Analysing the possible influence of transposon TCl4.7 insertion on the function of the genome of Cydia pomonella granulovirus [Elektronische Ressource] / Wael Hassan Ali Elmenofy

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• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2008
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

“Analysing the possible influence of transposon TCl4.7 insertion on
the function of the genome of Cydia pomonella granulovirus”


Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaften


am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz

Wael Hassan Ali Elmenofy
geb. in Kairo, Ägypten

Mainz, 2008






























Tag der mündlichen Prüfung: 08. Dezember 2008


CONTENTS
CONTENTS............................................................................................................................... 1
ABBREVIATIONS.................................................................................................................... 1
I. SUMMARY............................................................................................................................ 2
II. INTRODUCTION ................................................................................................................. 5
1. Baculovirus structure and taxonomy...................................................................................... 5
2. Baculovirus infection cycle.................................................................................................... 7
3. Host range, specificity and virulence of baculovirus ............................................................. 9
4. Baculoviruses as biocontrol agents ...................................................................................... 11
5. Baculovirus replication and gene regulation........................................................................ 12
6. Open reading frames (ORFs) and gene conservation........................................................... 12
7. Homologous repeated sequences (hrs)................................................................................. 13
8. Transposable elements and baculoviruses............................................................................ 14
9. Transposons in CpGV .......................................................................................................... 17
10. Influence of TCl4.7 transposon integration in CpGV-M ................................................... 19
Aim of work and outline of thesis............................................................................................ 19
III. MATERIALS AND METHODS....................................................................................... 21
1. MATERIALS ....................................................................................................................... 21
1.1. Insects................................................................................................................................ 21
1.2. Virus genotypes................................................................................................................. 21
1.3. Chemicals and laboratory materials .................................................................................. 21
Enzymes ................................................................................................................................... 21
Antibodies ................................................................................................................................ 22
Bacterial strains ........................................................................................................................ 22
Plasmids ................................................................................................................................... 22
Oligonucleotides....................................................................................................................... 23
2. METHODS........................................................................................................................... 25
2.1. Insects and viruses............................................................................................................. 25
2.1.1. Virus stock production ................................................................................................... 25
2.1.2. Virus OB purification from infected larvae ................................................................... 25
2.1.3. Virus OBs counting........................................................................................................ 26
2.1.4. Infection of CM in a time course for RT-PCR and qRT-PCR....................................... 26
2.1.5. Injection experiments of CM larvae using CpGV-M bacmid DNA (CpBAC).............. 26 2.1.6. Peroral infection of CM larvae (L4) using CpBAC OBs ............................................... 27
2.1.7. Determination of LC and ST values of CpBAC and its mutants.............................. 27 50 50
2.1.8. Statistical analysis .......................................................................................................... 28
hr3-kan-hr42.1.9. Competition experiment between CpBAC and CpBAC ................................... 28
2.1.10. Injection using mutant bacmids DNAs into CM larvae hemocoel............................... 28
2.1.11. Peroral infection of CM larvae (L4) using the generated mutant viruses .................... 29
2.2. DNA molecular analysis ................................................................................................... 29
2.2.1. DNA isolation of virus OBs ........................................................................................... 29
2.2.2. DNA restriction endonuclease analysis (REN).............................................................. 29
2.2.3. Low melting agarose extraction ..................................................................................... 30
2.2.4. Dephosphorylation of DNA 5' ends ............................................................................... 30
2.2.5. Ligation of DNA REN fragments .................................................................................. 30
2.2.6. Preparation of electro competent E. coli cells................................................................ 31
2.2.7. Transformation of E. coli cells....................................................................................... 31
2.2.8. Blue/white selection of recombinant E. coli colonies .................................................... 31
2.2.9. Plasmid DNA preparation .............................................................................................. 32
2.2.10. Agarose gel electrophoresis of nucleic acids ............................................................... 32
2.2.11. Polymerase Chain Reaction (PCR) .............................................................................. 33
2.2.12. Sequencing analysis ..................................................................................................... 33
2.2.13. Direct cloning of CpGV-M as a bacmid ...................................................................... 33
2.2.14. Deletion of Cp15 and Cp16 ORFs using Red/ET-Recombination .............................. 34
R
2.2.14.1. Generation of a Tn5-neo (Kan ) PCR product.......................................................... 34
2.2.14.2. Transformation of pRedET plasmid DNA into EPI300 E. coli cells........................ 36
2.2.14.3. Replacing of the target region with the Tn5-neo cassette ......................................... 36
2.3. RNA Molecular analysis ................................................................................................... 37
2.3.1. Total RNA extraction and RT-PCR analysis ................................................................. 37
2.3.2. RNA (glyoxal) electrophoresis....................................................................................... 37
2.3.3. Quantitative Real Time RT-PCR (qRT-PCR)................................................................ 38
2.4. Protein expression analysis ............................................................................................... 39
2.4.1. Induction of Cp15 and Cp16 His-Tagged fusion proteins ............................................. 39
2.4.2. SDS-PAGE..................................................................................................................... 39
2.4.3. Generating of anti-Cp15 and anti-Cp16 polyclonal antibodies...................................... 40
2.4.4. Infection of CM in a time course for protein expression ............................................... 41
2.4.5. Western blot ................................................................................................................... 41 IV. RESULTS .......................................................................................................................... 43
1. Transcription and translation analysis of ORFs Cp15, Cp16 and F protein ........................ 43
1.1. Preparation of MCp5 and CpGV-M virus stocks.............................................................. 43
1.2. Temporal transcriptional analysis of Cp15, Cp16 and F protein ...................................... 45
1.2.1. RT-PCR.......................................................................................................................... 46
1.2.2. Quantitative Real-Ti