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

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Identification of putative odorant receptors from Epiphyas postvittana 114 4.3.7 EpOR34 – a putative PR The only putative E. postvittana OR for which a full length coding region has been obtained from the 49 new ORs identified is EpOR34. The predicted coding region of EpOR34 (Figure 4.7) was assembled from the partial transcript of EST 1032444, a 2033 bp genomic scaffold and 3‟RACE-PCR. The predicted coding region was 1185 bp and encodes 395 amino acids from the start methionine to the stop codon. From the genomic sequence, two introns were identified, as indicated by red arrowheads in Figure 4.7. The first intron occurred after amino acid 258 and was 87 bp while the second intron was after amino acid 291 and was 351 bp long. The second intron could be even longer as the genomic scaffold encoding EpOR34 did not have the 3‟ end of the gene and the sequence after the second intron was obtained from the transcriptome sequence data and the poly A+ tail obtained by 3‟RACE-PCR. The amino acid sequence was also confirmed by homology to known B. mori ORs in GenBank. Seven different freely available membrane prediction programs were used for predicting the transmembrane domains of EpOR34 from its protein coding region. Moth ORs are predicted to have 7TM domains (Benton et al., 2006), however not all the prediction programs used here gave 7TMs. Since the exact placement of the start and finish of the TM domains and the number of TMs predicted varied between the different prediction programs, a consensus of the predicted domain regions was developed and is shown in roman numerals on Figure 4.8. This consensus found EpOR34 to have 7TMs and TMPred predicted an intracellular N-terminus, consistent with the recent evidence that suggest insect ORs as having intracellular N-terminus (Benton et al., 2006; Lundin et al., 2007; Smart et al., 2008; Jordan et al., 2009).
Identification of putative odorant receptors from Epiphyas postvittana 115 1 M E D K L V F E E V Y K I N M T C L R L N L S H 24 1 ATCTTTCAGTAAATTATAATGGAAGACAAATTAGTTTTTGAAGAAGTCTATAAGATCAACATGACCTGCCTCCGATTAAACCTCTCACAC 90 25 P S V P R D L K W L L T F I L M Q G L Y T A F N A I C L Y N 54 91 CCTTCTGTGCCGAGGGATCTCAAATGGCTCTTGACTTTTATCTTAATGCAGGGACTGTATACCGCATTCAATGCGATCTGTCTCTACAAC 180 55 L I F I N V K E N D F P N A C S N G V Y M V I Y F V V T F K 84 181 TTGATATTTATCAACGTAAAAGAAAATGATTTTCCCAATGCATGCAGCAACGGTGTTTATATGGTTATTTACTTTGTAGTAACATTTAAA 270 85 Y G V M V W Y Q K D I K D V I R Y Q Q E Y F D S F R E Y T V 114 271 TATGGTGTGATGGTGTGGTATCAGAAGGATATAAAGGATGTTATACGATATCAGCAGGAATACTTTGATTCTTTCCGAGAATATACTGTT 360 115 E E Q A V V K D Y I Q R G Q W V S K L W L R S T I V T A G M 144 361 GAAGAGCAAGCTGTAGTAAAAGATTACATCCAAAGAGGGCAGTGGGTATCGAAGTTATGGCTTAGATCCACTATCGTCACTGCGGGTATG 450 145 F P V K S F I D S A Y S A Y A G D F R L H S F N E N S Y G P 174 451 TTTCCTGTTAAGAGTTTCATTGATAGCGCTTATTCAGCCTACGCTGGAGATTTCAGGCTACATAGCTTCAATGAGAACAGTTATGGTCCG 540 175 Y I D E I K G R V D V F I L M Y A I F S V Y T T Y T A I M Y 204 541 TATATTGACGAAATCAAGGGCAGGGTAGATGTATTCATTTTAATGTACGCCATATTCAGTGTGTATACTACTTACACCGCAATCATGTAT 630 205 S G F A P F G P L C I L N A C A Q M D I V M M R V N H L F D 234 631 TCGGGCTTTGCGCCTTTTGGGCCACTTTGTATACTGAACGCCTGTGCTCAGATGGACATAGTAATGATGAGAGTCAATCACCTTTTCGAC 720 235 E G F D K E K S P K Q L Q N L V K F T Q N I Y G ▼ F V D Q I N 264 721 GAAGGTTTCGACAAGGAAAAATCACCGAAGCAGTTACAAAACTTAGTAAAGTTTACACAAAATATATACGGGTTTGTTGATCAAATTAAT 810 265 D I F Q V L Y E M C L K A S A I L I P I S L Y L I I E ▼ G F S 294 811 GATATATTTCAAGTTTTGTACGAGATGTGTTTAAAAGCCTCTGCGATATTAATACCTATTTCACTGTATTTGATAATTGAGGGTTTCAGT 900 295 E G K L Y Y D Y V M F S Y M A S L L C F V P C Y Y S D Y L K 324 901 GAGGGCAAGCTGTACTATGACTACGTGATGTTTTCTTACATGGCGTCTTTACTCTGCTTTGTGCCTTGCTATTATAGCGATTATCTCAAG 990 325 E K G D D L R C A I Y A S G W E K F Y D R N T R V T L R I M 354 991 GAAAAGGGTGACGATCTGCGTTGCGCAATATACGCGTCGGGCTGGGAGAAATTCTACGACAGAAATACCAGAGTCACCCTCCGCATAATG 1080 355 L I R A T R S L S I K T V F R A V C L E A F S D L C K E A Y 384 1081 CTGATACGAGCGACGAGGTCCCTCTCCATAAAGACGGTGTTCAGGGCTGTCTGCCTGGAAGCCTTCTCTGATTTGTGTAAAGAAGCTTAC 1170 385 V I F N M M Y A V L H 396 1171 GTTATTTTCAATATGATGTATGCTGTTTTGCATTAAGTCAGCTTGATGATTTAAATTTTATTCGGGCACAAACGGTACAATACTCATACT 1260 1261 TATAATTGCACAATTTAGTTTGGGTCACCTTGAGAGCTAAAAGCTAAAGAATCCTTTCAGCGGAATCCAACAAACTTTACTTGTACTAAT 1350 1351 TATCCACACACTTTGCAAATTTTATTGTTGTCATAATTCAAGTTCTTCACATATTTTAATAAACTCTTTCACACCACAAAAAAAAAA 1437 Figure 4.7: EpOR34 cDNA nucleotide sequence showing the predicted start methionine in green, the theoretical protein coding region above the nucleotide sequence and the stop codon at the end of the coding region as well as upstream of the translation initiation site in purple. The predicted polyadenylation site and the poly A+ tail are shown in red.
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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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3.1 Introduction 3 The roles of Epi
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The roles of Epiphyas postvittana G
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The roles of Epiphyas postvittana G
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Page 97 and 98: Identification of putative odorant
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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
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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,
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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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