Functional characterization of transgenic Arabidopsis thaliana plants co-over expressing aldehyde dehydrogenases and genes for soluble osmolytes [Elektronische Ressource] / vorgelegt von Hasnain Raza

Hasnain Raza

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2010
• Nombre de lectures: 45
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Functional Characterization of Transgenic Arabidopsis thaliana
Plants Co-over expressing Aldehyde dehydrogenases and Genes for
Soluble Osmolytes







Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn





vorgelegt von
Hasnain Raza
Aus
Multan, Pakistan


Bonn, 2009

Angefertigt mit Genehmigung
der Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn.





















1. Referentin: Prof. Dr. Dorothea Bartels
2. Koreferent: Priv. Doz. Dr. Hans-Hubert Kirch

Tag der Promotion: 21.09.2009
Erscheinungsjahr: 2010 Eidesstattliche Erklärung

Ich versichere, dass ich die von mir vorgelegte Dissertation selbstständig
angefertigt, die benutzten Quellen und Hilfsmittel vollständig angegeben und die
Stellen der Arbeit – einschließlich Tabellen, Karten und Abbildungen –, die
anderen Werken im Wortlaut oder dem Sinn nach entnommen sind, in jedem
Einzelfall als Entlehnung kenntlich gemacht habe; dass diese Dissertation noch
keiner anderen Fakultät oder Universität zur Prüfung vorgelegen hat; dass sie
noch nicht veröffentlicht worden ist sowie, dass ich eine solche Veröffentlichung
vor Abschluss des Promotionsverfahrens nicht vornehmen werde. Die Bestim-
mungen der Promotionsordnung sind mir bekannt. Die von mir vorgelegte
Dissertation ist von Prof. Dr. Dorothea Bartels betreut worden.
Table of contents
A Table of Contents ................................................................................................... i
B Abbreviations ........................................................................................................ v
C Summary (English) ............................................................................................. vii
1 Introduction........................................................................................................... 1
1.1 Aldehyde Drhydrogenases (ALDH) Genes .......................................................... 1
1.1.1 Ion Homeostasis .......................................................................................... 2
1.1.2 Reactive Oxygen Species ............................................................................. 2
1.1.3 Transcription Factors ................................................................................. 2
1.1.4 Stress Signaling ......................................................................................... 3
1.1.5 Aldehydes .................................................................................................. 4
1.2 Soluble Omolytes, Ectoine Genes ....................................................................... 7
1.3 Objectives ............................................................................................................... 12
2 Materials and Methods ....................................................................................... 13
2.1 Plant Material ................................................................................................. 13
2.1.1 Arabidopsis thaliana ................................................................................ 13
2.1.2 Nicotiana tabacum ................................................................................... 13
2.1.3 Lycopersicon esculantum ......................................................................... 14
2.2 Bacterial Species and Strains .......................................................................... 14
2.2.1 E. coli DH10B ......................................................................................... 14
2.2.2 E. coli XL1-Blue ...................................................................................... 14
2.2.3 Agrobacterium tumifaciens LA 4404 (pAL 4404) .................................. 14
2.2.4 Agrobacterium tumifaciens GV3101 ....................................................... 14
2.3 Plasmid Vectors ..................................................................................................... 14
2.4 Primers (Sequences 5´ 3´) ........................................................................ 14
2.5 Chemicals, Radioactive and other Supporting Materials ................................ 15
2.6 Enzymes and Size Markers ............................................................................ 16
2.7 Membranes and Films .................................................................................... 16
2.8 Kits ................................................................................................................. 16
2.9 Equipment and Instruments ............................................................................ 16
2.10 Softwares ........................................................................................................ 17
2.11 Media, Buffers and Solutions ......................................................................... 17
2.11.1 Media ....................................................................................................... 17
2.11.2 Buffers and Solutions .............................................................................. 18
2.12 Culture of Arabidopsis thaliana ..................................................................... 18
2.13 Tobacco Culture ............................................................................................. 19
2.14 Tomato Culture .............................................................................................. 19
2.15 Culture of Eschericia coli ............................................................................... 19
2.16 Culture of Agrobacterium tumifaciens ........................................................... 19
2.17 Plant Genomic DNA Extraction (Urea Method) ............................................ 19
2.18 Plant Genomic DNA Extaction from A. thaliana ........................................... 21
2.19 (Southern Blots) DNA Transfer to Nitrocelulose Membranes ........................ 22
i
32
2.19.1 Preparation of α P-dCTP Radio Labelled Probe ..................................... 22
2.19.2 Hybridizaton and Autoradiography .......................................................... 23
2.20 DNA Ectraction from E. coli at small scale .................................................... 24
2.21 Maxi/Midi Prep for Plasmid DNA ................................................................. 24
2.22 Extraction of Plasmid DNA from Agrobacterium tumifaciens ....................... 24
2.23 Total RNA Extraction .................................................................................... 25
2.24 RNA transfer to Nylon Membrane (Northern Blot) ....................................... 26
2.24.1 Staining of the membrane with methylene blue ....................................... 27
32
2.24.2 Preparation of α P-dCTP Radio Labeled Probe ...................................... 28
2.24.3 Hybridizaton and Autoradiography .......................................................... 28
2.25 Protein Extraction ........................................................................................... 29
2.25.1 Gel Electrophoresis of Denatured Proteins (SDS-PAGE) ........................ 29
2.25.2 Ponceau Staining ..................................................................................... 30
2.26 Purification of DNA Fragments from Agarose Gels ....................................... 30
2.27 Qualitative and Quantitative Estimation of Macromolecules ......................... 30
2.27.1 Qualitative and Quantitative Estimation of DNA and RNA ..................... 30
2.27.2 Qualitative and Quantitative Estimation of Protein Extracts .................... 31
2.28 Physiological and Biochemical Assays .......................................................... 31
2.28.1 Lipid Peroxidation Assay (MDA Analysis) ............................................. 31
2.28.2 Determination of Chlorophyll Content .................................................... 32
2.28.3 Gas Exchange and Chlorophyll Florescence ............................................ 32
2.28.4 Free Proline Content ................................................................................ 32
2.28.5 Electrolyte Leakage Percent .................................................................... 33
2.28.6 In vivo Detection of H O by the DAB-Uptake Method .......................... 33 2 2
2.28.7 H O Quantification ................................................................................. 33 2 2
2.29 Cloning of DNA Fragments ........................................................................... 34
2.29.1 Primer Design for Cloning ....................................................................... 34
2.29.2 Polymerase Chain Reaction (PCR) ......................................................... 34
2.29.3 Restriction Endonuclease Treatments ...................................................... 35
2.29.4 Dephosphorylation ......................................