Gene expression analysis in Candidatus Phytoplasma mali-resistant and -susceptible Malus genotypes [Elektronische Ressource] / Mirko Moser

Mm

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

“Gene Expression Analysis in „Candidatus
Phytoplasma mali‟-resistant and -susceptible
Malus genotypes”



Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaften


Am Fachbereich Biologie
Der Johannes Gutenberg-Universität Mainz


Mirko Moser
geb. am 29/01/1976 in Bozen (Italien)





Mainz, 2010












































Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 06.09.2010























to
my parents






























Abbreviations ............................................................................................................................. 1
1 Summary ............................... 3
2 Introduction ......................................................................................................................... 5
2.1 Apple proliferation ........................................................ 5
2.2 Phytopathogenic mollicutes: phytoplasmas and spiroplasmas ..................... 6
2.2.1 Phytoplasma classification ........................................................................................... 7
2.2.2 „Candidatus Phytoplasma mali‟ ................. 10
2.2.3 Transmission of „Ca. P. mali‟ .................... 11
2.2.4 Phytoplasma plant-host interaction ............................................................................ 13
2.3 A strategy for the development of apple proliferation resistant rootstocks . 15
2.3.1 Use of resistance in the control of AP ........ 15
2.3.1.1 Identification of a natural resistance to AP............................................................ 15
2.3.1.2 Breeding of new Malus hybrids for the development of a resistant rootstock ...... 16
2.4 In vitro system .............................................................................................. 18
2.5 Individuation of differentially expressed genes in phytoplasmas diseases . 19
2.5.1 cDNA-AFLP: a suitable technique for the selection of differentially expressed genes
in „Ca. P. mali‟ infected AP-resistant and –susceptible genotypes ............................ 20
2.6 Gene expression analysis by real-time PCR ................................................ 22
2.7 Aims of the work .......................................................... 23
3 Materials and methods ............................................................... 25
3.1 Materials ....................................................................................................... 25
3.1.1 Plant material .............. 25
3.1.2 Chemicals and radioisotopes ...................... 26
3.1.3 Enzymes ..................................................................................................................... 26
3.1.4 Bacterial strains .......... 26
3.1.5 Vectors ........................ 26
3.1.6 Buffers and media ...................................................................................................... 26
3.2 Methods ........................................ 27
3.2.1 Plant tissue culture ...... 27
________________________________________________________________________________________________________________________
I 3.2.1.1 In vitro grafting ...................................................................................................... 28
3.2.1.2 Rooting and acclimatization .................. 28
3.2.2 RNA isolation from plant tissue ................................................................................. 29
3.2.2.1 Preparation of phloem tissue from roots and branches .......... 29
3.2.2.2 Collection of the material from in vitro and ex vitro plants .................................. 29
3.2.2.3 RNA extraction procedure ..................................................................................... 29
3.2.3 DNA isolation from plant tissue ................. 30
3.2.4 General PCR ............................................... 31
3.2.5 Gradient PCR ............................................................................. 32
3.2.6 RT-PCR ...................... 32
3.2.7 Phytoplasma detection ................................................................................................ 32
3.2.8 Restriction reactions ... 33
3.2.9 Cloning of PCR products ........................... 34
3.2.9.1 TA cloning ............................................................................................................. 34
3.2.9.2 USER PCR product cloning method ..... 34
3.2.9.3 Preparation of chemically competent E. coli cells................. 35
3.2.9.4 Heat shock transformation of E. coli ..................................................................... 35
3.2.10 Colony PCR ................................................ 36
3.2.11 Plasmid miniprep ........ 36
+3.2.12 Poly (A) mRNA isolation ......................................................................................... 37
3.2.13 cDNA synthesis .......................................................................................................... 38
3.2.14 cDNA-AFLP .............. 38
3.2.15 Sequencing ................. 42
3.2.16 Sequencing data analysis and software ...................................................................... 43
3.2.17 Macro-array production .............................................................. 43
3.2.17.1 Low density macro-array production for the first hybridisation screening
experiment ............................................................................................................. 43
3.2.17.2 High density macro-array production for the second hybridisation screening
experiment ............. 45
3.2.18 Hybridisation and differential screening .................................................................... 47
3.2.19 Primer design .............................................. 49
3.2.20 Real-time PCR analysis .............................................................. 49
3.2.20.1 Real-time PCR on RNA ........................................................ 50
3.2.20.2 Real-time PCR on cDNA....................... 50
________________________________________________________________________________________________________________________
II 3.2.20.3 Preparation of a dilution series for the real-time qPCR primer efficiency test ..... 51
3.2.20.4 Real-time PCR data analysis ................................................................................. 51
3.2.21 Agarose gel, polyacrylamide gel preparation and electrophoresis parameter ............ 52
4 Results ................................................................................................................................... 53
4.1 Analysis of constitutively expressed genes in healthy AP-resistant and -
susceptible Malus genotypes ........................................................................ 53
4.1.1 cDNA-AFLP on healthy field-grown plants .............................. 53
4.1.2 Macroarray production and hybridisation .................................................................. 57
4.1.2.1 Cloning and sequencing of the PCR products of the selected cDNA AFLP
fragments ............................................................................................................... 59
4.1.2.2 Development of specific primers for the evaluation of the first set of candidate
genes ...................... 62
4.1.3 Cloning of all selected and isolated cDNA-AFLP fragments from 4551 and H0909 63
4.1.3.1 Production and analysis of a high density macroarray .......................................... 66
4.1.3.2 Sequence analysis of cDNA AFLP bands individuated by high density macroarray
............................................................................................................................... 70
4.1.3.3 Development of specific primers for the evaluation of the second set of candidate
genes ...................................................................................................................... 72
4.2 Analysis of induced genes in AP-resistant and susceptible plants after
infection with „Ca. P. mali‟ .......... 73
4.2.1 Production of homogenous in vitro shoot cultures of AP-resistant and susceptible
Malus genotypes ......................................................................................................... 73
4.2.2 Production of „Ca. P. mali‟-infected plants by in vitro graft-inoculation .................. 73
4.2.3 RNA extraction from healthy and „Ca. P. mali‟-infected in vitro plants ................... 75
4.2.4 cDNA-AFLP analysis of healthy and „Ca. P. mali‟-infected in vitro plants .............. 76
4.3 Selection of the differentially expressed cDNA-AFLP bands ........