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Identification of putative odorant receptors from Epiphyas postvittana 84 the sequences from a H. virescens cDNA library. This identified nine OR genes in H. virescens and further screening of the genomic sequences as well as transcriptome sequencing have led to the identification of a total of 21 ORs in H. virescens so far (Krieger et al., 2004). ORs have also been identified from transcriptome sequences of A. polyphemus, A. pernyi, and M. sexta. In B. mori, 68 ORs have been identified from transcriptome and genomic sequences, the highest number of ORs identified in a moth so far (Krieger et al., 2005; Nakagawa et al., 2005; Wanner et al., 2007; Tanaka et al., 2009). Degenerate PCR has also been used to identify moth ORs, for example, Mitsuno et al. (2008) identified 10 ORs from three different moth species using degenerate PCR. However, due to the highly divergent nature of ORs, with only 10- 75% amino acid sequence identity between and within them, degenerate PCR has been least successful in identifying new ORs, indicating the need for more high throughput sequencing in order to identify more members of this highly divergent group. From glomeruli studies in mammals, it has been observed that each OSN very likely express only a single OR. OSNs expressing the same OR are scattered throughout the epithelium, however they merge to the same glomerulus in the olfactory bulb. Hence a close estimation of the number of ORs in a given organism can be predicted from the number of glomeruli it possess. Glomeruli studies in tortricids have shown the presence of anywhere between 48 and 72 glomeruli (Masante-Roca et al., 2005; Varela et al., 2009). No glomeruli studies have yet been done in E. postvittana so the number of ORs could be anywhere between 48 and 72. From an EST library of male E. postvittana antennae, three ORs have been identified (Jordan et al., 2009) (refer to section 1.7 for more details). ORs and PRs are expressed at low levels in tissues therefore are under-represented in EST collections, especially in “shallow” Sanger based collections.
Identification of putative odorant receptors from Epiphyas postvittana 85 4.1.5 Aims The aim of this research chapter is to increase the receptor repertoire of E. postvittana. Does E. postvittana contain a similar set of ORs and PRs compared with B. mori in terms of the number and sequences of ORs, that is, are the E. postvittana ORs one-to- one orthologs of the B. mori ORs; or are the Tortricidae ORs distinct and different from those of the Bombycidae, having radiated independently? Based on the 65% amino acid identity (90% similarity) of BmOR49J and EpOR3, we might expect other E. postvittana ORs to follow a similar pattern. Does E. postvittana PR also fall in the PR clade, as has been shown for some other PRs? Do some E. postvittana ORs show no sex-bias expression while some are more highly expressed in males and some in females, as is the case with B. mori ORs? To address these questions multiple approaches are undertaken, each with increasing power to isolate ORs and PRs. First further screening of the Sanger 1800 ESTs is done to identify potential PRs by differential screening with male and female antennal mRNA on a microarray chip. Such an approach will allow the identification of differentially expressed genes even if their sequences are only represented by 3‟UTRs on the array. An attempt is be made at designing degenerate primers to the PR clade of moths and attempting to amplify homologous genes from E. postvittana antennal cDNA. If the above does not result in the identification of more receptors, then high throughput sequencing of E. postvittana will be conducted. ORs are expressed at very low levels so even deep transcriptome sequencing might not identify them all. It is hypothesized that normalisation of cDNA before sequencing increases the chance of identifying lowly expressed genes. Further ESTs will be obtained from transcriptome sequencing of male antennal cDNA and genomic DNA will also be sequenced for potential ORs using a low coverage whole genome sequencing method.
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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 .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 15 Tanaka et a
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General Introduction 17 Table 1.1:
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General Introduction 21 et al., 200
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General Introduction 23 the attract
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General Introduction 25 leaves and
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General Introduction 29 Expression
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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 and 76: The roles of Epiphyas postvittana G
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Page 109 and 110: Microarray OR 454 Transcriptome OR
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Page 149 and 150: 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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