Analysis of transcription factors under sulphur deficiency stress [Elektronische Ressource] / von Monika Bielecka

Monika Bielecka

Sujets

• Biologie

Informations

• Publié par: universitat_potsdam
• Publié le: 01 janvier 2007
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Max-Planck-Institute of Molecular Plant Physiology Golm





Analysis of transcription factors under sulphur
deficiency stress


Dissertation

zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
(Dr. rer. nat.)

Wissenschaftsdisziplin: Molekulare Pflanzenphysiologie

vorgelegt an der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität Potsdam

von
Monika Bielecka






Potsdam, März 2007













































Elektronisch veröffentlicht auf dem
Publikationsserver der Universität Potsdam:
http://opus.kobv.de/ubp/volltexte/2007/1481/
urn:nbn:de:kobv:517-opus-14812
[http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-14812]





















This Ph.D. thesis is the result of my own work and was done between October 2002 and
December 2005 in the department of Prof. Dr. Lothar Willmitzer at the Max-Planck-Institute of
Molecular Plant Physiology in Golm, Germany. It has not been submitted for any degree or Ph.D. at
any other university.


Die Dissertation ist das Ergebnis praktischer Arbeit, welche von Oktober 2002 bis Dezember
2005 durchgeführt wurde im Department von Prof. Dr. Lothar Willmitzer im Max-Planck-Insitut für
Molekulare Pflanzenphysiologie, Golm, Deutschland. Ich versichere, daß ich die vorliegende Arbeit
selbständig verfaßt und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe.
Diese Dissertation wurde an keiner anderen Hochschule zu Prüfung eingereicht.


Potsdam, den 05.03.2007


Monika Bielecka

34 CONTENT

1 INTRODUCTION ………………………………………………………………………………………..….....9
1.1 Sulphate metabolism in higher plants ………………………………………………………………………...…...9
1.1.1 The biochemical role of sulphur ………………………...………………………………….…………..…..9
1.1.2 Sulphate transport and assimilation ……….………………………………………………………..….…10
1.1.3 Methionine synthesis and metabolism …………………………………………………………….…..…13
1.1.4 SMM and SAM synthesis and metabolism .......................................................................................13
1.1.5 Sulphate regulation of transport and metabolism .............................................................................14
1.1.5.1 Development ..........................................................................................................................14
1.1.5.2 Reductant supply ....................................................................................................................14
1.1.5.3 Stress induced demand for sulphate .......................................................................15
1.1.5.4 Sulphur/nitrogen balance .......................................................................................................18
1.1.5.5 Methionine regulation .............................................................................................................19
1.2 Transcriptional control ................................................................................................................................20
1.2.1 Sulphate signalling and transcriptional regulation in lower organisms .............................................20
1.2.1.1 Algae ......................................................................................................................................20
1.2.1.2 Fungi .......................................................................................................................23
1.2.1.3 Bacteria ..................................................................................................................................26
1.2.2 Sulphur signaling in plants – an unexplored territory .......................................................................27
1.2.3 Transcription factors in Arabidopsis .................................................................................................27
1.2.4 AP2/EREBP family ...........................................................................................................................30
1.2.5 WRKY family ....................................................................................................................................35
1.2.6 MYB family .......................................................................................................................................36
1.2.7 cis-acting components involved in transcriptional control of sulphate status in Arabidopsis ............37
1.2.8 Regulation of nitrate and phosphate metabolism by transcription factors .......................................38
1.3 Aims of the thesis .......................................................................................................................................40

2 MATERIALS AND METHODS ....................................................................................................41
2.1 Commonly used equipment, kits and consumables ...................................................................................41
2.1.1 Equipment ........................................................................................................................................41
2.1.2 Consumables ...................................................................................................................................41
2.1.3 Kits ...................................................................................................................................................42
2.2 Media, growing conditions and plant lines .................................................................................................42
2.2.1 Plant material ...................................................................................................................................42
2.2.1.1 Wild type ................................................................................................................................42
2.2.1.2 35S overexpressor lines ........................................................................................................42
2.2.1.3 T-DNA knockout lines ............................................................................................................43
2.2.2 Seed sterilisation .............................................................................................................................43
2.2.3 Sterile liquid cultures .......................................................................................................................43
2.2.4 Growth on agar plates .....................................................................................................................44
2.2.4.1 Selection on BASTA-plates ...................................................................................................45
2.2.4.2 Selection on canamicin-plates ...............................................................................................45
52.2.4.3 Vertical plates ........................................................................................................................45
2.2.5 Hydroponic system ..........................................................................................................................46
2.2.6 Growth on soil ..................................................................................................................................47
2.3 Methods of molecular biology .....................................................................................................................48
2.3.1 RNA isolation procedures ................................................................................................................48
2.3.1.1 TRIzol maxi-prep protocol ......................................................................................................48
2.3.1.2 RNA extraction using TRIzol mini-prep protocol ....................................................................48
2.3.2 cDNA synthesis .................................................................................................................. .............48
2.3.3 Real time PCR conditions and analysis ...........................................................................................49
2.3.3.1 Real time reversed transcription (RT)-PCR-based platform ..................................................49
2.3.3.2 Real-time PCR primer design ................................................................................................50
2.3.4 DNA isolation ...................................................................................................................................50
2.3.5 PCR – based screening for homozygous knock-out (KO) lines .......................................................50
2.3.6 DNA cloning ................................................................