Transgenic plants as tool to study the evolution of pyrrolizidine alkaloids [Elektronische Ressource] / von Mohamed Ibrahim Saleh Mohamed Abd Elhady

Mohamed Mokeddem

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• Gesundheit

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2006
• Nombre de lectures: 56
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Transgenic Plants as Tool to Study the
Evolution of Pyrrolizidine Alkaloids




Von der Fakultät für Lebenswissenschaften
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer.nat.)
genehmigte
D i s s e r t a t i o n







von Mohamed Ibrahim Saleh Mohamed Abd Elhady
aus Kairo, Ägypten
























1. Referent: Prof. Dr. Dietrich Ober
2. Referent: Prof. Dr. Thomas Hartmann
eingereicht am: 09 Mai 2006
mundliche Prüfung (Disputation) am: 30 Juni 2006
2006
(Druckjahr)





Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät
für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in
folgenden Beiträgen vorab veröffentlicht:

Tagungsbeiträge:
Abd Elhady, Mohamed; Ober, Dietrich 2004. Transgenic plants as tool to
study the evolution and chemical ecology of pyrrolizidine alkaloids.
(Poster). Botanikertagung 05 bis 10. September in Braunschweig.








































ACKNOWLEDGMENT


I refer my great indebtedness to Professor Dr. Dietrich Ober, for his kind
supervision, indispensable advice, constructive discussion, great patience, and
for his efforts throughout this work.

I would like to express my sincere gratitude to Professor Dr. Thomas
Hartmann for his valuable scientific guidance, encouragement and for
agreeing to act as a co-referee.

I am deeply thankful to all members of the institute of Pharmaceutical biology,
TU-Braunschweig, especially Dr. Reiner Lindigkeit, Dr. Till Beuerle, Dr. James
Timbilla, Amala Jager, Anita Backenköhler, Claudine Theuring, Loretta Heise,
Daniel Niemüller, Daniela Gonde, Dorothee Langel, Hoda Mohagheghi for
their kindly help.

My deep thanks to all my former and current colleagues and co-workers of the
institute of Pharmaceutical biology, TU-Braunschweig, Dr. Niknik Nurhayati,
Dr. Sven Anke, Andreas Reimann, Sabine Denker, Sven Sehlmeyer, Dagmar
Enss, Elisabeth Kaltenegger, Linzhu Wang, Jenny Stiller, Helga Scharnhop,
Ina Martin, Burkhard Bohne, Ines Rahaus, Doris Glindemann.

I wish to extend my gratitude to the Egyptian government for financial support.

Finally I would like to express my deepest thanks and profound gratitude to my
family in Egypt, my father Ibrahim Saleh, my mother Ayda Hamouda, and my
brothers Hassan and Ahmed for their encouragement during the course of this
study.













Contents___________________________________________________________________ i
Contents
Abbreviations .......................................................................................v
1. INTRODUCTION .............................................................................1
1.1. Secondary metabolism................................................................................. 1
1.2. Polyamines................................................................................................... 1
1.3. Pyrrolizidine alkaloids (PAs)......................................................................... 4
1.4. Hspd and the evolutionary origin of HSS from DHS..................................... 6
1.5. Plant genetic engineering............................................................................. 8
1.6. Objectives of this work ............................................................................... 11
2. MATERIAL AND METHODS.........................................................12
2.1. Plant materials............................................................................................ 12
2.1.1 Nicotiana tabacum .............................................................................. 12
2.1.2 Senecio jacobaea................................................................................ 12
2.1.3 Senecio vernalis and Eupatorium cannabinum ................................... 12
2.1.4 Tagetes erecta .................................................................................... 12
2.2. Chemicals .................................................................................................. 13
2.2.1 Medium and constituents .................................................................... 13
2.2.2 Gel electrophoresis ............................................................................. 13
2.2.2.1 Enzyme assays ............................................................................... 14
2.2.2.2 Western blotting............................................................................... 14
2.2.3 Enzymes ............................................................................................. 14
2.2.3.1 Reverse transcription....................................................................... 14
2.2.3.2 Polymerase chain reaction (PCR) ................................................... 14
2.2.3.3 Endonucleases................................................................................ 15
2.2.3.4 Plasmid and genomic DNA preparation........................................... 15
2.2.3.5 Ligation............................................................................................ 15
2.2.4 Primers................................................................................................ 15
2.2.5 Others ................................................................................................. 15
2.3. Kits ............................................................................................................. 16
2.3.1 Isolation of nucleic acids ..................................................................... 16
2.3.2 Purification of DNA.............................................................................. 16
2.3.3 Isolation of plasmid DNA..................................................................... 17
2.3.4 Cloning of DNA fragments .................................................................. 17
2.4. Cloning materials........................................................................................ 17
2.4.1 Plasmids ............................................................................................. 17
® ®2.4.1.1 pCR2.1 -TOPO -Vector (Invitrogen)............................................... 17
® ®2.4.1.2 pCR -XL-TOPO -Vector (Invitrogen) .............................................. 18
2.4.1.3 pET3a Vector (Novagen) and pET3a-XhoI...................................... 18
2.4.1.4 pET-22b (Novagen) ......................................................................... 19
@ @2.4.1.5 pGEM -T and pGEM -T Easy vectors (Promega) ......................... 19
2.4.1.6 pCAMBIA vectors ............................................................................ 19
2.4.1.6.1 Binary vector pCAMBIA 1304..................................................... 20
2.4.1.6.2 Binary vector pCAMBIA 1305.2.................................................. 20
2.4.1.6.3 Binary vector pCAMBIA 1304K .................................................. 20
2.4.1.7 Binary vectors pGPTV-BAR and pGPTV-BARB.............................. 20
2.4.2 Escherichia coli strains........................................................................ 21
2.4.2.1 E. coli TOP10 Cells (Invitrogen) ...................................................... 21
TM2.4.2.2 E. coli DH5α (Invitrogen) and E. coli XL1 Blue (Stratagene)...... 21 ii ____________________________________________________________ Contents

2.4.2.3 E. coli BL21(DE3) (Invitrogen)........................................................ 21
2.4.2.4 E. coli JM109 (Promega) ................................................................ 22
2.4.2.5 E. coli HB101.................................................................................. 22
2.4.3 Agrobacterium tumefaciens strains..................................................... 22
2.4.3.1 A. tumefaciens GV3101/MP90 ........................................................ 22
2.4.3.2 A. tumefaciens AGL1....................................................................... 22
2.4.3.3 A. tumefaciens EHA105 .................................................................. 23
2.5. Medium ...................................................................................................... 23
2.5.1 Murashige skoog (MS) medium (Murashige and Skoog, 1962) .......... 23
2.5.2 Luria-Bertani (LB) Medium .................................................................. 25
2.5.3 SOC Medium....................................................................................... 25
2.5.4 YEB medium .............................................................