Gene expression profiling in different stages of development of Arabidopsis thaliana leaftrichomes at the single cell level [Elektronische Ressource] / von Sergiy Kryvych

Sergiy Kryvych

Sujets

• Biologie

Informations

• Publié par: universitat_potsdam
• Publié le: 01 janvier 2007
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

Gene expression profiling in different stages of development of
Arabidopsis thaliana leaf trichomes at the single cell level Dieses Werk ist unter einem Creative Commons Lizenzvertrag lizenziert:
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http://opus.kobv.de/ubp/volltexte/2008/1747/
urn:nbn:de:kobv:517-opus-17474
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Gene expression profiling in different stages of development of Arabidopsis thaliana leaf
trichomes at the single cell level






Dissertation
zur Erlangung des akademischen Grades
"doctor rerum naturalium"
(Dr. rer. nat.)
in der Wissenschaftsdisziplin Molekularbiologie





eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät
der Universität Potsdam




von
Sergiy Kryvych




Potsdam
Oktober 2007
2































For my father


















3














“Publish or perish“
Popular saying among scientists today


“Knowledge of some principles easily compensates ignorance of some facts”
Claude-Adrien Helvetius, French philosopher


“Electronics is the science of contacts”
Saying


















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CONTENTS

FIGURES………………………………………………………………………………… 6
COMMONLY USED ABBREVIATIONS……………………………………………… 7
1. INTRODUCTION…………………………………………………………………… 8
1.1. Trichomes of Arabidopsis thaliana……………………………………………... 8
1.1.1 Trichome development………………………………………………….. 9
1.1.2 Regulation of trichome development………………………………….... 10
1.1.3 Cell cycle alterations during trichome development……………………. 11
1.1.3.1 Cell cycle regulation in plants………………………………………. 11
1.1.3.2 Regulation of the switch between mitotic and endoreduplication
cycles in developing trichomes……………………………………... 14
1.2 Single cell analysis…………………………………………………………….. 15
1.2.1 Methods of the single cell sampling……………………………………... 16
1.2.1.1 Non-invasive methods………………………………………………. 17
1.2.1.2 Minimally invasive methods………………………………………… 17
1.2.1.3 Invasive sampling methods…………………………………………… 18
1.2.2 Amplification methods used for single cell transcript profiling…………….. 19
1.3 Aims of this thesis………………………………………………………………… 21
2. MATERIALS AND METHODS…………………………………………………….... 22
2.1 Commonly used equipment, kits and consumables………………………………. 22
2.1.1. Equipment………………………………………………………………… 22
2.1.2. Consumables……………………………………………………………... 22
2.1.3. Kits……………………………………………………………………….. 23
2.2. Plant material……………………………………………………………………. 23
2.3. Plant growth……………………………………………………………………… 23
2.4. Single Cell Sampling……………………………………………………………… 24
2.5. Single cell PCR amplification……………………………………………………. 24
2.6. Single cell sampling control reactions……………………………………………. 25
2.7. Probe generation for array hybridization…………………………………………. 26
2.8. Filter array hybridization…………………………………………………………. 26
2.8.1 Array hybridization and evaluation………………………………………….. 26
2.8.1.1.Reference hybridization……………………………………………….. 26
2.8.1.2.Complex hybridization………………………………………………… 27
2.8.1.3.Data processing……………………………………………………….. 27
2.9. Affymetrix GeneChip hybridization…………………………………………….. 27
2.10. Real-time RT-PCR………………………………………………………………. 28
2.11. Genomic DNA isolation………………………………………………………… 28
2.12. RNA extraction using TRIzol protocol…………………………………………. 29
2.13. Construction of promoter:: β-glucuronidase fusions……………………………... 29
2.14. Bacteria transformation and growth……………………………………………... 32
2.15. Arabidopsis thaliana transformation…………………………………………….. 32
2.16. Histochemical analysis of plants transformed with promoter::GUS constructs…. 32
2.17. Promoter analysis……………………………………………………………….. 32
2.18. PCR-based screening for homozygous knockout (KO) plant lines……………. 33
2.19. Microscopic analysis of the knockout plant lines……………………………….. 34
2.20. Gas chromatography-mass spectrometry (GC-MS)…………………………….. 34
3. RESULTS……………………………………………………………………………... 36
3.1. Workflow…………………………………………………………………………. 36
3.2. Single cell sampling and array hybridization……………………………………... 36
3.3. Affymetrix GeneChip hybridization……………………………………………… 40 5
3.4. Candidate gene selection and validation of single cell gene expression profiling.. 41
3.4.1. Real-time RT-PCR…………………………………………………………. 43
3.4.2. The construction of promoter::GUS plant lines………………………….. 44
3.5. Knockout and RNAi plant lines…………………………………………………. 50
3.5.1. Phenotypic analysis of knockout and RNAi plant lines …………………. 53
3.6. Metabolite profiling of gh3.5 mutants (SALK_151766 T-DNA insertion line)… 55
3.7. In silico analysis of promoter cis-regulatory elements of candidate genes……… 57
4. DISCUSSION………………………………………………………………………… 61
4.1. Single cell sampling and array hybridization…………………………………….. 61
4.2. Candidate gene selection and validation of single cell gene expression profiling.. 63
4.2.1. GASA4……………………………………………………………………. 64
4.2.2. At3g16980 …………………………………………………………………. 65
4.2.3. GH3.5 ……………………………………………………………………… 67
4.3. Analysis of knockout and RNAi plant lines……………………………………… 69
4.4. In silico analysis of promoter cis-regulatory elements of candidate genes……….. 72
4.5. Investigating potential functions for candidate genes in developing trichomes…... 73
5. SUMMARY AND CONCLUSIONS………………………………………………… 75
6. OUTLOOK……………………………………………………………………………. 77
REFERENCES……………………………………………………………………………. 78
APPENDICES…………………………………………………………………………….. 89
APPENDIX A…………………………………………………………………………. 89
APPENDIX B…………………………………………………………………………. 91
APPENDIX C…………………………………………………………………………. 92
LIST OF PUBLICATIONS AND PRESENTATIONS……………………………... 104
ACKNOWLEDGEMENTS………………………………………………………….. 105























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FIGURES

Figure 1.1 Leaf trichomes of Arabidopsis………………………………………………... 8
Figure 1.2 Scheme of trichome development stages……………………………………... 9
Figure 1.3 Schemee patterning…………………………………………………. 10
Figure 1.4 Model for G1–S and G2–M transitions in plants………………………………. 12
Figure 1.5 Endoreduplication in trichomes………………………………………………… 15
Figure 1.6 Single cell sampling procedure using glass microcapillary……………………. 17
Figure 2.1 Principle of the SMART technology…………………………………………… 25
TMFigure 2.2 Principle of the GATEWAY cloning technology…………………………… 31
Figure 3.1 Visualization of trichome initial cells using the pGL2::GFP localization……... 37
Figure 3.2 Sampling of single leaf epidermal cells……………………………………….. 37
Figure 3.3 Functional categorization of genes expressed in pavement, trichome initial
and mature trichome cells…………………………………………………………… 38
Figure 3.4 Visualisation of differential expression of the genes between two cell types
using MAPMAN…………………………………………………………………… 39
Figure 3.5 Biotin-labelled cRNA derived from 10 single pavement cells…………………. 41
Figure 3.6 Results of the single cell real-time RT-PCR…………………………………… 44
Figure 3.7 GUS staining in pAt3g16980::GUS and pGASA4::GUS transformants
at day 6 and day 9………………………………………………………………. 45
Figure 3.8 GUS staining in pGH3.5::GUS transformants at days 6 to 21………………… 46
Figure 3.9 GUS staining in and pGASA4::GUS transforma
at day 14 and day 21 …………………………………………………………… 47
Figure 3.10 GUS staining in iflorescence of pAt3g16980::GUS, pGASA4::GUS, and
pGH3.5::GUS transformants……………………………………………………….. 48
Figure 3.11 GUS staining in leaf trichomes of pAt3g16980::GUS, pGASA4::GUS and
pGH3.5::GUS transformants………………………………………………………… 49
Figure 3.12 T-DNA insertion sites in At3g16980 and At4g27260 (GH3.5)………………. 50
Figure 3.13 Verification of the T-DNA insertion position in the genomes of
SALK_151766 (gh3.5) and SALK_002430 (At3g16980) plant lines using PCR…… 51
Figure 3.14 Verification of specific GST presence in the plants transformed with
RNAi construct for GH3.5……………………………………