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....................................................................................................... 12 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 DNA..................................................................................................... 18 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. Electrophoresis ....................................................................................................... 19 3.5.1. Protein polyacrylamide gel electrophoresis (PAGE) ........................................... 19 3.5.2. RNA agarose gel .................................................................................................. 20 3.5.3. DNA agarose gel .................................................................................................. 21 I Content 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 bacteriophage 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.....................................................................