Molecular attempts to alter carbon partitioning towards the synthesis of phenolic compounds in transgenic tobacco plants [Elektronische Ressource] / von Li Ding

Mpp

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2005
• Nombre de lectures: 27
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

MOLECULAR ATTEMPTS TO ALTER CARBON PARTITIONING
TOWARDS THE SYNTHESIS OF PHENOLIC COMPOUNDS IN
TRANSGENIC TOBACCO PLANTS





Dissertation






zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
-Dr. rer. nat.-






vorgelegt der

Mathematisch-Naturwissenschaftlich-Technischen Fakultät
(mathematisch-naturwissenschaftlicher Bereich)
der Martin-Luther-Universität Halle-Wittenberg



von Herrn Li Ding

geb. am. 03. 08. 1972
in Heilongjiang, Volksrepublik China





1. Gutachter: Prof. Dr. Sonnewald (IPK Gatersleben)
2. Gutachter: Prof. Dr. W. Roos (Universität Halle)
3. Gutachter: Prof. Dr. A. Gierl (TU München)

Halle (Saale), den 18.02.2005
urn:nbn:de:gbv:3-000008055
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000008055]
Contents I
1. INTRODUCTION.........................................................................................................1
1.1 Phenolic compounds....... 1
1.2 The shikimate pathway ........................................................................................................... 1
1.2.1 Enzymes of the shikimate pathway.................................................................................... 2
1.2.1.1 3-deoxy-o-arabino-heptulosonate-7-phosphate synthase........................................... 2
1.2.1.2 3-dehydroquinate synthase.......................................................................................... 4
1.2.1.3 3-dehydroquinate dehydratase- shikimate dehydrogenase ........................................ 4
1.2.1.4 Shikimate kinase ......................................................................................................... 5
1.2.1.5 5-enolpyruvyl-shikimate-3-phosphate synthase......................................................... 5
1.2.1.6 Chorismate synthase.................................................................................................... 6
1.2.2 Regulation of the shikimate pathway................................................................................. 6
1.2.3 Subcellular localization of the shikimate pathway............................................................ 7
1.2.4 The shikimate pathway and the quinate pathway.............................................................. 8
1.2.5 Carbon resources of the shikimate pathway ...................................................................... 9
1.2.5.1 Erythrose-4-phosphate .............................................................................................. 10
1.2.5.2 Phosphoenolpyruvate ................................................................................................ 11
1.3 Scientific aims of the work.... 13
2 MATERIALS AND METHODS ...............................................................................15
2.1 Chemicals and enzymes ........................................................................................................ 15
2.2 Plant materials ....................................................................................................................... 15
2.3 Bacterial strains, plasmids and oligonucleotides ............................................................... 15
2.4 Tobacco transformation........................................................................................................ 16
2.5 Molecular cloning techniques............................................................................................... 17
2.5.1 RNA preparation and Northern blot analysis .................................................................. 17
2.5.2 Protein extraction and Western blot analysis................................................................... 17
2.5.3 Reverse transcription PCR (RT-PCR) ............................................................................. 18
2.5.4 Rapid Amplification of cDNA Ends (RACE)................................................................. 18
2.5.5 Production of 6 ×His-tagged fusion protein in E. coli...................................................... 18
2.5.6 Activity determination of 6 ×His-tagged DHD/SHD fusion protein............................... 19
Contents II
2.6 Biochemical methods .............................................................................................................19
2.6.1 Plant extracts for enzyme activity assays .........................................................................19
2.6.2 Enzyme activity measurements ........................................................................................19
2.6.2.1 DHD/SHD activity.....................................................................................................19
2.6.2.2 Enolase activity ..........................................................................................................19
2.6.2.3 PGM activity ..............................................................................................................20
2.7 Metabolite analysis.................................................................................................................20
2.7.1 Trichloroacetic acid (TCA) extraction .............................................................................20
2.7.2 Ethanol extraction .............................................................................................................20
2.7.3 Methanol extraction ..........................................................................................................21
2.7.4 Determination of metabolites............................................................................................21
2.7.4.1 Glucose, fructose, and sucrose contents....................................................................21
2.7.4.2 Starch..........................................................................................................................21
2.7.4.3 Chlorophyll ................................................................................................................21
2.7.4.4 Total soluble phenolic compound and lignin............................................................21
2.7.4.5 Anthocyanin...22
2.7.4.6 PEP and pyruvate.......................................................................................................22
2.7.4.7 3-PGA.........................................................................................................................22
2.7.4.8 Enzymatic assay of shikimate and dehydroquinate ..................................................22
2.7.4.9 IC-MS assay of the shikimate pathway intermediates..............................................23
2.7.4.10 Amino acid...............................................................................................................24
2.7.4.11 Chlorogenic acid ......................................................................................................24
2.8 Ethanol induction ...................................................................................................................25
2.9 Floating experiment ...............................................................................................................25
2.10 Isolation of chloroplasts.......................................................................................................25
3 RESULTS .................................................................................................................. 26
3.1 Molecular characterization of tobacco DHD/SHD (Nt-DHD/SHD-1).............................26
3.1.1 Cloning a full-length Nt-DHD/SHD-1 cDNA .................................................................26
3.1.2 Expressing Nt-DHD/SHD-1 in E. coli .............................................................................27
3.1.3 Kinetic properties of Nt-DHD/SHD-1..............................................................................29
3.1.4 A novel enzymatic assay for shikimate and dehydroquinate...........................................30
Contents III
3.2 Constitutive silencing of Nt-DHD/SHD-1 in transgenic tobacco ..................................... 31
3.2.1 Plasmid construction and plant transformation ............................................................... 31
3.2.2 Screening the transgenic plants........................................................................................ 32
3.2.3 Growth characteristics of DHD/SHD RNAi plants......................................................... 33
3.2.4 Transcript analysis............................................................................................................ 35
3.2.5 cDNA macroarray analysis .............................................................................................. 35
3.2.6 Inhibition of DHD/SHD leads to reduced chlorogenate and lignin content................... 39
3.2.7 Carbohydrates and chlorophyll content in DHD/SHD RNAi plants .............................. 40
3.2.8 Silencing of DHD/SHD leads to an accumulation of the pathway intermediates.......... 41
3.2.9 Shikimate feeding experiment ......................................................................................... 42
3.3 Inducible silencing of Nt-DHD/SHD-1 in transgenic tobacco.