Identification and analysis of wheat ( Triticum aestivum)
MADS-box genes with high expression levels in embryos
Inaugural-Dissertation
zur Erlangung der Doktorwürde der Fakultät für Biologie
der Albert-Ludwigs-Universität Freiburg im Breisgau
Vorgelegt von
Sabine Nadler
Freiburg im Breisgau, Oktober 2014
Abbreviations.........................................................................
V
1
Introduction............................................................................
1
1.1
Triticum aestivum..................................................................................................
1
1.2
MADS-box genes...................................................................................................
2
1.2.1
MADS-box gene evolution and function..........................................................
4
1.2.2
ABODE model........................................................................................................
7
1.2.3
MADS - MADS interactions................................................................................
9
1.2.4
MADS - non-MADS interactions......................................................................
12
1.2.5
Crosstalk between MADS-domain proteins and hormone signaling
pathways regulating plant development..........................................................
14
1.2.5.1
Auxin signaling pathway and MADS proteins..................................................
14
1.2.5.2
Brassinosteroid signaling pathway and MADS proteins...............................
15
1.3
Aim of the work......................................................................................................
18
2
Material and Methods........................................................
21
2.1
M aterials
2.1.1
Bacterial strains and media................................................................................
21
2.1.2
Yeast strains and media.....................................................................................
21
2.1.3
Plant material and media....................................................................................
23
2.1.4
Oligonucleotides, Plasmids and Antibodies....................................................
24
2.1.5
Enzymes and Reagents............................................................................
26
2 .2
2.2.1
M ethods
Microbiological methods....................................................................................
27
2.2.1.1
Transformation of electrocompetent E.coli cells.............................................
27
2.2.1.2
Transformation of chemocompetent BL21 (DE3) cells...................................
28
2.2.1.3
T ransformation of Agrobacterium tumefaciens................................................
28
2.2.2
Bacterial methods.................................................................................................
28
2.2.2.1
Preparation of Agrobacterium competent cells..............................................
28
2.2.2.2
Plasmid extraction (Mini/Midi DNA Preparation)............................................
28
2.2.2.3
Digestion/Vector dephosphorylation/Ligation.................................................
28
2.2.3
Yeast methods......................................................................................................
29
2.2.3.1
Small-scale yeast transformation......................................................................
29
2.2.3.2
Yeast mating..........................................................................................................
29
2.2.3.3
Yeast plasmid extraction and purification........................................................
29
2.2.4
Protein methods....................................................................................................
29
2.2.4.1
Protein expression and purification....................................................................
29
2.2.4.2
Pull down approach...............................................................................................
30
2.2.4.3
SDS gel electrophoresis......................................................................................
30
2.2.4 4
Western blot............................................................................................................
31
Plant methods.......................................................................................................
31
2.2.5.1
Transformation of Arabidopsis thalianavia floral-dip method......................
31
2.2.5.2
Analysis of transgenic lines.................................................................................
32
2.2.5.3
Germination assay................................................................................................
32
2.2.5 4
Plant DNA extraction............................................................................................
32
2.2.5.5
Nicotiana tabacum protoplast isolationand transformation...........................
32
DNA methods.........................................................................................................
33
2.2.5
2.2.6
2.2.6.1
PCR (Polymerase-Chain-Reaction)..................................................................
33
2.2.6 2
Semi-quantitative R T-P C R ...................................................................................
33
2.2.6.3
genomic DNA extraction.......................................................................................
34
2.2.6.4
Agarose gel electrophoresis...............................................................................
34
2.2.7
RNA methods.........................................................................................................
34
2.2.7.1
RNA isolation and quantification.........................................................................
34
2.2.7 2
RNA MOPS gel electrophoresis.........................................................................
35
2.2.7.3
First-strand cDNA synthesis................................................................................
35
2.2.7.4
SM ART RACE cDNA amplification....................................................................
35
2.2.7 5
Quantitative Real-time PC R .................................................................................
35
3
Results...................................................................................
36
3.1
TaMADS sequence analysis..............................................................................
36
3.2
Phylogenetic analysis..........................................................................................
40
3.3
Expression levels in different tissues..............................................................
43
3.4
Regulation of TMBs at the transcriptional level.............................................
46
3.5
Interplays between TMBs and OsMADS32 at the protein level................
48
3.5.1
Dimerization pattern among members of the OsMADS32 clade................
48
3.5.2
Search for interaction partners by screening a rice embryo cDNA
expression library..............................................................................................
55
3.5.2.1
OsMADS32 library screening.............................................................................
56
3.5.2.2
TMB1 library screening........................................................................................
60
3.5.2.3
Verification of two selected putative interactions from the
library screening...................................................................................................
62
3.5.2.4
via yeast-two-hybrid approach...........................................................................
63
3.5.2.5
via split-YFP approach.........................................................................................
66
3.5.2.6
via pull down approach........................................................................................
3.5.3
Investigation of interactions between selected GSK3-like kinases
and OsMADS32 clade members......................................................................
3.5.3.1
67
69
Investigation of interactions between selected GSK3-like kinases
and the two selected rice proteins OsMYB and OsARF2...........................
73
3.6
Overview of interaction patterns identified in this work...............................
75
3.7
Overexpression of TMB1 in Arabidopsis.........................................................
77
3.7.1
TMB1 transgenic line 9 2 .....................................................................................
78
3.7.2
TMB1 transgenic line 100...................................................................................
81
3.8
Interaction pattern between BIN2 and OsMADS32 clademembers........
83
4
Discussion.............................................................................. 87
4.1
Sequence analysis...............................................................................................
87
4.2
Expression levels in different tissues............................................................
91
4.3
Interplays between TMBs and OsMADS32 at the protein level................
92
4.3.1
Dimerization behavior of OsMADS32 dade members..............................
93
4.3.2
Search for interaction partners of OsMADS32 and TM B 1.........................
95
4.3.2.1
Verification of selected putative interactions..............................................
98
4.3.2.2
Interaction patterns between selected GSK3-like kinases and
OsMADS32 clade members............................................................................
4.3.2.3
100
Interactions between selected GSK3-like kinases and the two
selected rice proteins OsMYB and OsARF2.............................................
102
4.4
TMB1 overexpressing lines.............................................................................
103
4.5
Proposed working model.................................................................................
109
4.6
Outlook.................................................................................................................
112
Summary................................................................................................ 114
Deutsche Zusammenfassung............................................................ 116
Danksagungen...................................................................................... 118
References............................................................................................. 119