Novel isolates of Cydia pomonella granulovirus (CpGV): deciphering the molecular mechanism for overcoming CpGV resistance in codling moth (Cydia pomonella) [Elektronische Ressource] / Karolin Elisabeth Eberle

Eberle

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 60
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Novel isolates of Cydia pomonella granulovirus
(CpGV): deciphering the molecular mechanism for
overcoming CpGV resistance in codling moth
(Cydia pomonella)




Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaften

am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz

Karolin Elisabeth Eberle
geb. am 22.07.1981 in Frankenthal

Mainz, 2010 PAGE II

Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 23.09.2010



















PAGE III


Ever tried. Ever failed. No matter.
Try again. Fail again. Fail better.
(Samuel Beckett) CONTENTS PAGE IV
Contents
Contents ....................................................................................................................................... IV
List of abbreviations ...................................................................................................................... 8
Summary ..................................................................................................................................... 10
1 Introduction ........................................................................................................................... 13
1.1 Baculoviruses .............................................................................................................. 14
1.1.1 Baculovirus structure and infection cycle ....................................................... 14
1.1.2 Classification of baculoviruses ....................................................................... 17
1.1.3 Baculovirus genome and gene expression .................................................... 18
1.1.4 Baculovirus host range determination............................................................ 20
1.1.5 Cydia pomonella Granulovirus (CpGV).......................................................... 23
1.2 Cydia pomonella: a pest in apple orchards ................................................................. 24
1.2.1 Codling moth life cycle and damage .............................................................. 24
1.2.2 Codling moth control strategies ..................................................................... 25
1.2.3 Occurrence of codling moth resistance to CpGV ........................................... 26
1.3 Aim of this thesis ......................................................................................................... 28
2 Materials ............................................................................................................................... 30
2.1 Chemicals ................................................................................................................... 30
2.2 Bacterial strains and plasmids .................................................................................... 31
2.3 Media and buffers ....................................................................................................... 32
2.4 Insect strains ............................................................................................................... 33
2.5 Origin of CpGV Isolates .............................................................................................. 34
2.6 Software ...................................................................................................................... 36
2.7 Oligonucleotides .......................................................................................................... 36
3 Overcoming CpGV resistance in codling moth with new CpGV isolates .............................. 43 CONTENTS PAGE V
3.1 Introduction ................................................................................................................. 43
3.2 Methods ...................................................................................................................... 46
3.2.1 Virus propagation ........................................................................................... 46
3.2.2 Estimation of virus concentration ................................................................... 47
3.2.3 Bioassays: Estimation of the LC .................................................................. 47 50
3.2.4 Statistical analysis .......................................................................................... 48
3.2.5 DNA isolation of a virus solution .................................................................... 48
3.2.6 Restriction enzyme analysis (REN) ............................................................... 49
3.2.7 Gelelectrophoresis ......................................................................................... 49
3.3 Results: Bioassays with CpS, CpR and CpRR1 ......................................................... 50
3.3.1 Activity testing of different CpGV isolates at a given OB concentration ........ 50
3.3.2 Activity testing of different CpGV isolates in full range bioassays ................. 51
3.4 Results: RFLP analysis of 14 CpGV isolates .............................................................. 61
3.5 Discussion ................................................................................................................... 72
4 Molecular characterisation of CpGV isolates: partial sequencing of genomic differences ... 77
4.1 Introduction ................................................................................................................. 77
4.2 Methods ...................................................................................................................... 80
4.2.1 PCR amplification ........................................................................................... 80
4.2.2 Sequencing of selected marker genes........................................................... 81
4.2.3 Partial sequencing of CpGV-I01 and -E2 ....................................................... 81
4.2.4 Phylogenetic analysis..................................................................................... 81
4.3 Results: partial sequencing of CpGV-I01 and -E2 ...................................................... 82
4.4 Phylogenetic analysis of 11 CpGV isolates ................................................................ 87
4.5 Discussion ................................................................................................................... 90
5 Comparative genomics of different CpGV isolates ............................................................... 94
5.1 Introduction ................................................................................................................. 94 CONTENTS PAGE VI
5.2 Methods ...................................................................................................................... 96
5.2.1 Whole genome sequencing ........................................................................... 96
5.2.2 Genome analysis using DNAStar .................................................................. 96
5.3 Results: Genome comparison of CpGV-M, -I12, and -S ............................................. 97
5.3.1 Whole genome sequence of CpGV-M ........................................................... 97
5.3.2 Whole genome sequencing of CpGV-I12 .................................................... 105
5.3.3 Whole genome sequencing of CpGV-S ....................................................... 112
5.4 Discussion ................................................................................................................. 122
6 Investigations on the role of pe38 in overcoming codling moth resistance using CpGV
bacmids ..................................................................................................................................... 128
6.1 Introduction ............................................................................................................... 128
6.2 Methods .................................................................................................................... 133
6.2.1 Transformation of E.coli cells ....................................................................... 133
6.2.2 BAC DNA Isolation of E.coli cells ................................................................. 133
6.2.3 Deletion of pe38 using Red/ET- Recombination .......................................... 133
6.2.4 Insertion of CpGV-I12 pe38 into CpBacpe38 using Red/ET-Recombination ..
..................................................................................................................... 137
6.2.5 Biological activity of the different constructs ................................................ 140
6.2.6 Transfection of Cp14 cells ........................................................................... 140
6.2.7 Estimation of DNA concentration by qPCR ................................................. 140
6.3 Results ..................................................