Functional characterization of the STICHEL gene in Arabidopsis thaliana:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2008
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| Beschreibung: | VI, 80 S. Ill. |
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| 245 | 1 | 0 | |a Functional characterization of the STICHEL gene in Arabidopsis thaliana |c vorgelegt von Cho-Chun Huang |
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Datensatz im Suchindex
| _version_ | 1804140673644888064 |
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| adam_text | Titel: Functional characterization of the STICHEL gene in Arabidopsis thaliana
Autor: Huang, Cho-Chun
Jahr: 2008
Table of Contents
Table of contents
Table of contents .................................................................................I
Index of figures................................................................................TV
Index of tables...................................................................................VI
Summary.............................................................................................I
Zusammenfassung...................................................................................2
Abbreviations........................................................................................4
1. Introduction....................................................................................5
1.1 Cell shape formation.............................................................................................5
1.2 Cytoskeleton and trichome cell development..........................................5
1.3 Cytoskeleton related genes...............................................................6
1.3.1 The microtubule related gene CLASP and 0FP1.................................6
1.3.2 The DISTORTED genes..............................................................8
1.4 The STICHEL gene......................................................................10
Aim of this study.................................................................................14
2. Results..........................................................................................15
2.1 Subcellular localization of STI.........................................................15
2.1.1 Confocal laser scanning and Delta Vision micrographs of STI.................15
2.1.2 Characterization of the STI localization at the branch points....................16
2.1.2.1 Raising STI antibody and detecting STI by S2 antibody...................16
2.1.3 The C-terminus of STI mediates the localization to the plasma membrane 18
2.1.4 Identifying interactions of STI. and STI fragments by yeast two- hybrid and
BiFC assays..................................................................................20
2.1.5 The localization of STI protein shows time frame differences................22
2.2 Molecular Function of the STICHEL Protein........................................23
2.2.1 The AAA+ ATPase domain is necessary for trichome branching.............23
2.2.2 The STI homologue.................................................................25
2.3 Identification of the interacting factors of STI.......................................26
2.3.1 Screening for the proteins interacting with STI by large-scale yeast
two-hybrid assays....................................................................26
2.3.1.1 The interaction between STI and the chromosome segregation ATPases
ENS1.......................................................................................................27
2.3.1.2 The interaction between STt and AtOFP 1...................................29
2.3.1.3 The interaction between STI and PIPKs......................................30
I
Table of Contents
2.3.1.4 The interaction between STI and ArpC4.....................................30
2.3.1.4.1 Mapping of the STI domain which interacts with ArpC4.............32
2.3.1.4.2 The interaction between STI and Arp2/3 related proteins............34
2.3.1.5 The interaction between STI and CLASP....................................35
2.3.1.5.1 Mapping of the STI domain which interacts with CLASP............37
2.3.2 The STI horn also interact with ArpC4 and CLASP............................38
2.3.3 The STI localization and the cytoskeleton drugs.................................39
2.3.4 YFP-ArpC4 and YFP-CLASP in .vri mutant......................................41
3 Discussion.....................................................................................42
3.1 The functional domains of STI.........................................................42
3.2 STI may bind STI-hom and form a hetero-oligomer, and the oligomer might be
movable via microtubule.................................................................44
3.3 STI protein is expressed with little amount in the early stage of cell
development..............................................................................46
3.4 Is STI one of the cytoskeleton linker proteins in plant?.....................................46
3.5 STI and actin microfilaments............................................................47
3.6 STI and microtubule.....................................................................49
Perspectives......................................................................................52
Material...........................................................................................53
Chemicals......................................................................................53
Enzymes.......................................................................................53
Primers.........................................................................................53
Cloning vectors................................................................................53
Constructs were created or used in this study.............................................54
Bacteria strains................................................................................56
Yeast strains...................................................................................56
Plant lines......................................................................................56
Suspension culture...........................................................................57
Microscopes...................................................................................57
Software.......................................................................................57
Methods...........................................................................................58
Standard Molecular biology method........................................................58
Plasmid DNA preparation....................................................................58
Sequencing....................................................................................58
II
Table of Contents
Protein expression and extraction from bacteria...........................................58
Protein expression and extraction from yeast.............................................59
Protein extraction from whole seedlings...................................................59
Protein extraction from Arabidopsis protoplasts.........................................59
Western blot and antibody detection.......................................................59
Protoplast isolation...........................................................................60
Protoplast transformation with PEG.......................................................60
Bimolecular fluorescence complementation (BiFC)....................................61
Large scale yeast two-hybrid screens......................................................61
Gap repair......................................................................................62
Plant growth condition.......................................................................62
Plant transformation..........................................................................62
Seeds sterilization............................................................................62
Particle bombardment........................................................................62
Cytoskeleton Drugs treatment for young seedlings......................................63
Appendix.........................................................................................64
References....................................................................................................................71
Erklarung.........................................................................................78
Acknowledgements......................................................................................................79
Lebenslauf...................................................................................................................80
Index of figures
Index of figures
Figure I The cytoskeleton and trichome cell differentiation............................5
Figure 2 The distinctive trichomes of wild type and clasp mutant.....................6
Figure 3 Cell polarity cues can stabilize microtubules through +TIPs.................7
Figure 4 The distinctive trichomes of wild type and distorted mutant.................8
Figure 5 The Arp2/3 complex and its function in actin branching......................9
Figure 6 Phylogenetic tree of STI, the DNA polymerase III y-subunits, and the RFC
small subunits family.............................................................10
Figure 7 Single, double and triple mutant phenotypes of combinations with different
47/ alleles............................................................................11
Figure 8 Conserved motifs of AAA and AAA+- proteins...............................12
Figure 9 The diverse cellular functions of the AAA and AAA+ proteins............13
Figure 10 Localization of GFP-STI and STI-YFP.......................................15
Figure 11 Schematic map and predicted functional domains of STI protein.........16
Figure 12 The His-S2 expression from bacteria and Western blot analysis of purified
His-S2 protein.....................................................................17
Figure 13 Western blots analysis of S2-GFP.............................................18
Figure 14 Localization of SI-YFP, S2-YFP, GFP-S3, and STI-YF.....................19
Figure 15 The BiFC assays by transient transformation of STI, and STI fragments SI,
S2, and S3 in Arabidopsis protoplasts........................................21
Figure 16 STI-YFP locates differently in different time frame in Arabidopsis
protoplast..........................................................................22
Figure 17 The protein sequence alignment results between STI and AAA or AAA+
proteins............................................................................23
Figure 18 The BiFC assays by transient transformation of mutated version of STI and
STI in onion epidermal cells...................................................24
Figure 19 The localization of YFP-STI-hom and the BiFC assays by transient
transformation of STI-hom with SI. S2, S3, and STI in onion epidermal
cells................................................................................25
Figure 20 The amino acid alignment results of the group of chromosome segregation
ATPase (Atlg50660) and ENS1...............................................27
Figure 21 The BiFC assays by transient transformation of STI with ENS1 in onion
epidermal cells...................................................................28
IV
Index of figures
Figure 22 The BiFC by transient transformation of STI and AtOFPl in onion
epidermal cells...................................................................29
Figure 23 The BiFC by transient transformation of STI and ArpC4 in different cell
types................................................................................31
Figure 24 Schematic map of STI and SI.................................................32
Figure 25 The BiFC by transient transformation of the small fragments of SI and
ArpC4 in onion epidermal cells................................................33
Figure 26 The BiFC by transient transformation of Arp2/3 complex subunits and STI
in onion epidermal cells.........................................................34
Figure 27 The BiFC by transient transformation of STI and CLASP in different cell
types...............................................................................36
Figure 28 The BiFC by transient transformation of STI small fragments and CLASP
in onion epidermis...............................................................37
Figure 29 The BiFC assays by transient transformation of STI-hom and Arpc4 or
CLASP in onion epidermal cells...............................................38
Figure 30 The localization of GFP-STI after latrunculin B treatmen...................39
Figure 31 The localization of GFP-STI after oryzalin treatment......................40
Figure 32 Confocal images of the localization of YFP-ArpC4 and YFP-CLASP in sti
mutant..............................................................................41
Figure 33 The functional domain structure of STI protein.............................43
Figure 34 STI and its interacting factors..................................................45
Figure 35 STI may play an important role in actin dynamics..........................48
Figure 36 A model for cortical MT attachment through CLASPs, LL5p. and ELK.49
Figure 37 Model of how STI regulate trichome branch with microtubule...........51
Index of tables
Index of tables
Table 1 Summary of different determining factors which applied for plant protein
extraction in this study................................................................18
Table 2 The interaction results between STI, and STI fragments SI, S2, and S3 by yeast
two-hybrid assays.....................................................................20
Table 3 The rescue results of different mutated versions of STI by plant
transformation.......................................................................24
Table 4 The interaction results between STI hom and different STI fragments by yeast
two-hybrid assays......................................................................26
Table 5 The interaction results between ENS1 and STI by yeast two-hybrid assays.....28
Table 6 The yeast two-hybrid assays of STI and the OFP1..............................29
Table 7 The yeast two hybrid assay of STI and the PIPKs...............................30
Table 8 The yeast two-hybrid assays of STI, SI and the ArpC4...........................31
Table 9 Summary of the yeast two-hybrid and BiFC assays for STI small fragment and
ArpC4...................................................................................33
Table 10 The yeast two hybrid assay of STI and the subunit of Arp2/3 complex........34
Table 11 The yeast two-hybrid assay of STI and the CLASP............................35
Table 12 Summary of the yeast two hybrid assay and BiFC for the small fragment of
SI and CLASP.......................................................................37
VI
|
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| spelling | Huang, Cho-Chun 1978- Verfasser (DE-588)138489416 aut Functional characterization of the STICHEL gene in Arabidopsis thaliana vorgelegt von Cho-Chun Huang 2008 VI, 80 S. Ill. txt rdacontent n rdamedia nc rdacarrier Köln, Univ., Diss., 2008 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=018613740&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Huang, Cho-Chun 1978- Functional characterization of the STICHEL gene in Arabidopsis thaliana |
| subject_GND | (DE-588)4113937-9 |
| title | Functional characterization of the STICHEL gene in Arabidopsis thaliana |
| title_auth | Functional characterization of the STICHEL gene in Arabidopsis thaliana |
| title_exact_search | Functional characterization of the STICHEL gene in Arabidopsis thaliana |
| title_full | Functional characterization of the STICHEL gene in Arabidopsis thaliana vorgelegt von Cho-Chun Huang |
| title_fullStr | Functional characterization of the STICHEL gene in Arabidopsis thaliana vorgelegt von Cho-Chun Huang |
| title_full_unstemmed | Functional characterization of the STICHEL gene in Arabidopsis thaliana vorgelegt von Cho-Chun Huang |
| title_short | Functional characterization of the STICHEL gene in Arabidopsis thaliana |
| title_sort | functional characterization of the stichel gene in arabidopsis thaliana |
| topic_facet | Hochschulschrift |
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