Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...

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Contents 2.3 Results ........................................................................................................... 45 2.3.1 EpOR1 functional characterisation in Sf9 cells ..................................... 46 2.4 Discussion ..................................................................................................... 51 3 The roles of Epiphyas postvittana GOBP2 in odour detection by EpOR1 .... 57 3.1 Introduction ................................................................................................... 57 3.1.1 Aim ........................................................................................................ 60 3.2 Materials and methods .................................................................................. 61 3.2.1 Recombinant Expression of EpGOBP2 ................................................. 61 3.2.1.1 Purification of recombinant EpGOBP2.............................................. 62 3.2.1.2 Delipidation of recombinant EpGOBP2 ............................................ 62 3.2.2 Volatile odorant binding assay............................................................... 63 3.2.3 Reconstituted cell assay ......................................................................... 65 3.3 Results ........................................................................................................... 65 3.3.1 Purification of recombinant GOBP2 ...................................................... 65 3.3.2 Volatile Odorant Binding Assay ............................................................ 68 3.3.3 Reconstituted EpOR1 receptor activation assays .................................. 70 3.4 Discussion ..................................................................................................... 74 4 Identification of putative odorant receptors from Epiphyas postvittana ....... 79 4.1 Introduction ................................................................................................... 79 4.1.1 Transcriptome sequencing – EST approach .......................................... 79 4.1.2 Genome sequencing ............................................................................... 80 4.1.3 Recent progress in sequencing field ...................................................... 81 4.1.3.1 454 Sequencing .................................................................................. 82 4.1.3.2 Illumina Genome Analyzer ................................................................ 82 4.1.3.3 Bioinformatics .................................................................................... 82 4.1.4 Odorant Receptor Identification in Moths ............................................. 83 4.1.5 Aims ....................................................................................................... 85 4.2 Materials and Methods .................................................................................. 86 4.2.1 Materials ................................................................................................ 86 4.2.2 Total RNA Extraction ............................................................................ 86 4.2.3 mRNA Isolation ..................................................................................... 86 4.2.4 cDNA Synthesis ..................................................................................... 86 vi
Contents 4.2.5 Degenerate PCR ..................................................................................... 86 4.2.6 Cloning and Sequencing ........................................................................ 87 4.2.7 Microarray Screening............................................................................. 88 4.2.8 Microarray Data Analysis ...................................................................... 89 4.2.9 Transcriptome Sequencing..................................................................... 89 4.2.10 Genome Sequencing .............................................................................. 90 4.2.11 Nuclear DNA isolation and mitochondrial DNA contamination test .... 90 4.2.11.1 Isolation of insect nuclei ................................................................. 90 4.2.11.2 Genomic DNA extraction ............................................................... 91 4.2.11.3 Mitochondrial DNA contamination test ......................................... 91 4.2.12 qRT-PCR primer design ........................................................................ 92 4.2.13 qRT-PCR primer test ............................................................................. 94 4.2.14 Quantitative Real-Time PCR ................................................................. 94 4.2.15 Rapid Amplification of cDNA Ends (RACE) ....................................... 96 4.2.16 Bioinformatics........................................................................................ 98 4.3 Results ........................................................................................................... 99 4.3.1 Microarray Analysis............................................................................... 99 4.3.2 Deep Transcriptomics .......................................................................... 100 4.3.2.1 454 Sequencing Statistics ................................................................. 101 4.3.3 Mitochondrial DNA contamination ..................................................... 102 4.3.4 Illumina Sequencing ............................................................................ 103 4.3.5 Data Mining ......................................................................................... 103 4.3.6 Quantitative Real-Time PCR ............................................................... 110 4.3.7 EpOR34 – a putative PR ...................................................................... 114 4.4 Discussion ................................................................................................... 119 5 Concluding Discussion ..................................................................................... 125 5.1 Introduction ................................................................................................. 125 5.2 Summary of results and discussion ............................................................. 126 5.2.1 EpOR1 characterisation ....................................................................... 126 5.2.2 EpGOBP2 reconstitution of the Sf9 cell assay .................................... 126 5.2.3 Identification of putative ORs from E. postvittana .............................. 127 5.3 Current hypothesis and future directions .................................................... 128 A. Appendix A – Dose response profile of pIB-V5 His..................................134 vii
Page 1 and 2: http://researchspace.auckland.ac.nz
Page 3 and 4: Abstract Abstract Olfaction or the
Page 5 and 6: Acknowledgements Acknowledgements I
Page 7: Contents v Contents Abstract……
Page 11 and 12: List of Figures List of Figures Fig
Page 13 and 14: List of Tables List of Tables Table
Page 15 and 16: List of Abbreviations gDNA Genomic
Page 17 and 18: 1.1 General overview 1 General Intr
Page 19 and 20: General Introduction 3 moth in loca
Page 21 and 22: General Introduction 5 Figure 1.1:
Page 23 and 24: General Introduction 7 1.5 Componen
Page 25 and 26: General Introduction 9 OBPs have al
Page 27 and 28: General Introduction 11 A model has
Page 31 and 32: General Introduction 15 Tanaka et a
Page 33 and 34: General Introduction 17 Table 1.1:
Page 37 and 38: General Introduction 21 et al., 200
Page 39 and 40: General Introduction 23 the attract
Page 41 and 42: General Introduction 25 leaves and
Page 45 and 46: General Introduction 29 Expression
Page 47 and 48: General Introduction 31 BmOr2 HvOr2
Page 49 and 50: General Introduction 33 for lepidop
Page 51 and 52: Functional characterisation of Epip
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Functional characterisation of Epip
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3.1 Introduction 3 The roles of Epi
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The roles of Epiphyas postvittana G
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4.1 Introduction 4 Identification o
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Identification of putative odorant
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Microarray OR 454 Transcriptome OR
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5.1 Introduction 5 Concluding Discu
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Concluding Discussion 127 VOBA sett
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Concluding Discussion 129 redundant
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Concluding Discussion 131 against t
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Concluding Discussion 133 sequencin
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Appendix B 135 Buffered TB (Sambroo
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Appendix C 137 C. Appendix C - Clus
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Appendix D 139 D. Appendix D - Solu
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Appendix E 141 I II
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Appendix E 143 V
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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