Untersuchungen zur Biosynthese der pflanzlichen Zellwand [Elektronische Ressource] / von Björn Usadel

Sujets

• Biologie

Informations

• Publié par: universitat_potsdam
• Publié le: 01 janvier 2005
• Nombre de lectures: 17
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Max Planck Institut für molekulare Pflanzenphysiologie
AG Pauly




Untersuchungen zur Biosynthese der Zellwand










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











eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät
der Universität Potsdam




von
Björn Usadel





Table of Contents
Table of Contents
1 Introduction....................................................................................................................................... 1
1.1 The plant cell wall..................................................................................................................... 1
1.2 St ructures in the cell wall.......................................................................................................... 2
1.2.1 Cellulose............................................................................................................................ 2
1.2.2 Hemicelluloses................................................................................................................... 2
1.2.3 Pectic polysaccharides....................................................................................................... 3
1.2.4 W all associated proteins .................................................................................................... 5
1.2.5 Lignins............................................................................................................................... 6
1.3 Cell wall networks 7
1.4 Synthesis of cell wall components............................................................................................. 8
1.4.1 Cellulose synthesis........................................................................................................... 10
1.4.2 Hemicelluloses synt hesis................................................................................................. 10
1.4.3 Pectin synthesis................................................................................................................ 11
1.4.4 Nucleotide sugar metabolism.......................................................................................... 12
1.5 St ructural analysi s of cell walls............................................................................................... 17
1.6 Biotechnology of cell walls..................................................................................................... 18
1.7 Aims of the thesis .................................................................................................................... 20
2 Materials and Methods 21
2.1 Materials.................................................................................................................................. 21
2.1.1 Chemicals........................................................................................................................ 21
2.1.2 Plasmids........................................................................................................................... 21
2.1.3 Primers............................................................................................................................. 21
2.2 Organisms and cultivation conditions..................................................................................... 22
2.2.1 Escherichia coli................................................................................................................ 22
2.2.2 Agrobacterium tumefaciens............................................................................................. 23
2.2.3 Plant growth..................................................................................................................... 23
2.2.4 Insect cells 23
2.2.5 In vi tro pollen germination.............................................................................................. 23
2.3 Molecular biology techniques................................................................................................. 24
2.3.1 Standard molecular biology techniques........................................................................... 24
2.3.2 Polymerase Chain Reaction (PCR).................................................................................. 24
2.3.3 Transformation of bacteria 24
2.3.4 DNA isolation.................................................................................................................. 24
2.3.5 RNA isolation 25
2.3.6 Sou thern blotting............................................................................................................. 25
2.3.7 Northern blotting 25
2.3.8 Radioactive probe synthesis............................................................................................ 26
2.3.9 Hybridization of radioactive probes................................................................................ 26
2.3.10 cDNA synt hesis............................................................................................................ 26
2.3.11 Semiquantitative RT-PCR 26
2.3.12 Real time RT-PCR ........................................................................................................ 27
2.3.13 Transformation of plants.............................................................................................. 27
2.3.14 Molecular analysis of Arabidopsis T-DNA tagged mutant lines................................. 27
2.3.15 Generation of pRRHM................................................................................................. 28
2.3.16 Generation of pVXGAE6............................................................................................. 29
2.3.17 Generation of pIXGA E6............................................................................................... 29
2.3.18 Generation of pA XGA E6 29
iTable of Contents
2.3.19 Generation of pRGAE.................................................................................................. 30
2.4 Biochemical Methods.............................................................................................................. 30
2.4.1 Protein Expression in insect cells.................................................................................... 30
2.4.2 Protein expression in Pichia pastoris............................................................................... 31
2.4.3 GA E6 activ ity essay......................................................................................................... 31
2.4.4 SDS polyacrylamid gel electrophoresis........................................................................... 32
2.4.5 W estern blot analysis....................................................................................................... 32
2.5 Analytical and preparational techniques................................................................................. 32
2.5.1 Isolation of cell wall material.......................................................................................... 32
2.5.2 Isolation of mu cilage material from Arabidopsis thaliana seeds..................................... 32
2.5.3 Isolation of pectic fragments from cell wall material...................................................... 33
2.5.4 Gel permeation chromatography of polysaccharidic samples......................................... 33
2.5.5 Monos accharide composition 33
2.5.6 Determination of crystalline cellulose content................................................................ 34
2.5.7 Uronic acid content.......................................................................................................... 34
2.5.8 Hexose content................................................................................................................. 35
2.5.9 Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) mass
spectrometry.............................................................................................................................. 35
2.5.10 High performance anion exchange chromatography with pulsed amperometric or UV
detection (HPAEC-PAD) 35
2.6 Microscopy and Histochemis try.............................................................................................. 35
2.6.1 Seed ultrastructure analysis............................................................................................. 35
2.6.2 Sectioning and bright field microscopy of seeds............................................................. 36
2.6.3 Seed staining.................................................................................................................... 36
2.7 Bioinformatics......................................................................................................................... 36
2.7.1 Identification of candidate genes..................................................................................... 36
2.7.2 Characterization of genes................................................................................................ 37
2.7.3 Softwa re development..................................................................................................... 37
3 Results...............................................................