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

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Functional characterisation of Epiphyas postvittana odorant receptor 1 46 glycosylated form of the receptor protein while the lower band observed at „c‟ could be degraded protein or background noise, as seen in the vector only lane 3. Figure 2.2: Western blot of myc-tagged EpOR1 in Sf9 cells transfected with pIB- EpOR1. Lane 1 is the magic marker western ladder (Invitrogen), lane 2 is the pIB- EpOR1 protein membrane fraction and lane 3 is the pIB-V5/His protein membrane fraction from cells transfected with vector only. Three bands of 60 (a), 50 (b) and 25 (c) kDa were observed in lane 2, the 50 kDa band corresponding to the molecular weight of myc-EpOR1. 2.3.1 EpOR1 functional characterisation in Sf9 cells In a typical calcium assay experiment, no change in fluorescence intensity of the Sf9 cells is observed upon addition of the control saline solution. An increase in the intracellular calcium levels, which is measured as an increase in fluorescence of the pIB-EpOR1 expressing cells is observed upon the addition of the ligand, and the maximum fluorescence intensity of the cells is achieved upon addition of the ionophore, ionomycin as depicted in Figure 2.3. Sf9 cells transfected with the empty
Functional characterisation of Epiphyas postvittana odorant receptor 1 47 pIB-V5/His vector were also tested in a dose-dependent manner with the methyl salicylate and the data obtained is given in Appendix A. Figure 2.3: The change in response of a single Sf9 cell transfected with pIB-EpOR1 over the timecourse of a calcium assay experiment. Arrow on the images indicates the change in fluorescence intensity of the responding cell plotted on the graph over the course of the assay: six initial images are acquired followed by the addition of the control assay saline buffer represented in frames 6–12, then 10 -5 M citral is added and six more images are acquired (frames 12–18). Frames 18–24 are acquired after the addition of the ionophore, ionomycin. Of the 33 compounds tested (Table 2.1), ten putative ligands were identified for EpOR1, based on the ability of the compounds to elicit a calcium response at a concentration of 10 -5 M in Sf9 cells transfected with pIB-EpOR1. These putative ligands belong to a wide range of chemical groups ranging from monoterpenes through to alcohols, esters and benzoates. The average normalised change in fluorescence of the cells upon interaction with each ligand at 10 -5 M was calculated and is given in Table 2.1.
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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
Page 61: Functional characterisation of Epip
Page 73 and 74: 3.1 Introduction 3 The roles of Epi
Page 75 and 76: The roles of Epiphyas postvittana G
Page 95 and 96: 4.1 Introduction 4 Identification o
Page 97 and 98: Identification of putative odorant
Page 109 and 110: Microarray OR 454 Transcriptome OR
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Identification of putative odorant
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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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