Evolution of phage-type RNA polymerases in higher plants [Elektronische Ressource] / Chang Yin. Gutachter: Thomas Börner ; Volker Knoop ; Wolfgang Schuster

Yin Chang

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 65
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Evolution of phage-type RNA polymerases
in higher plants
DISSERTATION

Zur Erlangung des akademischen Grades
Doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Master der Landwirtschaftswissenschaften
Chang Yin


Präsident der Humboldt-Universität zu Berlin
Prof. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Andreas Herrmann

Gutachter: 1. Prof. Dr. Thomas Börner
2. Prof. Dr. Volker Knoop
3. Prof. Dr. Wolfgang Schuster

Datum der Promotion: 29.11.2010 1

Table of contents
Table of contents ........................................................................................................................ 1
Zusammenfassung...................................................................................................................... 5
Summary .................................................................................................................................... 6
1 Introduction................................................................................................................. 7
1.1 Organellar RNA polymerases in plants and their functions ....................................... 7
1.1.1 Mitochondrial RNA polymerases ........................................................................... 7
1.1.2 Plastid RNA polymerases ....................................................................................... 8
1.1.3 RpoT genes encode both mitochondrial and plastid RNAPs in angiosperms......... 9
1.1.4 Structure, promoter recognition and functions of RpoT in plants ........................ 12
1.1.4.1 Structure of RpoT 12
1.1.4.2 Promoter recognition by RpoT ................................................................... 13
1.1.4.3 Specific functions and regulation of RpoT polymerases in plants ............. 15
1.1.5 Origin and evolution of RpoT polymerases.......................................................... 17
1.2 Gene duplication and molecular evolution................................................................ 20
1.3 Lower plant species for investigation of RpoT evolution ......................................... 22
1.3.1 Selaginella moellendorffii..................................................................................... 22
1.3.2 Nuphar advena...................................................................................................... 24
2 Materials and Methods.............................................................................................. 26
2.1 Plants......................................................................................................................... 26
2.2 Laboratory equipments and disposables.................................................................... 26
2.3 Chemicals, Biochemicals, Biologicals...................................................................... 27
2.4 Kits............................................................................................................................. 28
2.5 Enzymes..................................................................................................................... 29
2.6 Buffers and solutions................................................................................................. 30
2.7 Primers and other oligonucleotides ........................................................................... 31
2.8 Isolation of nucleic acids ........................................................................................... 37
2.8.1 Isolation of genomic DNA from Sm and Na ........................................................ 37
2.8.2 Isolation of BAC DNA......................................................................................... 37
2.8.3 Small-scale plasmid DNA Isolation from E. coli ................................................. 37
2.8.4 Isolation of total RNA from Selaginella moellendorffii and Nuphar advena....... 37
2.9 cDNA synthesis......................................................................................................... 37
2.10 Determination of nucleic acid concentration............................................................. 38
2
2.11 Agarose gel electrophoresis of DNA......................................................................... 38
2.12 Agarose gel electrophoresis of RNA 38
2.13 PCR............................................................................................................................ 38
2.14 Colony PCR............................................................................................................... 39
2.15 Cloning and sequencing of PCR products................................................................. 39
2.16 3’- and 5’- RACE reactions....................................................................................... 39
2.17 Genomic cloning from BACs .................................................................................... 41
2.17.1 BAC Filters........................................................................................................... 41
2.17.2 Hybridization of BAC Filters................................................................................ 42
2.17.2.1 Preparation of probe 42
2.17.2.2 Filter hybridization...................................................................................... 42
2.17.3 BAC DNA fingerprinting analysis........................................................................ 42
2.18 Southern hybridization of genomic DNA.................................................................. 43
2.18.1 Restriction cleavage of genomic DNA ................................................................. 43
2.18.2 Southern transfer and radioactive labeling of probe............................................. 43
2.18.3 Hybridization and image analysis......................................................................... 43
2.19 Generation of GFP targeting constructs .................................................................... 44
2.19.1 pOL-GFP S65C plasmid....................................................................................... 44
2.19.2 RecA-GFP and CoxIV-GFP ................................................................................. 44
2.19.3 GFP targeting constructs 44
2.19.3.1 SmRpoT-GFP constructs............................................................................ 44
2.19.3.2 NaRpoT-GFP ............................................................................. 46
2.20 Transient expression of GFP constructs .................................................................... 48
2.20.1 Isolation of protoplast from Arabidopsis thaliana................................................ 48
2.20.2 Selaginella moellendorffii........................................ 49
2.20.3 PEG transformation .............................................................................................. 49
2.20.4 Confocal microscopy............................................................................................ 49
2.21 Computational phylogenetic analysis........................................................................ 49
3 Results....................................................................................................................... 51
3.1 Identification of RpoT genes...................................................................................... 51
3.1.1 Molecular cloning of the RpoT cDNA and gene of Selaginella moellendorffii ... 52
3.1.1.1 Identification of the full length SmRpoT cDNA sequence.......................... 52
3.1.1.2 Isolation of the genomic DNA sequence of SmRpoT ................................. 53
3.1.2 Molecular cloning of the RpoT genes and cDNAs of Nuphar advena ................. 56
3
3.1.2.1 Southern hybridization of the N. advena BAC filters................................. 57
3.1.2.2 Identification of the genomic DNA sequences of three NaRpoT genes ..... 59
3.1.2.3 Identification of the full length NaRpoT cDNA sequences ........................ 60
3.2 Sequence comparison of the RpoT polymerases from Selaginella and Nuphar
with other plant RpoT polymerases .......................................................................... 63
3.3 RpoT gene copy number............................................................................................ 64
3.3.1 RpoT gene copy number in Selaginella moellendorffii......................................... 64
3.3.1.1 Selection of probe and restriction cleavage sites ........................................ 64
3.3.1.2 Southern hybridization................................................................................ 67
3.3.2 RpoT gene copy number in Nuphar advena ......................................................... 68
3.3.2.1 Selection of probe ....................................................................................... 68
3.3.2.2 Restriction cleavage sites............................................................................ 69
3.3.2.3 Southern hybridization................................................................................ 70
3.4 Subcellular localization