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

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Contents B. Appendix B – EpGOBP2 expression buffer recipe and protein details ...... 135 C. Appendix C – ClustalX multiple sequence alignment of moth PR clade .... 137 D. Appendix D – Solutions for nuclear DNA isolation ...................................... 139 E. Appendix E – Multiple sequence alignment of E. postvittana ORs.............. 140 References ................................................................................................................. 146 viii
List of Figures List of Figures Figure 1.1: Structure of a single insect olfactory sensillum ......................................... 5 Figure 1.2: Odorant receptor activation ...................................................................... 10 Figure 1.3: Model of insect pheromone detection ...................................................... 13 Figure 1.4: Topologies of insect and mammalian odorant receptors .......................... 19 Figure 1.5: The ion channel–GPCR model of odorant signalling .............................. 20 Figure 1.6: The sex pheromone blend of E. postvittana. ............................................ 26 Figure 1.7: Scanning electronmicrograph of adult male (left) and female (right) antennaes of E.postvittana. .......................................................................................... 27 Figure 1.8: Phylogenetic tree constructed from lepidopteran odorant receptors with the three E. postvittana ORs ........................................................................................ 31 Figure 2.1: Reverse transcription PCR analysis ......................................................... 45 Figure 2.2: Western blot of myc-tagged EpOR1 ........................................................ 46 Figure 2.3: The change in response of a single Sf9 cell transfected with pIB-EpOR1 over the timecourse of a calcium assay experiment. .................................................... 47 Figure 2.4: Dose response curves ............................................................................... 50 Figure 3.1: Schematic representation of the VOBA setting. ...................................... 64 Figure 3.2: Purification of His6-EpGOBP2 by HiTrap chelating HP column. ....... 66 Figure 3.3: Purification of His6-EpGOBP2 from the pooled HiTrap fractions by Q- sepharose anion exchange chromatography................................................................. 67 Figure 3.4: Purification of AcTEV cleaved EpGOBP2 .............................................. 68 Figure 3.5: Overnight VOBA of EpGOBP2 ............................................................... 69 Figure 3.6: Dissociation constants ( ) for ten odorants against EpGOBP2 ............. 69 Figure 3.7: Dose response of EpOR1 expressing Sf9 cells to EpGOBP2 .................. 71 Figure 3.8: Dose response curves of EpOR1 to geranyl acetate ................................. 72 Figure 3.9: Dose response curves of EpOR1 to methyl salicylate.............................. 73 Figure 4.1: Quantitative RT-PCR of four candidates PRs .......................................... 99 Figure 4.2: E. postvittana male antennal SMART-amplified cDNA ....................... 101 Figure 4.3: Semi-quantitative PCR on nuclear (Takeout 3) and mitochondrial (cytochrome oxidase I) genes .................................................................................... 103 Figure 4.4: Multiple sequence alignment in ClustalX of E. postvittana ORs.......... 110 Figure 4.5: Expression levels of the putative E. postvittana ORs ............................ 111 ix
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Page 3 and 4: Abstract Abstract Olfaction or the
Page 5 and 6: Acknowledgements Acknowledgements I
Page 7 and 8: Contents v Contents Abstract……
Page 9: Contents 4.2.5 Degenerate PCR .....
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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