WWW.BOOK.DISLIB.INFO
FREE ELECTRONIC LIBRARY - Books, dissertations, abstract
 
<< HOME
CONTACTS



Pages:   || 2 | 3 | 4 | 5 |   ...   | 21 |

«Nitrogen signalling in Arabidopsis thaliana vorgelegt an der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Potsdam von Tomasz ...»

-- [ Page 1 ] --

Dissertation

Nitrogen signalling in Arabidopsis thaliana

vorgelegt an der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Potsdam

von Tomasz Czechowski

geb. am 30.09.1978 in Wroclaw (Polen)

zur Erlangung des akademischen Grades

Dr. rer. nat.

Wissenschaftsdisziplin: Molekulare Pflanzenphysiologie

28.Februar 2005

Content

Figures

Tables

COMMONLY USED ABBREVIATIONS

1. INTRODUCTION

1.1 Nitrogen in the environment (nitrogen cycle) and plants biology

1.2 Nitrogen transport and assimilation by plants

1.3 Nitrogen regulation of transport and metabolism

1.4 Nitrogen control of plant development

1.5 Nitrogen signalling in prokaryotes and lower eukaryotes

1.5.1 Bacteria

1.5.2 Fungi

1.5.3 Algae

1.6 Nitrogen signalling in plants

1.7 Arabidopsis transcription factors

1.8 Aims of this thesis

2. MATERIALS AND METHODS

2.1 Commonly used equipment, kits and consumables

2.1.1 Equipment

2.1.2 Consumables

2.1.3 Kits

2.2 Plant material

2.3 Plant growth media and conditions

2.3.1 Sterile liquid cultures

2.3.2 Growth on agar plates

2.3.3 Growth on soil

2.4 RNA isolation procedures

2.4.1 TRIzol maxi-prep protocol

2.4.2 RNA extraction using TRIzol mini-prep protocol

2.5 cDNA synthesis

2.6 Real-time PCR primer design

2.7 Real–time PCR conditions and analysis

2.8 Northern blotting

2.8.1 RNA electrophoresis and transfer according to Roche manual

2.8.2 Probe labelling with Dioxygenin-11-dUTP

2.8.3 Pre hybridisation and hybridisation conditions

2.8.4 Detection

2.9 DNA isolation

2.10 DNA cloning and sequence analysis

2.11 PCR – based screening for homozygous knock-out (KO) lines

2.12 Southern blotting

2.13 Transformations

2.13.1 Transformation of bacteria

2.13.2 Plant transformations

2.14 Selection of TF over-expressing plants and EtOH induction experiments.................. 43

2.16 EMS mutagenesis

2.17 Bioinformatics tools and computer analysis

3. RESULTS

3.A. Identification of TF’s involved in N-regulation – A reverse genetic approach............... 47

3.A.1 Development and testing of a resource for qRT-PCR profiling of Arabidopsis TF genes (in collaboration with Dr Wolf-Ruediger Scheible and Rajendra Bari from Molecular Genomics Group, MPI-MP, Golm, Germany)

3.A.1.1 PCR primer design and reaction specificity

3.A.1.2 Dynamic range, sensitivity and robustness of real-time PCR

3.A.2.3 Precision of real-time RT-PCR

3.A.1.4 Efficiency of PCR reactions

3.A.1.5 Comparison of technologies: qRT-PCR versus Affymetrix chips

3.A.1.6 Identification of root and shoot-specific TF genes by real-time RT-PCR.......... 54

3.A.1.7 Further development of the TF RT-PCR platform (in collaboration with Dr Wolf-Ruediger Scheible)

3.A.2 Nitrogen regulated transcription factors: needles in a haystack

3.A.2.1 Physiological responses to N-deprivation and nitrate re addition in Arabidopsis seedlings grown in liquid cultures

3.A.2.2 Transcriptional regulators

3.A.2.3 Candidate genes selection

3.A.3 Further characterisation of N-regulated TF genes

3.A.3.1 TF transcript changes in response to changes in nitrate or glutamine in the growth medium.

3.A.3.2 Nitrate regulation of TF genes in a nia1nia2 double mutant

3.A.4 Functional characterisation of the N-regulated TF genes

3.A.4.1 Genes cloning with the GATEWAY™ system

3.A.4.2 Selection of over expressing lines

3.A.4.4 Characterisation of TF knock-out mutants

3.A.4.5 Selection of homozygous T-DNA KO lines

3.A.4.6 Visible phenotypes in some of the selected lines

3.B Identification of N-regulators of AtNRT2.1 expression – A forward genetic approach.... 91

3.B.1 Preparation of PNRT2-1-LUC lines for EMS mutagenesis

3.B.2 N-regulation of PNRT2.1-LUC expression in line 9

3.B.3 Pilot EMS mutagenesis

3.B.4 Full-scale EMS mutagenesis experiment

3.B.5 Screening of the M2 generation on the plates under nitrate induction conditions..... 95

3.B.6 Confirmation of mutant phenotypes in the M3 generation

3.B.7 qPCR analysis of the expression of the other genes in selected mutant lines............ 98

4. DISCUSSION

4.1 Development of a qPCR platform for profiling all Arabidopsis transcription factors. 100

4.2 Identification of N-regulated TF genes

4.3 Characterisation of selected N-regulated transcription factors

4.4 Isolation of novel mutants affected in nitrate-induction of gene expression............... 116

5. SUMMARY AND CONCLUSIONS

6. FUTURE OUTLOOK

7. REFERENCES

APPENDIX A

APPENDIX B





APPENDIX C

CURRICULUM VITAE

ACKNOWLEDGEMENTS

Figures FIGURE 1. 1 THE NITROGEN CYCLE

FIGURE 1. 2 WORLD NITROGEN FERTILIZER USE IN 1996 (TAKEN FROM KAWASHIMA H, 2000).

.. 8

FIGURE 1. 3 POSSIBLE PATHWAYS FOR THE ASSIMILATION OF INORGANIC NITROGEN INTO

ORGANIC COMPOUNDS

FIGURE 1. 4 NITRATE-REGULATED GENES FROM PRIMARY METABOLISM (TAKEN FROM STITT,

1999)

FIGURE 1. 5 DEVELOPMENTAL PROCESSES CONTROLLED BY NITRATE

FIGURE 1. 6 DUAL PATHWAY MODEL FOR REGULATION OF LR GROWTH AND DEVELOPMENT

BY NITRATE (TAKEN FROM ZHANG ET AL., 1999)

FIGURE 1. 7 LONG DISTANCE NITROGEN SIGNALLING PROCESSES IN ARABIDOPSIS

FIGURE 1. 8 MODEL OF BACTERIAL NTR REGULATORY SYSTEM (TAKEN FROM MOORHEAD

AND SMITH, 2003)

FIGURE 1. 9 TOR SIGNALLING PATHWAYS IN YEASTS (TAKEN FROM KURUVILLA ET AL.

, 2001)21

FIGURE 1. 10 THE ARABIDOPSIS COMPLEMENT OF TRANSCRIPTION FACTORS (TAKEN FROM

RIECHMANN, 2002)

FIGURE 2. 1 SCHEME OF CLONING TF GENES, USING GATEWAY™ TECHNOLOGY

FIGURE 3. 1 SPECIFICITY OF QRT-PCR

FIGURE 3. 2 SENSITIVITY AND ROBUSTNESS OF QRT-PCR

FIGURE 3. 3 TECHNICAL PRECISION OF QRT-PCR AND AFFYMETRIX FULL GENOME ARRAYS.. 52

FIGURE 3.4 COMPARISON OF SHOOT TF TRANSCRIPT LEVELS MEASURED BY QRT-PCR AND

AFFYMETRIX WHOLE GENOME ARRAYS

FIGURE 3. 5 COMPARISON OF TF TRANSCRIPT LEVELS IN SHOOTS AND ROOTS

FIGURE 3. 6 COMPARISON OF SHOOT TO ROOT EXPRESSION RATIOS OBTAINED FROM QRT-PCR

AND AFFYMETRIX DATA

FIGURE 3. 7 PHENOLOGY OF NINE-DAY OLD N-LIMITED AND N-REPLETE ARABIDOPSIS

SEEDLINGS GROWN IN STERILE LIQUID CULTURE (TAKEN FROM SCHEIBLE ET AL. 2004) 59

FIGURE 3. 8 TRANSCRIPTIONAL RESPONSE TO N – DEPRIVATION AND TO NITRATE

REPLENISHMENT FOR SELECTED MARKER GENES.

FIGURE 3. 9 COMPARISON OF TF TRANSCRIPT LEVELS UNDER N DEPRIVATION AND 30

MINUTES AFTER NITRATE REPLENISHMENT

FIGURE 3. 10 COMPARISON OF TF GENE EXPRESSION RATIOS, AS DETERMINED BY QRT-PCR

AND AFFYMETRIX TECHNOLOGY (TAKEN FROM SCHEIBLE ET AL., 2004)

FIGURE 3. 11 THE EXPERIMENTAL SET-UP FOR TESTING VARIOUS ABIOTIC STRESSES IN

ARABIDOPSIS LIQUID CULTURES, USED FOR SELECTION OF NITROGEN-REGULATED TF

GENES.

FIGURE 3. 12 CHANGES IN GENE EXPRESSION OF 6 MARKER GENES AFTER NITRATE /

GLUTAMINE STARVATION AND RE-ADDITION

FIGURE 3. 13 CHANGES IN EXPRESSION OF MARKER GENES IN NIA1NIA2 MUTANT AFTER N

DEPRIVATION AND NITRATE RE-ADDITION

FIGURE 3. 14 OVERVIEW OF THE APPROACHES USED FOR FUNCTIONAL CHARACTERISATION

OF N-REGULATED TF GENES

FIGURE 3. 15 PCR AMPLIFICATION OF SOME TF GENES

FIGURE 3. 16 RESTRICTION ANALYSIS OF THE RECOMBINANT DESTINATION VECTORS CLONED

INTO E.COLI (A) OR A.TUMEFACIENS. (B)

FIGURE 3. 17 NORTHERN BLOT ANALYSIS OF PLANTS CONSTITUTIVELY OVEREXPRESSING TF

GENE AT4G38340 (LEFT) AND INDUCIBLE OVEREXPRESSING TF GENE AT2G45300 (RIGHT)

FIGURE 3. 18 PHENOTYPIC VARIATION IN THE 35S-AT1G01530

FIGURE 3. 19 FLOWER PHENOTYPE OF 35S-AT2G33720

FIGURE 3. 20 PHENOTYPIC VARIATION IN THE 35S -AT1G33550A

FIGURE 3. 21 SEQUENCE OF AT2G33550

FIGURE 3. 22 FLOWER PHENOTYPE OF 35S-AT2G33550

FIGURE 3. 23 OVER-EXPRESSION OF AT2G33550 LEADS TO SEVERELY DWARFED PHENOTYPE.

82

FIGURE 3. 24 ROOT ARCHITECTURE OF COL-0 SEEDLINGS GROWN UNDER VARIOUS NITRATE

REGIMES

FIGURE 3. 25 DEVELOPMENT OF LATERAL ROOTS OF 35S-AT2G22200

FIGURE 3. 26 KINETICS OF PRIMARY ROOT GROWTH OF 35S-AT2G22200

FIGURE 3. 27 PCR SCREENING FOR THE HOMOZYGOUS T-DNA INSERTION LINE FOR AT3G51910

FIGURE 3. 28 GERMINATION RATIO FOR HOMOZYGOUS T-DNA KO LINES FOR TWO TF GENES. 89

FIGURE 3. 29 FLOWERING TIME POINT FOR THREE T-DNA KNOCK-OUT MUTANT LINES FOR

AT3G51910 AND WT

FIGURE 3. 30 SCHEME OF THE REPORTER CONSTRUCT USED FOR LUC ACTIVITY SCREENING. 91

FIGURE 3. 31 SOUTHERN BLOT ANALYSIS OF PNRT2.1-LUC LINE NUMBER 9 HYBRIDISED WITH

LUC GENE PROBE

FIGURE 3. 32 NITROGEN INFLUENCE OF LUC REPORTER GENE ACTIVITY UNDER CONTROL OF

ATNRT2.1 PROMOTER

FIGURE 3. 33 TYPICAL VIEW OF THE MATURE SILIQUE FROM M1 PLANTS TREATED WITH 0.3%

EMS

FIGURE 3. 34 SCREENING FOR THE MUTANT PHENOTYPE UNDER THE INDUCIBLE CONDITIONS

FIGURE 3. 35 LUC ACTIVITY UNDER INDUCIBLE CONDITIONS IN CONFIRMED PUTANT LINE 54/3

FIGURE 3. 36 EXPRESSION LEVEL OF ATNRT2-1 IN THE PUTANT LINES

FIGURE 3. 37 EXAMPLE GROWTH OF PUTANT SEEDLINGS

Tables

TABLE 1. 1 CONTENT AND DISTRIBUTION OF TFS IN EUKARYOTIC ORGANISMS (FROM

RIECHMANN 2002)

TABLE 2. 1 STERILE FULL NUTRITION AND LOW-NITRATE MEDIUM COMPOSITION

TABLE 2. 2 MEDIUM USED FOR ROOT ARCHITECTURE STUDIES ON AGAR PLATES

TABLE 3. 1 SHOOT-SPECIFIC AND ROOT-SPECIFIC TF GENES IDENTIFIED BY REAL-TIME RT-PCR

TABLE 3. 2 N-REGULATED TF GENES OF ARABIDOPSIS

TABLE 3. 3 QPCR RESULTS OF VARIOUS ABIOTIC STRESSES FOR ALL NITROGEN REGULATED TF

GENES

TABLE 3. 4 N –REGULATION OF SELECTED TF GENES

TABLE 3. 5 N-REGULATION OF TF GENES IN WT AND NIA1NIA2 MUTANT PLANTS

TABLE 3. 6 CURRENT STATUS OF CLONING AND PLANT TRANSFORMATION FOR 17 NREGULATED TF GENES

TABLE 3. 7 OVERVIEW OF THE SELECTED KO LINES, USED FOR “LOSS OF FUNCTION”

APPROACH

TABLE 3. 8 EXPRESSION OF THE NITRATE TRANSPORTER GENES IN THE SELECTED MUTANT

LINES

TABLE 3. 9 EXPRESSION OF THE GENES FROM PRIMARY NITROGEN AND CARBON ACQUISITION

IN THE SELECTED MUTANT LINES

COMMONLY USED ABBERVIATIONS 6

COMMONLY USED ABBREVIATIONS

–  –  –

1. INTRODUCTION

1.1 Nitrogen in the environment (nitrogen cycle) and plants biology Nitrogen is the fourth most abundant element in plants, after carbon, oxygen, and hydrogen. It is an essential component of many biological compounds, including: amino acids, purines and pyrimidines which are the building blocks of proteins, nucleotides, and nucleic acids.



Pages:   || 2 | 3 | 4 | 5 |   ...   | 21 |


Similar works:

«WertVoll entscheiden in Beruf und Alltag WertVoll entscheiden in Beruf und Alltag Bibliografische Information der Deutschen Bibliothek Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.© Copyright der deutschen Ausgabe 2009: Börsenmedien AG, Kulmbach Gestaltung und Satz: Jürgen Hetz, denksportler Grafikmanufaktur, Bad Homburg v. d. Höhe, www.denksportler.com Druck:...»

«FP Tunneldiode NanoWiki http://www.nano.uni-hannover.de/local/NanoWiki/index.php?titl. FP Tunneldiode aus NanoWiki, der freien Wissensdatenbank Autor: Lüdtke, 19.10.2006 Inhaltsverzeichnis 1 Theorie 1.1 Die Einzelbarriere 1.2 Die Doppelbarriere: Resonantes Tunneln 2 Herstellung 2.1 Molekularstrahlepitaxie 2.2 Aufbau der Proben 2.3 Ohmsche Kontakte und naßchemisches Ätzen 2.4 Probenstrukturierung 3 Versuchsdurchführung 4 Versuchsauswertung 5 Versuchsbeschreibung zum Download Theorie Die...»

«arqus Arbeitskreis Quantitative Steuerlehre www.arqus.info Diskussionsbeitrag Nr. 118 Rainer Niemann / Martina Rechbauer Wie können Unternehmen mit steuerlichen Verlustvorträgen identifiziert werden? – Ergebnisse einer Replikationsstudie – Mai 2011 arqus Diskussionsbeiträge zur Quantitativen Steuerlehre arqus Discussion Papers in Quantitative Tax Research ISSN 1861-8944 Wie können Unternehmen mit steuerlichen Verlustvorträgen identifiziert werden? – Ergebnisse einer...»

«Cölsch, Nele Potential and limitations of peace education in Israel. A case study on parents’ perspectives on the Hand in Hand school in Jerusalem Freiburg, Br. : Centaurus-Verl. 2011, 119 S. (Gender and Diversity; 3) urn:nbn:de:0111-opus-52936 in Kooperation mit / in cooperation with: http://www.centaurus-verlag.de Nutzungsbedingungen / conditions of use Gewährt wird ein nicht exklusives, nicht übertragbares, persönliches und beschränktes Recht auf Nutzung dieses Dokuments. Dieses...»

«Laboratoire de Constructions Hydrauliques Ecole Polytechnique Fédérale de Lausanne Communication 27 Symposium érosion et protection des rives lacustres Fachtagung Erosionsprozesse und Schutzmassnahmen an Seeufern Sutz-Lattrigen, jeudi 18 mai 2006 Bases de dimensionnement des mesures de protection des rives lacustres Bemessungsgrundlagen für Massnahmen zum Schutz von Flachufern an Seen Editeur : Prof. Dr A. Schleiss Lausanne, 2006 Communications du Laboratoire de constructions hydrauliques...»

«OTTO-FRIEDRICH-UNIVERSITÄT BAMBERG ZENTRUM FÜR MITTELALTERSTUDIEN INFORMATIONSHEFT FÜR DAS WINTERSEMESTER 2009/10 LEHRVERANSTALTUNGEN – PUBLIKATIONEN – AKTIVITÄTEN BAMBERG 2009 Anschrift: Universität Bamberg, Zentrum für Mittelalterstudien, 96045 Bamberg Tel.: 0951/863-2451 – Fax: 0951/863-2306 E-Mail: zemas@uni-bamberg.de Internet: www.uni-bamberg.de/zemas/ Besucheradresse: Am Kranen 10, Raum 304, 96045 Bamberg Leitungsgremium: Prof. Dr. Ingrid Bennewitz (geschäftsführend), Prof....»

«Akademievorträge an Brandenburger Schulen vom 17. bis 21. November 2003 VORTRAG 1 REFERENT: PROF. DR. RUDOLF BENTZINGER Die deutschsprachige Bibel im Mittelalter VORTRAG 2 REFERENT: DR. MONIKA BÖNING Erforschung und Dokumentation mitelalterlicher Glasmalereien VORTRAG 3 REFERENT: PROF. DR. THOMAS BÖRNER Genetische Manipulation von Pflanzen – nützlich oder gefährlich Gefahr? VORTRAG 4 REFERENT: PROF. DR. ADOLF BORBEIN Die Darstellung des Menschen in der griechischen Kunst VORTRAG 5...»

«BAROCKE PUTZE IM BÜRGERSCHLOSS LANDSCHAFT | GÄR TEN | STADT | KUNST | ARCHITEKTUR | SKULPTUR MALEREI | MENSCH | GESELLSCHAFT | GLAUBEN | WISSENSCHAFT L I T E R AT U R | M U S I K | T H E AT E R | J A G D | F O R S T | L A N D W I R T S C H A F T INDUSTRIE | HANDWERK | HANDEL | VERKEHR | KÜCHE | KELLER S P U R E N S U C H E E N T L A N G D E R B A R O C K S T R A S S E S A A R P FA L Z DIE BAROCKSTRASSE SAARPFALZ LÄDT EIN ZUR SPURENSUCHE....in die saarländische Landeshauptstadt nach...»

«1 23 Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is...»

«Jahreshauptversammlung Geschäftsstelle: Turnhallenstrasse 13 • 35435 Wettenberg Tel. 06 41 / 8 65 40 • Fax 0641 / 9 84 89 48 email: geschaeftsstelle@tsv-krodorf-gleiberg.de Inhaltsverzeichnis 1. Basketball 2. Fußball 3. Handball 4. Judo 5. Leichtathletik 5.1 LG Wettenberg 5.2 Volkslauf, Laufund Walkingtreff 6. Ski 7. Triathlon 8. Turnen 8.1 Allgemeine Wettkämpfe 8.2 Aquqjogging – Aquafitness 8.3 Bodyforming 8.4 Eltern-Kind-Turnen / Kleinkinderturnen 8.5 Frauengymnastik I 8.6...»





 
<<  HOME   |    CONTACTS
2016 www.book.dislib.info - Free e-library - Books, dissertations, abstract

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.