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Identification of putative odorant receptors from Epiphyas postvittana 88 competent cells in a 1.5 mL microcentrifuge tube and incubated on ice for 30 minutes. The cells were heat shocked at 42°C for 1 minute, and immediately placed on ice. After three minutes on ice, 225 µL Luria Broth media was added to the tubes and left to incubate for one hour at 37°C with shaking at 150 rpm. Fifty microlitres of media solution was then plated on LB agar plates containing 100 µg/mL ampicillin and 40 µg/mL X-galactose (for blue/white screening). After overnight incubation at 37°C white colonies were screened by PCR with plasmid specific M13 primers by transferring a small portion of the colony with a pipette tip and dipping it in a PCR reaction mix containing 1x PCR buffer including 1.5 mM magnesium, 0.2 mM of each dNTP, 2 units of Taq DNA polymerase and water up to 20 µL. The PCR cycling conditions were as follows: initial denature at 96°C for 2 minutes, then 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for one minute and a final extension of 72°C for 10 minutes. The resulting products were visualised on agarose gel as before and colonies containing an insert greater than 500 bp were picked for sequencing. Briefly, the colonies were grown in 10 mL LB media supplemented with 100 µg/mL ampicillin overnight at 37°C in a shaking incubator at 220 rpm. The plasmid was isolated using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced in both directions using BigDye TM Terminator Version 3.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems) with capillary analysis performed on the ABI3730 DNA Analyzer (Applied Biosystems) at the Allan Wilson Centre Sequencing Services, Palmerston North. The sequences obtained were analysed in Sequencher v4.2 software (Gene Codes) by aligning both the sequence strands and to confirm that the overlapping regions matched. These sequences were then searched against all the available sequences in the NCBI BLAST server (Altschul et al., 1990) using the tblastx algorithm to identify homologous genes and confirm the identity of the cloned genes. 4.2.7 Microarray Screening For differential microarray screening of the male antennal EST library to identify potential PR candidates, each of the 4472 male antennal EST oligonucleotides were checked manually to see if the oligonucleotide was in an acceptable region of the sequence trace file. Candidates for spotting onto a microarray chip were selected based on their trace file being good and the putative function of the EST from
Identification of putative odorant receptors from Epiphyas postvittana 89 homology searches in NCBI databases yielding proteins with olfactory function or for whom a function could not be determined. 1809 candidates were selected for which oligos were designed, synthesized and spotted onto a microarray chip for hybridisation screening with male and female E. postvittana antennae mRNA. 250 ng of male and female antennae mRNA each, were labelled with both Cy3 and Cy5 to be used in a dye swap hybridisation (Cy3 male vs Cy5 female and Cy3 female vs Cy5 male). The microarray hybridisation of the oligonucleotides spotted on the chip with male and female antennae mRNA was performed at the microarray facility at Plant and Food Research, Mt Albert, Auckland, as outlined in Janssen et al. (2008). 4.2.8 Microarray Data Analysis The fluorescence scores from the microarray hybridisation were checked for male- biased expression and confirmed by checking the corresponding spots by eye. An oligo was considered to have male antennae biased expression if the same results were obtained for the dye swap. Genes with oligos showing differential (if fluorescence for male was ≥ to 2 times that of female) expression were further tested for male-biased expression by qRT-PCR. The primer design, primer testing and qRT-PCR is outlined in sections 4.2.12 – 4.2.14 and the primer pairs are given in Table 4.2. 4.2.9 Transcriptome Sequencing 5 µg of male E. postvittana antennal total RNA with an OD260/280 of 1.96 was sent to Evrogen (www.evrogen.com) for synthesising directionally normalised cDNA, using the SMART approach and the enzyme DSN, as outlined in section 4.1.1. The normalised sample was then sequenced on the Roche 454 GS FLX Titanium at the Department of Anatomy and Structural Biology at The University of Otago. The sequence data was assembled using the Newbler assembler by the Bioinformatics department at Plant and Food Research Ltd, Mt Albert, Auckland. The contigs and singletons were deposited into BioView, a privately held database at Plant and Food Research Ltd. B. mori OR amino acid sequences were used to perform tblastn searches of the E. postvittana Insect Database (containing the male antennal contigs and singletons) and best hit ESTs with e-value less than 0.5 were taken as significant and selected for further 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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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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