Functional characterization of AtSP1, a nutrient-induced receptor-like kinase:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 112 Bl. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Functional characterization of AtSP1, a nutrient-induced receptor-like kinase |c von Xu-Na Wu |
| 264 | 1 | |c 2012 | |
| 300 | |a 112 Bl. |b Ill., graph. Darst. | ||
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| 502 | |a Potsdam, Univ., Diss., 2012 | ||
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Datensatz im Suchindex
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| adam_text | IMAGE 1
TABLE OF CONTENT
ABSTRACT 6
ABSTRAKT 8
FIGURES AND TABLES 10
ABBREVIATION 12
1 INTRODUCTION . 13
1.1 SUCROSE IN PLANTS 13
1.2 CARBON AND NITROGEN SIGNALING IN PLANTS 14
1.2.1 CARBON SIGNALING 14
1.2.2 NITROGEN SIGNALING 15
1.2.3 CARBON AND NITROGEN CROSS TALK 16
1.3 PHOSPHORYLATION IN CELLULAR PROCESSES 18
1.4 MASS SPECTROMETRY BASED PROTEOMICS 20
1.5 AIM OF THE WORK 22
2 MATERIAL 24
2.1 CHEMICAL 24
2.2 EQUIPMENT AND SOFTWARE 25
2.3 ENZYMES 26
2.4 OLIGONUCLEOTIDES 26
2.5 PLASMID 28
2.5.1 PGEM-T EASY VECTOR (PROMEGA, U.S.A.) 28
2.5.2 PEZR(H)-LN VECTOR (DAVID EHRHARDT, STANFORD, CA, U.S.A.) FOR GENE
AND GFP FUSION 28
2.6 PLANT MATERIALS 29
2.7 DATABASES UTILIZED 29
3 METHODS 30
3.1 IDENTIFICATION OF HOMOZYGOUS T-DNA INSERTION LINES 30
3.1.1 DNA EXTRACTION (TOTAL GENOMIC DNA FROM ARABIDOPSIS TISSUE) 30
3.1.2 PCR FOR IDENTIFICATION OF HOMOZYGOUS T-DNA INSERTION LINES 30
3.2 MOLECULAR CLONING 31
3.2.1 T-A CLONING 31
3.2.2 SELECTION OF POSITIVE CLONES 32
3.2.3 RESTRICTION DIGESTION OF DNA AND DEPHOSPHORYLATION OF 5 ENDS OF
LINEARIZED VECTORS 32
2
HTTP://D-NB.INFO/1028356153
IMAGE 2
3.2.4 LIGATION OF PEZR (H)-LN LINEARIZED VECTOR AND DNA FRAGMENTS 32
3.2.5 CONSTRUCTION OF TRANSGENIC ARABIDOPSIS LINES 33
3.2.5.1 AGROBACTERIA-TRANSFORMATION 33
3.2.5.2 ARABIDOPSIS-TRANSFORMATIONS BY DIPPING (CLOUGH AND BENT, 1998)
33
3.2.5.3 IDENTIFICATION OF HOMOZYGOUS TRANSGENIC LINES 34
3.3 SUCROSE STARVATION-RESUPPLY EXPERIMENT AND DATA ANALYSIS 34
3.3.1 S EED STERILIZATION 35
3.3.2 SEEDLINGS GROWTH, MEDIUM CHANGE, STARVATION, RESUPPLY AND HARVEST
35
3.3.3 MICROSOME AND SOLUBLE PROTEIN PREPARATION 36
3.3.4 IN-SOLUTION DIGESTION 37
3.3.5 IN-GEL PROTEIN REDUCTION AND ALKYLATION 37
3.3.6 IN-GEL DIGESTION 38
3.3.7 C18-STAGE TIPS FOR PEPTIDE DESALTING AND CONCENTRATION
(RAPPSILBERE/ AL., 2003) 39
3.3.8 ENRICHMENT OF PHOSPHOPEPTIDES WITH TI0 2 (LARSEN ET AL., 2005) 39
3.3.9 USE OF CI 8-STAGE TIPS FOR PHOSPHOPEPTIDE DESALTING AND
CONCENTRATION 40
3.3.10 LC-MS 40
3.3.11 PEPTIDE IDENTIFICATION AND VALIDATION 40
3.3.12 QUANTITATIVE PROTEIN ANALYSIS-LABEL FREE QUANTITATION 41
3.3.13 SRM (SELECTED REACTION MONITORING) 42
3.4 GENOTYPE AND GENE EXPRESSION ANALYSIS ON THE C/N MATRIX 44
3.4.1 RNA EXTRACTION: INVISORB SPIN PLANT RNA MINI KITS 44
3.4.2 DNASE I DIGESTION 44
3.4.3 CDNA SYNTHESIS: SUPERSCRIPT II KIT 44
3.4.4 REAL-TIME PCR 45
3.4.5 DATA ANALYSIS FOR REAL-TIME PCR 45
3.4.6 PULL-DOWN WITH GFP AS BAIT (GFP-TRAP METHOD) 45
3.5 TOTAL PROTEIN EXTRACTION 46
4 RESULTS 48
4.1 INTRODUCTION TO CANDIDATE GENE: ATSPL 48
4.2 ATSPL COULD BE LOCALIZED TO THE PLASMA MEMBRANE 48
4.3 IDENTIFICATION OF HOMOZYGOUS T-DNA INSERTION LINE ( SPL ) 49
4.4 GENE TRANSFORMATION CONSTRUCTS 50
4.5 SUCROSE STARVATION-RESUPPLY EXPERIMENT (SHORT TERM EFFECTS) 51
4.5.1 SUCROSE-INDUCED PHOSPHORYLATION OF ATSPL 52
3
IMAGE 3
4.5.2 COMPARISON OF THE RELATIVE PHOSPHORYLATION STATUS OF DIFFERENT
PROTEINS
BETWEEN WT AND THE SPL MUTANT 53
4.5.2.1 REDUCED SUCROSE-INDUCED PHOSPHORYLATION OF IT 1 -ATPASES, CA +
-ATPASES, TRANSPOTERS, AND AQUAPORINS IN THE SPL MUTANT 57
4.5.2.2 REDUCED SUCROSE-INDUCED PHOSPHORYLATION OF PROTEINS RELATED TO
PROTEIN SYNTHESIS IN SPL MUTANT 58
4.5.2.3 REDUCED SUCROSE-INDUCED PHOSPHORYLATION OF SIGNALING,
MISCELLANEOUS (MISC), METABOLISM, RNA, AND CELL WALL RELATED PROTEINS IN
SPL MUTANT...58
4.5.2.4 REDUCED SUCROSE-INDUCED PHOSPHORYLATION OF UNKNOWN PROTEINS AND
PROTEINS WITH OTHER FUNCTIONS IN THE SPL MUTANT 59
4.5.3 FUNCTIONAL CONFIRMATION OF THE ROLE OF ATSPL IN THE REGULATION OF
SUCROSEINDUCED PHOSPHORYLATION 60
4.5.4 ROLE OF SER-744 OF ATSPL IN SUCROSE-INDUCED PHOSPHORYLATION 62
4.6 GROWTH EFFECTS UNDER THE C/N MATRIX (LONG TERM EFFECTS) 65
4.6.1 PHENOTYPE SCREEN 66
4.6.1.1 COMPARISON OF PRIMARY ROOT LENGTH BETWEEN WT AND SP 1 MUTANT 66
4.6.1.2 UNDER THE HIGH C/HIGH N CONDITION, THE OX AND OX-CTERM LINES
SHOW ENHANCED PRIMARY ROOT GROWTH 69
4.6.1.3 THE PUTATIVE ACTIVE PROTEIN ATSPL S744D CAN COMPLEMENT THE SPL
PHENOTYPE WHILE A PUTATIVE INACTIVE VERSION (ATSPL S744A ) CANNOT UNDER
THE LOW C/HIGH N CONDITION 70
4.6.2 REAL-TIME PCR REVEALS THE NUTRITIONAL STATUS OF SPL MUTANTS 71
4.6.2.1 SPL MUTANT DISPLAYS A CARBON STARVATION EXPRESSION PHENOTYPE
UNDER LOW C/HIGH N CONDITION AND HIGH C/LOW N CONDITIONS, WHILE SHOWING
A NITROGEN STARVATION EXPRESSION PHENOTYPE UNDER THE HIGH C/HIGH N
CONDITION (YELLOW BOXES IN TABLE III) 73
4.6.2.2 ATNRT2.5 EXPRESSION IS DOWN-REGULATED IN SPL MUTANT UNDER THE
MID C/MID N CONDITION (YELLOW BOX IN TABLE III) 74
4.6.2.3 SPL AND SPL+ATSPL S744D LINES ARE SENSITIVE TO LOW C/HIGH N
CONDITIONS WHILE SPL+ATSPL S744A LINES ARE NOT (GREEN BOX IN TABLE III)
75
4.6.2.4 ATSPL OVEREXPRESSION SUPPRESSES ASN1 EXPRESSION UNDER LOW C/LOW
N CONDITION (BLUE BOX IN TABLE III) 75
4.6.3 INTERACTION PARTNERS OF ATSPL AS DETERMINED THROUGH AP-MS 76
DISCUSSION 79
5.1 SHORT TERM EFFECTS OF ATSPL 79
5.1.1 PHOSPHOPROTEINS WERE REGULATED BY ATSPL IN RESPONSE TO SUCROSE
RESUPPLY AFTER STARVATION 80
5.1.1.1 TRANSPORT-RELATED PROTEINS 81
5.1.1.2 PROTEINS WITH SYNTHESIS OR SIGNALING FUNCTION 83
4
IMAGE 4
5.1.2 CONCLUSION OF SHORT TERM EFFECTS OF ATSPL 85
5.2 LONG TERM EFFECTS OF ATSPL 86
5.2.1 ANALYSIS OF PHYSIOLOGICAL ROLE OF ATSPL UNDER LOW C/HIGH N
CONDITION 87
5.2.2 ANALYSIS OF PHYSIOLOGICAL ROLE OF ATSPL UNDER HIGH C/HIGH N
CONDITION 88
5.2.3 AT3G02880 IS A POSSIBLE INTERACTION PARTNER OF ATSPL 89
5.2.4 CONCLUSION FOR THE LONG TERM EFFECTS OF ATSPL 90
6 OUTLOOK 91
N
7 ACKNOWLEDGEMENT 93
8 LITERATURE 94
9 SUPPLEMENTARY DATA 105
5
|
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| author | Wu, Xu-Na |
| author_facet | Wu, Xu-Na |
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| dewey-ones | 571 - Physiology & related subjects |
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| discipline | Biologie |
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| spelling | Wu, Xu-Na Verfasser aut Functional characterization of AtSP1, a nutrient-induced receptor-like kinase von Xu-Na Wu 2012 112 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Potsdam, Univ., Diss., 2012 Genexpression (DE-588)4020136-3 gnd rswk-swf Phosphoproteine (DE-588)4174416-0 gnd rswk-swf Pflanzenwachstum (DE-588)4045595-6 gnd rswk-swf Phosphorylierung (DE-588)4174459-7 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Phosphorylierung (DE-588)4174459-7 s Phosphoproteine (DE-588)4174416-0 s Pflanzenwachstum (DE-588)4045595-6 s Genexpression (DE-588)4020136-3 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025377680&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wu, Xu-Na Functional characterization of AtSP1, a nutrient-induced receptor-like kinase Genexpression (DE-588)4020136-3 gnd Phosphoproteine (DE-588)4174416-0 gnd Pflanzenwachstum (DE-588)4045595-6 gnd Phosphorylierung (DE-588)4174459-7 gnd |
| subject_GND | (DE-588)4020136-3 (DE-588)4174416-0 (DE-588)4045595-6 (DE-588)4174459-7 (DE-588)4113937-9 |
| title | Functional characterization of AtSP1, a nutrient-induced receptor-like kinase |
| title_auth | Functional characterization of AtSP1, a nutrient-induced receptor-like kinase |
| title_exact_search | Functional characterization of AtSP1, a nutrient-induced receptor-like kinase |
| title_full | Functional characterization of AtSP1, a nutrient-induced receptor-like kinase von Xu-Na Wu |
| title_fullStr | Functional characterization of AtSP1, a nutrient-induced receptor-like kinase von Xu-Na Wu |
| title_full_unstemmed | Functional characterization of AtSP1, a nutrient-induced receptor-like kinase von Xu-Na Wu |
| title_short | Functional characterization of AtSP1, a nutrient-induced receptor-like kinase |
| title_sort | functional characterization of atsp1 a nutrient induced receptor like kinase |
| topic | Genexpression (DE-588)4020136-3 gnd Phosphoproteine (DE-588)4174416-0 gnd Pflanzenwachstum (DE-588)4045595-6 gnd Phosphorylierung (DE-588)4174459-7 gnd |
| topic_facet | Genexpression Phosphoproteine Pflanzenwachstum Phosphorylierung Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025377680&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT wuxuna functionalcharacterizationofatsp1anutrientinducedreceptorlikekinase |