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Identification of putative odorant receptors from Epiphyas postvittana 98 Table 4.4: Primers used for amplifying 5‟ RACE products from the two candidate PRs identified from microarray screening and the three PR candidates identified from the 454 transcriptome sequencing of E. postvittana. EST Identified from: Microarray screening 454 Transcriptome sequencing Primer Nucleotide Sequence (5’ – 3’) 25174 (outer) AGATTTCTCTGATTATTTACACAATGTATA 25174 (nested) GTTGCACAGTGGCGACAGACCGGCACTTTT 25214 (outer) TTATTACTAAAATTACCCTCGTAACAACAA 25214 (nested) CCCGAATGATATTCCAAATGCTTACAAACT 1032444 (outer) GTGGCCCAAAAGGCGCAAAGCCCGAAT 1032444 (nested) GCCATAACTTCGATACCCACTGCCCTC 1037459 (outer) GCGGGAACAGCGCGCAGTTCACACAAC 1037459 (nested) GAAGTCATCCCCTTTATGTCGACAGCG 1042257 (outer) GTATGAGTATTGTACCGTTTGTGCCCCG 1042257 (nested) CTTCCAGGCAGACAGCCCTGAACACCG Abridged anchor primer (AAP) GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG Abridged universal amplification primer (AUAP) 4.2.16 Bioinformatics GGCCACGCGTCGACTAGTAC The predicted OR proteins from the low coverage genomic scaffolds were translated into the six reading frames and compared with the B. mori OR exons for determining the exons of the proteins. The program SplicePredictor (http://deepc2.psi.iastate.edu/cgi-bin/sp.cgi) was also used for predicting the exon/intron boundaries. A phylogenetic tree was constructed using the PHYLIP3.6a3 package (Felsenstein, 2005) of all the known and putative E. postvittana ORs together with the ORs from B. mori (Warren et al., 2007; Tanaka et al., 2009), H. virescens (Krieger et al., 2002; Krieger et al., 2004), P. xylostella, M. separata, and D. indica (Mitsuno et al., 2008). The protein sequence of EpOR34 was submitted to seven different transmembrane prediction programs including HMMTOP (Tusnady and Simon, 2001), TMPred (Hofmann and Stoffel, 1993), TMHMM (Krogh et al., 2001), DAS (Cserzo et al., 1997), SPLIT (Juretic et al., 1993), TMMOD (Kahsay et al., 2005) and TOPPRED (Claros and von Heijne, 1994). The predicted TM regions were compared and a
Identification of putative odorant receptors from Epiphyas postvittana 99 consensus of the different domains was made manually. A topology map of EpOR34 was then drawn in TOPO2 (http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl) using the consensus sequence above. 4.3 Results 4.3.1 Microarray Analysis A microarray analysis of 1809 ESTs from a male antennal cDNA library of E. postvittana was carried out to identify male-specific genes. From the microarray screening, four ESTs had more than two times higher expression in male than female antennae (EST numbers 23893, 25116, 25174 and 25214). The sex-biased expression of two (ESTs 25174 and 25214) of the four ESTs were confirmed by qRT-PCR (Figure 4.1). Figure 4.1: Quantitative RT-PCR of four candidates PRs as identified from a differential microarray screening of EST oligos. The error bars represent standard errors of three technical replicates for each tissue type. ND = not detectable. 3‟RACE-PCR was not done on ESTs 25174 and 25214 as the original sequences already had the poly A + tail sequence. The 5‟RACE-PCR was only able to extend the original EST sequences by 350–400 bp. Further 5‟RACE to extend the sequences
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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 63 and 64: Functional characterisation of Epip
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Page 163 and 164: 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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