Isolation and characterization of (S)-chelianthifoline synthase, (S)-stylopine synthase and two FAD oxidases from a cDNA library from Argemone mexicana [Elektronische Ressource] / von María Luisa Díaz Chávez

Maria Luisa

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 37
• Poids de l'ouvrage: 3 Mo

Extrait

Isolation and characterization of (S)-chelianthifoline synthase,
(S)-stylopine synthase and two FAD oxidases from a cDNA
library from Argemone mexicana


Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)


vorgelegt an der
Naturwissenschaftlichen Fakultät I-Biowissenschaften
der Martin-Luther-Universität Halle-Wittenberg
Fachbereich Biochemie / Biotechnologie


von
Frau María Luisa Díaz Chávez
geboren am 25.03.1974 in Mexiko




Gutachter:
1. Prof. Dr. Toni M. Kutchan
2. Prof. Dr. Jörg Degenhardt
3. Prof. Dr. Ute Wittstock

Halle, den 22. Januar 2009

urn:nbn:de:gbv:3-000015158
[ http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000015158 ]










This work was supported by DAAD/CONACyT and PROMEP/UAEM
Content

Content

CONTENT……………………………………………………………………………………. I
ABBREVIATIONS ………………………………………......………...………………….. IV
1. INTRODUCTION..................................................................................................1
1.1. Alkaloids.................................................................................................................... 1
1.1.1. Benzylisoquinoline alkaloids ................................................................................. 1
1.1.2. Biosynthesis of berberine and sanguinarine........................................................... 3
1.2. Cytochrome P450.....................................................................................................4
1.2.1. Plant cytochrome P450........................................................................................... 5
1.3. Berberine bridge enzyme (BBE) and BBE-like proteins ...................................... 7
1.4. (S)-tetrahydroprotoberberine oxidase (STOX) ..................................................... 9
1.5. Argemone mexicana L. 10

2. MATERIAL.........................................................................................................12
2.1. Enzymes................................................................................................................... 12
2.2. Proteins.................................................................................................................... 12
2.3. Nucleotides..............................................................................................................12
2.4. DNA fragments.......................................................................................................12
2.5. Cloning vectors
2.6. Synthetic Oligonucleotides.....................................................................................12
2.7. Organisms...............................................................................................................13
2.7.1. Plants .................................................................................................................... 13
2.7.2. Bacteria................................................................................................................. 13
2.7.3. Insect cells work................................................................................................... 13
2.8. Antibiotics 13
2.9. Internet searches and alignments ......................................................................... 13
2.10. Chemicals................................................................................................................ 14
2.11. Kits........................................................................................................................... 15
2.12. Consumables...........................................................................................................15
2.13. Instruments.............................................................................................................15

3. METHODS..........................................................................................................16
3.1. Alkaloids.................................................................................................................. 16
3.1.1. Alkaloid extraction from plant tissues.................................................................. 16
3.1.2. Analyses by High Performance Liquid Chromatography (HPLC) ...................... 16
3.1.3. Analyses by Liquid Chromatography- Mass Spectrometry (LC-MS, TOF)........ 16
3.2. Isolation of RNA.....................................................................................................17
3.2.1. RNA Isolation....................................................................................................... 17
+3.2.2. Poly-(A) RNA isolation ...................................................................................... 18
3.3. Isolation of DNA18
3.3.1. Plasmid DNA purification.................................................................................... 18
3.3.2. Purification of DNA fragments from agarose gel ................................................ 18
3.3.3. Baculovirus DNA from infected Sf9 cells............................................................ 18
3.4. Insect cell culture....................................................................................................19
3.4.1. Maintenance ......................................................................................................... 19
3.5. Electrophoresis19
3.5.1. Protein polyacrylamide gel electrophoresis (PAGE) ........................................... 19
3.5.2. RNA agarose gel .................................................................................................. 20
3.5.3. DNA agarose gel 21
IContent

3.6. cDNA library...........................................................................................................21
3.6.1. λ-cDNA library construction................................................................................ 21
3.7. Preparation of plating cells for library amplification......................................... 22
3.8. Plating bacteriophage  ......................................................................................... 22
3.9. Picking  plaques........................................................................... 22
3.10. Library screening...................................................................................................22
3.11. Northern Blot..........................................................................................................23
3.11.1. Blotting............................................................................................................. 23
3.11.2. Prehybridization ............................................................................................... 24
3.11.3. Random primer labelling of DNA.................................................................... 24
3.11.4. Hybridization.................................................................................................... 25
3.12. First-strand cDNA synthesis.................................................................................. 25
3.13. Rapid amplification of 5’-cDNA ends (5’-RACE) ............................................... 26
3.14. Polymerase chain reaction (PCR) ......................................................................... 26
3.14.1. Standard PCR reaction ..................................................................................... 26
3.14.2. Screening bacterial colonies by PCR ............................................................... 27
3.14.3. Sequencing of DNA ......................................................................................... 27
3.15. DNA modifications.................................................................................................28
3.15.1. Addition of a 3’-A Overhang ........................................................................... 28
3.15.2. Dephosphorylation of DNA fragments ............................................................ 28
3.15.3. Restriction enzyme digestion 29
3.16. DNA cloning............................................................................................................29
3.16.1. TA cloning........................................................................................................ 29
3.16.2. Subcloning 29
3.17. Transformation of competent cells ....................................................................... 30
3.18. Protein Expression.................................................................................................30
3.18.1. BaculoGold Expression Vector System ........................................................... 30
3.18.2. Co-transfection using BD Baculogold ............................................................. 31
3.18.3. BAC-to-BAC expression system...................................................................... 31
3.18.4. Bacmid transposition........................................................................................ 32
3.18.5. Transfection of Sf9 insect cells with recombinant bacmid DNA ..................... 32
3.18.6. Baculovirus amplification ................................................................................ 32
3.18.7. CYP450