF FEH RECOMBINANT PROTEINS 110
6.6.4 CARBOHYDRATE ANALYSIS VIA HPAEC-PAD 110
6.6.5 PME ACTIVITY MEASUREMENT ILL
6.7
MICROSCOPY.........................................................................................................
112
6.7.1 FLUORESCENCE MICROSCOPY. 112
6.7.2 CONFOCAL LASER SCANNING MICROSCOPY. 112
6.8 PLANT
TRANSFORMATION.........................................................................................
112
6.8.1 TRANSIENT EXPRESSION BY AGROBACTERIA LEAF INFILTRATION 112
6.8.2 TRANSIENT EXPRESSION BY PARTICLE GUN BOMBARDMENT 113
6.8.3 STABLE TRANSFORMATION BY FLORAL DIP 113
7 ABBREVIATIONS 114
8 BIBLIOGRAPHY. 117
9 ACKNOLOWLEDGEMENTS 147
IV
|
| any_adam_object | 1 |
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| spelling | Zhao, Hongbo Verfasser (DE-588)1054669295 aut Fructan exohydrolases in corn search for functions [presented by Hongbo Zhao] 2014 IV, 147 Bl. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache Heidelberg, Univ., Diss., 2014 (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=027475645&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Zhao, Hongbo Fructan exohydrolases in corn search for functions |
| subject_GND | (DE-588)4113937-9 |
| title | Fructan exohydrolases in corn search for functions |
| title_auth | Fructan exohydrolases in corn search for functions |
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| title_full | Fructan exohydrolases in corn search for functions [presented by Hongbo Zhao] |
| title_fullStr | Fructan exohydrolases in corn search for functions [presented by Hongbo Zhao] |
| title_full_unstemmed | Fructan exohydrolases in corn search for functions [presented by Hongbo Zhao] |
| title_short | Fructan exohydrolases in corn |
| title_sort | fructan exohydrolases in corn search for functions |
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