Biosynthesis of lignans in plant species of the section Linum: pinoresinol-lariciresinol reductase and justicidin B 7-hydroxylase [Elektronische Ressource] / by Shiva Hemmati

Shiva Hemmati

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2007
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Biosynthesis of lignans in plant species of the section Linum:
pinoresinol-lariciresinol reductase and justicidin B 7-hydroxylase





Inaugural-Dissertation
submitted to the Faculty of Mathematics and Natural Sciences
Heinrich-Heine University, Düsseldorf
in fulfillment of the requirement for doctoral degree
(Dr. rer. nat)






by
Shiva Hemmati
from Shiraz

Düsseldorf, 2007



From the Institut für Entwicklungs-und Molekularbiologie der Pflanzen,
Heinrich-Heine-Universität Düsseldorf,
Universitätsstr. 1, D-40225 Düsseldorf, Germany






















Printed with the permission of the
Faculty of Mathematics and Natural Sciences,
Heinrich-Heine University, Düsseldorf

Referee: Prof. Dr. A. W. Alfermann
Coreferee1: Prof. Dr. S. M. Li
Coreferee 2: Prof. Dr. D. Ober
Date of oral examination: 19.11.2007 Table of contents

Table of contents

1. Introduction
1.1. The genus Linum and its infrageneric taxa 1
1.1.1. Section Linum 1
1.1.1.1. Linum usitatissimum L. 2
1.1.1.2. Linum perenne L. 3
1.2. Primary and secondary metabolism 3
1.2.1. The building blocks of secondary metabolites 4
1.3. The shikimate-chorismate pathway 4
1.4. The general phenylpropanoid pathway 6
1.5. Lignans 8
1.5.1. Distribution of lignans 8
1.5.2. Structural diversity of lignans 10
1.5.3. Enantiomeric diversity of lignans 10
1.5.4. Pharmacological effects of lignans 11
1.6. Biosynthesis of lignans 12
1.6.1. Dimerization of two coniferyl alcohols 14
1.6.1.1. Stereoselective coupling by dirigent proteins 14
1.6.2. Pinoresinol-lariciresinol reductase (PLR) 16
1.6.2.1. Mechanism of hydride transfer 19
1.7. Plant cell cultures 21
1.8. Plant transformation by Agrobacterium rhizogenes 22
1.9. RNA interference (RNAi) 23
1.9.1. Mechanism of RNAi 25
1.10. Cytochrome P450 monooxygenases 26
1.10.1. The reaction mechanism of cytochrome P450s 28
1.11. Scope of the work 30

2. Materials and Methods
2.1. Materials
2.1.1. Oligonucleotides 31
I Table of contents

2.1.2. Enzymes for molecular biology 32
2.1.3. Vectors 33
2.1.4. Bacterial strains 33
2.1.5. General chemicals 33
2.1.6. Instruments 39
2.1.7. Media 41
2.1.8. Buffers and solutions 42
2.2. Methods
2.2.1. Plant materials 45
2.2.2. Isolation of RNA 45
2.2.3. First strand cDNA synthesis 45
2.2.4. PCR components and programs 46
2.2.5. Cloning of a partial cDNA sequence of L. usitatissimum and L. perenne 48
2.2.6. Generation of full-length cDNA 48
2.2.7. Cloning of the DNA fragment in cloning and expression vectors 49
2.2.8. Preparation of competent bacteria 50
2.2.9. Transformation of E. coli 50
2.2.10. Preparation of Plasmid DNA 50
2.2.10.1. Mini plasmid preparation with the TENS reagent 51
2.2.10.2. Plasmid preparation by using QIAgen Miniprep kit 51
2.2.11. Restriction hydrolysis of plasmid DNA 51
2.2.12. Agarose gel electrophoresis 52
2.2.13. Determination of purity and concentration of DNA and RNA 52
2.2.14. Sequence analysis 52
2.2.15. Southern blotting 52
2.2.15.1. Isolation of genomic DNA 52
2.2.15.2. Restriction hydrolysis of gDNA and electrophoresis 53
2.2.15.3. Blotting 53
2.2.15.4. Probe labelling and hybridization 54
2.2.15.5. Stringency washes and detection 55
2.2.16. Heterologous expression of PLR in E. coli 55
2.2.17. Extraction of soluble proteins from plant cells (PLR) 56
II Table of contents

2.2.18. Determination of protein concentrations according to Bradford 56
2.2.19. SDS polyacrylamide gel electrophoresis (SDS-PAGE) 57
2.2.20. Chemical synthesis of pinoresinol and dehydrodiconiferyl alcohol 57
2.2.21. Enzyme assays for PLR 58
2.2.21.1. Enzyme assays for P