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Identification of putative odorant receptors from Epiphyas postvittana 100 further were unsuccessful. Tblastx of the sequences of the 5‟RACE-PCR products of ESTs 25174 and 25214 in the NCBI BLAST server did not yield any significant hits. The original EST sequences were also used to search the blastable E. postvittana 454 transcripts as well as the assembled genomic scaffolds using the tblastx tool. For both the ESTs, the 454 transcripts hit were the same length and sequence as the original sequences hence deep transcriptomics was not able to extend these sequences any further. Fragments of the original ESTs were also found in the genomic scaffolds, however the scaffolds were not long enough to extend the EST sequences any further. 4.3.2 Deep Transcriptomics The male E. postvittana antennal cDNA was normalised prior to deep transcriptomics in an attempt to reduce to levels of highly expressed genes and increase the chance of sequencing the lowly expressed ORs. The male E. postvittana antennal cDNA received from Evrogen after normalisation had an average length of 1 kb. Figure 4.2 shows 500ng of the cDNA before (lane 1) and after (lane 2) normalisation. The highly fluorescing bands in lane 1 seemed to have been evened out after undergoing DSN treatment suggesting that the cDNA population in the sample had been successfully normalised.
Identification of putative odorant receptors from Epiphyas postvittana 101 Figure 4.2: E. postvittana male antennal SMART-amplified cDNA before (1) and after (2) normalisation. Approximately 500ng of cDNA were loaded on the gel. Lane M is 1kb DNA marker. 4.3.2.1 454 Sequencing Statistics A single whole plate of 454 titanium EST sequencing of the normalised E. postvittana male antennae cDNA yielded 1,091,459 EST sequences. This data underwent validation before any analysis was conducted. The validation process involved filtering the ESTs and trimming possible vector sequences, removal of low quality sequences as well as sequences shorter than 50 bp. After the validation process, the number of ESTs remaining were 903,121 totalling 188,511,228 bp. Contig assembly was done using Newbler software under default settings. This resulted in 21,627 contigs and 94,579 singletons. The longest contig was 1994 bp and the N50 (the contig length that produces half the bases in the genome) of the combined contigs and singletons was 279 bp. An analysis of the 454 sequence data is given in Table 4.5. The contigs and singletons were deposited into a privately held Bioview Insect Database at Plant and Food Research Ltd and automatically annotated using Gene Ontology (GO) analysis.
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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
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General Introduction 33 for lepidop
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Functional characterisation of Epip
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Page 65 and 66: Functional characterisation of Epip
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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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