Characterization of plant tocopherol cyclases [Elektronische Ressource] / vorgelegt von Rajeev Kumar

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Characterization Of Plant Tocopherol
Cyclases
Von der Fakult˜at fur˜ Mathematik, Informatik und Naturwissenschaften
der Rheinisch-Westf˜alischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades eines Doktors
der Naturwissenschaften genehmigte Dissertation
vorgelegt von
Rajeev Kumar, M.Sc.
aus
Kairana, Indien
Berichter: Universitatsprofessorin Dr. rer. nat. M. Frentzen˜
Universitatsprofessorin Dr. rer. nat. U. B. Priefer˜
Tag der mun˜ dlichen Prufu˜ ng: 09.06.2005
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfug˜ bar.Part of this work is published:
Rajeev Kumar, Mirella Raclaru, Thomas Schu…˜ eler, Jens Gruber, Radin Sadre, Wilfried
Luhs, Karim M. Zarhloul, Wolfgang Friedt, Dieter Enders, Margrit Frentzen, Dagmar Weier˜
(2005) Characterisation of plant tocopherol cyclases and their overexpression in
transgenic Brassica napus seeds. FEBS Letters. Vol. 579/6 pp 1357-1364
iContents
1 Introduction 1
1.1 Vitamin E: What is it? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Chemical structure, occurrence and subcellular localization of vitamin E . . 1
1.3 Functional roles of vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3.1 Antioxidant functions . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3.2 Membrane stability functions . . . . . . . . . . . . . . . . . . . . . . 6
1.3.3 Functions in photosynthetic electron transport . . . . . . . . . . . . . 6
1.3.4 Tocopherols in intracellular signaling . . . . . . . . . . . . . . . . . . 7
1.3.5 Transcriptional regulation functions . . . . . . . . . . . . . . . . . . 7
1.4 Vitamin E in human disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4.1 Neurological disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4.2 Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4.3 Cataract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4.4 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.5 Biosynthesis of tocopherols . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.6 General considerations and approaches for improvement of tocopherol levels
in transgenic plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.7 Tocopherol cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.8 Research objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2 Materials and Methods 19
2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.1.1 Chemicals and consumables . . . . . . . . . . . . . . . . . . . . . . . 19
2.1.2 Enzymes and kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
ii2.1.3 Primary antibodies, secondary antibodies and substrate for Western
blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1.4 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1.5 Solutions, bufiers and media . . . . . . . . . . . . . . . . . . . . . . . 21
2.1.6 Chromatography matrices and membranes . . . . . . . . . . . . . . . 25
2.1.7 Biological materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.1.8 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.1.9 Vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2 Microbial methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2.1 Culture of bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2.2 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.3 Molecular Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.3.1 Isolation of plasmid DNA from E. coli . . . . . . . . . . . . . . . . . 29
2.3.2 Isolation of plasmid DNA from Agrobacterium tumefaciens . . . . . . 30
2.3.3 Determination of DNA yield and quality (Sambrook et al., 1989) . . . 30
2.3.4 DNA restriction analysis and agarose gel electrophoresis . . . . . . . 30
2.3.5 DNA extraction from agarose gels . . . . . . . . . . . . . . . . . . . . 30
2.3.6 DNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.3.7 Isolation of mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3.8 Two-step Reverse transcriptase-PCR (RT-PCR) . . . . . . . . . . . . 31
2.3.9 Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . . 32
2.3.10 Phosphorylation and dephosphorylation of DNA . . . . . . . . . . . . 33
2.3.11 Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.12 Construction of TC expression chimeric plasmids . . . . . . . . . . . 33
2.4 Biochemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.4.1 Expression of recombinant protein in E. coli . . . . . . . . . . . . . . 35
2.4.2 Puriflcation of His-tagged TC proteins by Ni-NTA a–nity chromatog-
raphy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.4.3 Ammonium sulphate precipitation of recombinant TC . . . . . . . . . 36
2.4.4 Molecular weight determination by gel flltration chromatography. . . 36
2.4.5 Estimation of protein concentration by the Bradford method . . . . . 37
2.4.6 SDS-Polyacrylamide gel electrophoresis . . . . . . . . . . . . . . . . . 37
iii2.4.7 Western blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.4.8 Tocopherol cyclase assays . . . . . . . . . . . . . . . . . . . . . . . . 39
2.5 Computer programmes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3 Results and Discussion 41
3.1 Identiflcation of tocopherol cyclase sequences . . . . . . . . . . . . . . . . . . 41
3.2 Expression of tocopherol cyclases in E. coli . . . . . . . . . . . . . . . . . . . 46
3.3 Optimizationoftheexpressionconditionsforrecombinanttocopherolcyclases
in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.4 Puriflcation of recombinant tocopherol cyclases by a–nity chromatography . 54
3.5 Size exclusion chromatography of purifled tocopherol cyclase . . . . . . . . . 56
3.6 Characterization of tocopherol cyclases from plants . . . . . . . . . . . . . . 57
3.6.1 pH and temperature optimum . . . . . . . . . . . . . . . . . . . . . . 59
3.6.2 Kinetic parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.6.3 Protein and time linearity . . . . . . . . . . . . . . . . . . . . . . . . 61
3.6.4 Substrate speciflcity . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.7 Transgene expression of plant Tocopherol cyclases in seeds of Brassica napus 63
4 Summary 67
Appendix69
A.1 Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
A.2 The alignment of sequences using CLUSTAL X . . . . . . . . . . . . . . . . 71
A.3 Vectors maps for cloning and expression in E. coli . . . . . . . . . . . . . . . 73
A.4 Chimeric construct maps for E. coli expression . . . . . . . . . . . . . . . . . 74
A.5 Map for pPZP111 and chimeric TC construct for B. napus transformation . 75
ivList of Tables
1.1 Tocopherol levels and composition in selected crops and plant oils. . . . . . . 4
2.1 List of instruments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 Bufiers and media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1 Sequence homologs of the SLR1737 sequence from plants and cyanobacteria. 42
3.2 Sequence identity (%) index of putative tocopherol cyclase orthologs from
plants and cyanobacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.3 Tocochromanol content of transgenic rapeseed plants overexpressing chimeric
TC genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
vList of Figures
1.1 The structure of tocopherols and tocotrienols. . . . . . . . . . . . . . . . . . 2
1.2 The biosynthesis of tocopherols and plastoquinone in plants. . . . . . . . . . 12
1.3 The chromanol head ring formation of ?-tocopherol from DMPBQ catalyzed
by TC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.1 Semi-dry Western blot transfer stack assembly . . . . . . . . . . . . . . . . . 38
3.1 Phylogenic relationship amongst the putative TCs from cyanobacteria and
plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.2 Alignment of the N-terminal regions of TC orthologs from plants and Syne-
chocystis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.3 Functional expression of TC orthologs in E. coli . . . . . . . . . . . . . . . . 47
3.4 Western blot analysis of the expression levels of the recombinant TC proteins
from Arabidopsis, maize and Synechocystis. . . . . . . . . . . . . . . . . . . . 48
3.5 Western blot analysis of the expression levels of TC preproteins from Ara-
bidopsis and maize. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.6 TC activity in soluble and membrane fractions of E. coli cells overexpressing
the recombinant ¢76TC from Arabidopsis thaliana. . . . . . . . . . . . . . . 50
– –3.7 Western blot analysis of TC induction from Arabidopsis at 25 C, 20 C and
–15 C in E. coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.8 Western blot ana