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

More documents

Recommendations

Info

The roles of Epiphyas postvittana GOBP2 in odour detection 60 antennae hence postulated to have a female oriented role, perhaps in the binding of some as yet unknown male specific pheromone. The fourth PBP, called PBP3 showed higher expression in male than female antennae, and has been postulated to be involved in binding of the pheromone component(s) of E. postvittana. The two GOBPs are members of the GOBP1 and GOBP2 family of GOBPs in moths and GOBP2 has a similar level of expression in male and female antennae (Newcomb et al., 2002). No data on sensillum localisation of EpGOBPs is available yet, however, the expression of A. polyphemus, B. mori and H. armigera GOBPs have been shown to be restricted to sensilla basiconica, hence a role of GOBPs in binding plant volatiles of importance to moths is assumed (Steinbrecht et al., 1995; Wang et al., 2003). Very limited functional data is as yet available for any moth GOBP, with the only study revealing that M. sexta GOBP2 binds the plant volatiles (Z)-3-hexen-1-ol, geraniol, geranyl acetate and limonene (Feng and Prestwich, 1997) and a recent study revealed B. mori GOBP2 binds bombykol and is able to discriminate it from the antagonist, bombykal (Zhou et al., 2009). Both M. sexta and B. mori GOBP1 have so far failed to bind any of the ligands they have been tested with (Vogt et al., 2002; Zhou et al., 2009) leading to the conclusion that the proteins tested might be inactive allelic variants. 3.1.1 Aim The aim of this research chapter is to test hypotheses of possible roles for GOBPs. GOBPs could be non-specific solubilising agents for a range of plant volatiles, or they could be ligand specific, like that observed for moth PBPs. GOBPs could form a complex with the ligand, and interaction of this complex confers increased sensitivity and selectivity onto the OR, as shown for H. virescens and B. mori PBPs in heterologous cell assay systems. With this knowledge of roles of PBPs in heterologous systems, a similar approach of using a reconstituted heterologous assay system can be used to test the hypothesised roles of GOBPs with E. postvittana GOBP2 (EpGOBP2) as a model: can EpGOBP2 act as a solubilising agent for plant volatiles? GOBP2 homologues in M. sexta and B. mori have been shown to bind plant volatiles and sex pheromone components hence we assume EpGOBP2 to have a similar ligand binding range. As part of this PhD research, E. postvittana OR1 was
The roles of Epiphyas postvittana GOBP2 in odour detection 61 characterised in an Sf9 cell assay system and shown to bind a range of plant volatiles. Some of the plant volatiles identified as ligands of EpOR1 have also been shown to be ligands for M. sexta GOBP2 (geraniol and geranyl acetate); hence leading us to hypothesis that EpGOBP2 might share ligands with EpOR1. The odorants used for testing EpGOBP2 hence is limited to the binding repertoire of EpOR1, as determined in chapter two. The Sf9 cell assay system heterologously expressing EpOR1 forms a good starting point for testing the role(s) of EpGOBP2. 3.2 Materials and methods 3.2.1 Recombinant Expression of EpGOBP2 A pET30a (Novagen) plasmid clone of EpGOBP2 (pET30a-EpGOBP2) was obtained from Duncan Stanley at Plant & Food Research. This clone encodes a protein containing an N-terminal His-tag and TEV protease recognition site upstream of the EpGOBP2 sequence (411 bp, GenBank accession no. AF411460), which is missing its N-terminal 20 amino acid signal peptide (Newcomb et al., 2002). The pET30a-EpGOBP2 plasmid was transformed into Rosetta-Gami2 cells (Novagen) and positive clones selected on kanamycin and chloramphenicol luria broth (LB) plates. pET30a-EpGOBP2 was over-expressed in buffered terrific broth (TB) with isopropyl-beta-D-1 thiogalactopyranoside (IPTG) induction, as stated in Hamiaux et al. (2009). Refer to appendix A for recipe of TB. Briefly, a colony of the transformed cells was used to inoculate a starter culture for protein expression consisting of 10 mL LB media, 15 µg/mL kanamycin, 34 µg/mL chloramphenicol and 0.01% glucose. This was incubated at 37°C overnight with shaking at 220 rpm after which 5 mL of the culture was used to inoculate 500 mL of buffered TB with 15 µg/mL kanamycin and 34 µg/mL chloramphenicol. The culture was left to grow at 37°C, 220 rpm until an optical density (OD600) of 0.6 to 0.8 was attained. IPTG was then added to the culture to a final concentration of 0.5 mM, and the culture let to grow overnight at 20°C shaking at 220 rpm. The cells were harvested by centrifugation at 13,000g at 4°C for 30 minutes, with the resulting pellet stored at -20°C until purification.
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 and 8:
Contents v Contents Abstract……
Page 9 and 10:
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 29 and 30: General Introduction 13 Figure 1.3:
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 73 and 74: 3.1 Introduction 3 The roles of Epi
Page 75: The roles of Epiphyas postvittana G
Page 79 and 80: 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
Page 127 and 128:
Identification of putative odorant
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
5.1 Introduction 5 Concluding Discu
Page 143 and 144:
Concluding Discussion 127 VOBA sett
Page 145 and 146:
Concluding Discussion 129 redundant
Page 147 and 148:
Concluding Discussion 131 against t
Page 149 and 150:
Concluding Discussion 133 sequencin
Page 151 and 152:
Appendix B 135 Buffered TB (Sambroo
Page 153 and 154:
Appendix C 137 C. Appendix C - Clus
Page 155 and 156:
Appendix D 139 D. Appendix D - Solu
Page 157 and 158:
Appendix E 141 I II
Page 159 and 160:
Appendix E 143 V
Page 161 and 162:
Appendix E 145 VII continued Figure
Page 163 and 164:
References 147 Ansebo, L., Ignell,
Page 165 and 166:
References 149 Butenandt, A., Beckm
Page 167 and 168:
References 151 Fedurco, M., Romieu,
Page 169 and 170:
References 153 Große-Wilde, E., St
Page 171 and 172:
References 155 Hrdy, I., F. Kuldova
Page 173 and 174:
References 157 Kaissling, K. E. (19
Page 175 and 176:
References 159 Krieger, J., Große-
Page 177 and 178:
References 161 Lopez-Gomez, R. and
Page 179 and 180:
References 163 Miura, N., Nakagawa,
Page 181 and 182:
References 165 Pell, J. K., Macaula
Page 183 and 184:
References 167 Røstelien, T., Stra
Page 185 and 186:
References 169 Stowers, L. and Loga
Page 187 and 188:
References 171 Turner, C. T., Davy,
Page 189 and 190:
References 173 Vogt, R. G., Riddifo
Page 191 and 192:
References 175 Widmayer, P., Heifet
show all

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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?