Evaluation of a viral vector system, based on a defective interfering RNA of tomato bushy stunt virus, for protein expression and the induction of gene silencing [Elektronische Ressource] / Christian Naumer

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2005
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

"Evaluation of a viral vector system, based on a defective interfering
RNA of tomato bushy stunt virus, for protein expression and the
induction of gene silencing"
D i s s e r t a t i o n
zur Erlangung des Grades
"Doktor de r
N aturwissenschaften"
am Fachbereich B iologie
der Johannes Gutenberg- U niversität
in M ainz
Christian Naumer
geb. Am 14.12.1972 in Speyer
M ainz, 2005Dekan:
1.Berichterstatter:
2.Berichterstatter:
Tag der mündlichen Prüfung: 04. Oktober 2005T able of Contents
1 Table of Contents
1 T able of Contents....................................................................................................................................................I
2 A bbreviations .......................................................................................................................................................V
3 A bstract................................................................................................................................................................VI
4 Introduction............................................................................................................................................................1
4.1 M olecular farming.........................................................................................................................................1
4.1.1 Viral vectors in molecular farming.......................................................................................................3
4.2 Post transcriptional gene silencing (P T GS)..................................................................................................4
4.2.1 Virus-induced gene silencing (VIGS)..................................................................................................6
4.3 Effect of D I sequences on ge ne silencing.....................................................................................................7
4.4 Tomato bus hy stunt vi rus (T B SV)................................................................................................................8
4.4.1 Replication of TB SV............................................................................................................................9
4.4.2 D efective interfering pa rticles............................................................................................................10
4.5 Aim..............................................................................................................................................................11
5 M aterials and M ethods.........................................................................................................................................13
5.1 Chemicals....................................................................................................................................................13
5.2 Enzymes......................................................................................................................................................13
5.3 Antibodies....................................................................................................................................................13
5.4 Plants...........................................................................................................................................................13
5.5 Bacterial strains...........................................................................................................................................14
5.6 Plasmids.......................................................................................................................................................14
5.7 Cultivation media........................................................................................................................................15
5.8 Cultivation conditions of greenhouse m aterial...........................................................................................15
5.9 M olecular biology m ethods.........................................................................................................................16
5.9.1 M ini-preparation of pl asmid D N A w ith Qiaprep spin m iniprep kit .................................................16
5.9.2 M ini-preparation of plasmid DN A from Agrobacterium tumefaciens with W izard P lus DN A
purification system.......................................................................................................................................16
5.9.3 Cryopreservation of bacterial cells.....................................................................................................16
5.9.4 D etermination of D N A and RN A concentration by U V spectrometry..............................................16
5.9.5 T-tailing of pl asmid D N A ...................................................................................................................16
5.9.6 Restriction enzyme digests.................................................................................................................17
5.9.7 Agarose ge l electrophoresis................................................................................................................17
5.9.8 Agarose ge l extraction........................................................................................................................18
5.9.9 Klenow fragment “fill in” reaction.....................................................................................................18
5.9.10 Phenol/chloroform pur ification of nucleic acids..............................................................................18
5.9.11 Phosphorylation of nuc leic acids using T 4 pol ynucleotide ki nase (T 4 PN K ).................................19
5.9.12 D ephosphorylation of di gested pl asmid D N A w ith shrimp alkaline pho sphatase (SA P )...............19
5.9.13 Ligation with T4 ligase.....................................................................................................................19
IT able of Contents
5.9.14 Production of chemically competent E. coli cells............................................................................20
5.9.15 Production of electro competent Agrobacterium tumefaciens cells.................................................20
5.9.16 Transformation of chemically competent E. coli cells.....................................................................20
5.9.17 Transformation of electro competent Agrobacterium tumefaciens cells.........................................21
5.9.18 PCR and sequencing pr imers............................................................................................................21
5.9.19 PCR w ith Taq DN A pol ymerase......................................................................................................22
5.9.20 PCR w ith Pfx DN A pol ymerase.......................................................................................................23
5.9.21 Sequencing PCR...............................................................................................................................23
5.9.22 QuickChange™ site-directed m utagenesis ki t.................................................................................24
5.9.23 RN A extraction from plants.............................................................................................................24
5.9.24 RT -P CR us ing SuperScript One-Step RT -P CR kit from Invitrogen ..............................................25
5.9.25 Northern bl ot.....................................................................................................................................25
5.9.26 In vi tro transcription of RN A ...........................................................................................................27
5.10 P rotein analysis..........................................................................................................................................27
5.10.1 Histochemical GU S staining............................................................................................................27
5.10.2 Sample pr eparation for SDS-P A GE.................................................................................................28
5.10.3 SDS-P A GE.......................................................................................................................................28
5.10.4 Coomassie staining...........................................................................................................................29
5.10.5 Western bl ot......................................................................................................................................29
5.10.6 EL ISA (enzyme linked immuno sorbent assay)...............................................................................30
5.11 Inoculation of Nicotiana benthamiana w ith RN A transcripts..................................................................31
5.12 Agrobacterium infiltration.........................................................................................................................32
5.13 Fluorescence microscopy and phot ography of GFP .................................................................................32
5.14 ClustalX alignments..................................................................................................................................32
5.15 P rotoplast isolation and tran