The role of sulfite reductase in assimilatory sulfate reduction in Arabidopsis thaliana [Elektronische Ressource] / presented by Muhammad Sayyar Khan

Muhammad Sayyar Khan

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 62
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
M.Sc. (Hons) Muhammad Sayyar Khan
born in Mardan, NWFP, Pakistan
stOral-examination: November 21 , 2008The role of sulfite reductase in assimilatory
sulfate reduction in Arabidopsis thaliana
Referees: Prof. Dr. Rüdiger Hell
Prof. Dr. Thomas RauschTable of Contents
Summary...................................................................................................................................1
Zusammenfassung....................................................................................................................2
1 Introduction...........................................................................................................................3
1.1 Importance of sulfur for the plants and agriculture.........................................................3
1.2 An overview of uptake and assimilation of sulfur in higher plants.................................4
1.3 Role of ATP sulfurylase in sulfate reduction...................................................................7
1.4 Role of APS reductase in sulfate reduction.....................................................................8
1.5 Role of sulfite reductase in sulfate reduction..................................................................9
1.6 Comparison of Arabidopsis sulfite reductase with sulfite reductases from other
organisms ............................................................................................................................10
1.7 Sulfur and selenium: uneven twins in plant and human nutrition.................................10
1.8 Relevance of selenium metabolism in plants................................................................11
1.9 Goals in Se-metabolism.................................................................................................13
1.10 Aims of the project......................................................................................................14
2 Materials and methods.......................................................................................................15
2.1 Chemicals and Consumables.........................................................................................15
2.2 Enzymes........................................................................................................................17
3.3 Technical Equipment.....................................................................................................18
2.4 Biological Material........................................................................................................20
2.4.1 Bacterial Stains......................................................................................................20
2.4.2 Plant Material........................................................................................................20
2.5 Growth Conditions........................................................................................................20
2.5.1 Growth of Bacteria................................................................................................20
2.5.1.1 Preparation of competent cells.......................................................................21
2.5.1.1 Transformation of bacteria with DNA...........................................................21
2.5.2 Growth of Plants....................................................................................................22
2.5.2.1 Growth on soil................................................................................................22
2.5.2.2 Sterilization of seeds......................................................................................22
2.5.2.3 Hydroponical cultures....................................................................................22
2.5.2.4 Germination on agar-dishes...........................................................................23
2.5.2.5 Chemical complementation of sir1-1 mutants...............................................23
2.5.2.6 Stable transformation and screening of Arabidopsis.....................................23
2.6 Molecular Biological Methods.................................................................................24
2.6.1 Isolation of genomic DNA from plants.................................................................24
2.6.2 Isolation of total mRNA .......................................................................................24
2.6.3 Genotyping and molecular characterization of T-DNA insertions........................24
2.6.4 cDNA synthesis and semi-quantitative RT-PCR analysis......................................25
2.6.5 Determination of the transcript levels by using custom made microarrray...........25
2.6.6 Separation of nucleic acids by agarose gel electrophoresis...................................26
2.6.7 DNA sequencing....................................................................................................26
2.7 Biochemical Methods....................................................................................................26
2.7.1 Recombinant expression and purification of AtSiR under native
conditions........................................................................................................................26
2.7.2 Recombinant expression and purification of AtSiR under denatured
conditions........................................................................................................................27
2.7.3 Antibody production..............................................................................................28
I2.7.4 Antibody testing for SiR Protein...........................................................................28
2.7.5 Isolation of soluble proteins from plants...............................................................28
2.7.6 Determination of the protein concentration...........................................................29
2.7.7 Determination of enzymatic activities ..................................................................29
2.7.7.1 Determination of SiR activity........................................................................29
2.7.7.3 Determination of SAT activity.......................................................................30
2.7.7.4 Determination of sulfite oxidase activity.......................................................30
2.7.8 Separation of proteins by SDS-polyacrylamide gel electrophoresis.....................30
2.7.9 Immunological detection of SiR protein...............................................................31
2.7.10 Immunological detection of SAT and OAS-TL proteins.....................................32
2.7.11 Determination of metabolites and element contents............................................32
2.7.11.1 Quantification of the anions sulfate, phosphate, and nitrate........................33
2.7.11.2 Determination of OAS after derivatization with AccQ-Tag........................33
2.7.11.3 Determination of thiol metabolites after derivatization with
monobromobimane....................................................................................................33
2.7.11.4 Determination of leaf chlorophyll contents.................................................34
2.7.11.5 Determination of glucosinolates ...............................................................34
2.7.12.6 Determination of total CNS contents...........................................................34
2.7.12.7 Determination of sulfolipids.......................................................................35
2.7.12.8 Determination of total sulfur and selenium through Inductively Coupled
Plasma Emission Spectroscopy..................................................................................35
2.8 Physiological methods...................................................................................................36
2.8.1 Analysis of metabolic fluxes..................................................................................36
35 35 2-2.8.1.1 Incorporation of S into thiols and protein from SO ...............................364
3 32.8.1.2 Incorporation of H into OAS, thiols and protein from H-serine.................36
2.8.1.3 Isolation of radiolabeled metabolites ............................................................37
2.8.1.3.1 Isolation of OAS and thiol metabolites..................................................37
2.8.1.3.2 Isolation of the protein fraction after radiolabel feeding experiments...38
2.8.1.4 Determination of incorporated radioactivity by liquid scintillation counting
....................................................................................................................................38
2.9 Cloning..........................................................................................................................38
2.9.1 Vectors...................................................................................................................38
2.9.2 PCR for cloning.....................................................................................................39
2.9.3 DNA digestion with restriction enzymes...............................................................39
2.9.4 Constructs............................................................