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

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Identification of putative odorant receptors from Epiphyas postvittana 92 CCCGGTAAAATTAAAATATAAACTTC-3‟, specific for the cytochrome oxidase I gene of the mitochondrial genome of E. postvittana, were used in a PCR reaction to test for mtDNA contamination in the gDNA sample. Primers for Takeout 3, which is a nuclear gene, were used for a direct comparison of the nuclear versus mtDNA (5‟- AAAGGCGAGGGTCACTACAAG-3‟ TO3 forward primer and 5‟- GGTTCTTCAGTGCGTACACCT-3‟ TO3 reverse primer). The PCR reaction mix was prepared and the first round of PCR conducted, as stated in section 4.2.5. The products were analysed on 1% agarose gel with 1x SYBR safe DNA gel stain (Invitrogen) and visualised on the ImageQuant 300 system (GE Healthcare Life Sciences). One lane of 75 bp paired-end genome sequencing was done as stated in section 4.2.10 and Bowtie, a short read aligner (Langmead et al., 2009) was used for mapping mitochondrial sequences in GenBank and a 2,216 base pair region of the E. postvittana mitochondrial genome containing the cytochrome oxidase I and II genes to the 13 million paired-end reads obtained from one lane of Illumina sequencing. 4.2.12 qRT-PCR primer design Primers were designed using Oligo Explorer 1.1.0 (Kuulasmaa, 2000) and where possible primers were designed using the following stringent conditions: optimal temperature between 50-60°C, GC% content between 40-60%, and at least 3 GC dinucleotides at the 3‟end for primer stability. The primers were between 20–23 bp in length and the amplicon size was between 100–200 bp. The primers were made such that the formation of primer dimers are minimised and the primers do not self anneal. The primer pairs for the candidate OR sequences identified from the microarray screening and transcriptome sequencing are given in Table 4.2.
Microarray OR 454 Transcriptome OR candidate ESTs Identification of putative odorant receptors from Epiphyas postvittana 93 Table 4.2: Primer pairs for qRT-PCR amplification of products from OR candidates identified from microarray screening and 454 transcriptome sequencing of male E. postvittana antennae. candidate ESTs EST Number Primer Forward (5’ – 3’) Primer Reverse (5’ – 3’) 23893 AGGCATCACCAAAGGTAAG CGATAGAAACGCTGTCATTG 25116 GTAATACGCCTTCTGAATGC CCAAAGCGTATCAAAGTTCC 25174 GCAACTCATCAGAAAACTCG AGACAGCAGAGTGAAATCCAG 25214 GCCCCTTGTATTATTATTGGTC AGATAGGTCTGCCTTTTACACG 1032444 CACTGCGGGTATGTTTCCTG TACCCTGCCCTTGATTTCG 1037459 CGTGCTCATCCAAAGATCC ATGCCTTGCCGAAGTCATC 1051140 GCGAGCTGCGTAAAGTTGG TGGAATACAGGGGTCTGGTC 1041667 CTCGGCAACAAATAAAACGC ACCATTTGCTCTCATAAGGC 1043845 AGACGCAGCGTACAAAAGC AATAACCCATGGCAGACAAC 1042257 TTTATTCGGGGCACAAACG GGATTCCGCTGAAAGGATTC 1034291 AACTGGGCTTAAGTAAGTGC TAGCCCTGGAATGAAACAC 1037304 ATGCCTTTCGCTTTGATGC TCCCCACTTGCTATCGTAAC 1040652 CGAGACGTTTTCTTGTGTC ATAGGTCAGCCGACTGTAG 1034929 ACTACGATGCCACAGAAAGG GCCCAGAGGTAGCGATATG 1218954 TCCGTTTCCTTGACATAGG ATTGAGGCGAGGCTGATAG 1212362 TCAATGGGGGGAAGTAATAG TGAGCTGCGGTATATTTCTG 1209721 TTGGGAGTCGACATCTGTG CCACCGACATTGTTTTCTGC 1037495 CCAATACAATCAGGTGAAGC CGTTGTCTGTCGGTCTTAG 1234206 CAAACGCCCCAGTACCAAG CTCTCGCTCACCACTTTCAC 1233473 TAGCACGCTGACATTGATG TACTTCACTGGTTCTGTGGC 1039296 AACGAGGTTGCTGTTGAAG ACATACGCGCCTTACTACG 1212480 CAAACGGCATCCAAGAACTG GTGACGGTCACCACAACAC 1207127 GGACGAGTCACTGAACTGC CGGGATACCCATTAGAGAG 1226006 GCCCTTCTCTACACTCACTG CTGCGTCTAATCTTCGTTG 1043233 CTTCCCCAAGATGAAATGCC GACCCCTAACCCAACTGATG 1201411 TGGCTTCGCTTATATGGAC CCCTAATGGTGGTTGATGG 1049412 AGGTCTGGCAATCATCTCAG GGTGCCGTGTCTTATTATG 1041985 GCTCCAAGGTGAGTACAAC CTGGCTGAGCTAAATCTGC 1046227 ACCAAGGCCAAAGAGAGAAC CTTCGACTGGCAATAAGGTG 1039491 GCCAGCTGCACTCATCAAG GCTGGCGATTCTCCTATTC
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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 57 and 58: 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 97 and 98: Identification of putative odorant
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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
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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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