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

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Appendix C 138 EposOR1 YSNFKSGMLSS-EKPINGTYEHSVYYVLPFDHNNEV-WYPVVGLYNFYVSYNLGAMFSCH BmOR-6 LHNIK---HFEEIMNKSMEFQYYIYFWTPLDYKYNLRDYIIIHTLCIYLGATAVTVIVTF . : . * . MsOR1 DLALSLLIFTVSGHFKILIHNLNNFPLPAVVSDSSKVLKTDEIQ----------APLYNK BmOR-3 DLILSLMISTVSGHFRILIHNLLTFPLPEAITASKKFVDKHRCNGNRSEFVLEEAKLYSP HR6 NLFLSLLVFNLWGHFKILISTLNEFPRPSSKSVDTQESP----------------YKYTE HR16 DLFLSLMVFHLWGHFKILINTLNDFPRPSSKVEGAQ---------------------FSD HR14 DLFLSIMVFHVWGHFKILLHDLDHFPRPANLTTFKLDNSN--IT--------LTSEKFSS HR15 DFFLSLMVFHIWGHFKILLHDLDHFPRPLNKVNSVIEDS---IT--------ITNEMYSQ HR11 DLFLCLMIFNLWGHFKILIHNLEHFPRPASEIVDAEGAERSGRI--------IGSEMYSQ BmOR-5 ELSLSLMIICLWGHLRIFIHNLNHIPAPRNSFE------------------------YTK BmOR-7 DLLLSMMIIHLWGHIRIFIHNLNHIPAPRNSLE------------------------YTR HR13 DLTISLVIFHLWGHMRLLTYHLANFKKPASVLESNDNNKDEIKD-----------HSYTE BmOR-4 DLFLSLLILHLWGHLRIILNNLKTFPKPYTNNS-----------------------MYTE BmOR-1 DLLMCLMVMHVWGHLKILSHNLINFPRPKASHVITTPNGPTN-V-----------ETYTE EposOR1 DLLISVYVFHIWGHLNICEHNLNNFPRPSITRNSKTVPLR-----------------YSA BmOR-6 DIFNFIAVFHVVAHIQILKNNVKSNWSDD----------------------------FNE :: : : : .*:.: : :. MsOR1 TEKKDITLRLKQCIDYHREVLEFTQDISEAFGPMLFVYYLFHQVSGCLLLLECSQMDAAA BmOR-3 AEMWQVTDRLRQCIDYHRKLVEFTGDISEAFGPMLFVYYLFHQVSGCLLLLECSQLNTAA HR6 EELIEVAEKLKDCINYHREIKIFTNRMSDVFGPMLFIYYAFHQASGCLLLLECSQMTARA HR16 EELVDVAARLKDCIVYHREITLFTDRMSNVFGPMLFVYYSFHQASGCLLLLECSQMTAQA HR14 IELGQVSEKLKKCIEYHRKIVSFTDEMSEVFGPMLFVYYGFHQTSGCLLLLECSQMTVAA HR15 TELDQVFDRLGKCIDYHREIVSFTDKMSEVFGPMLFAYYGFHQASGCLLLLECSQMTVAA HR11 AELEQVAVLLRECIQYHMLIFDFTNNMSDAFGMALFIYYSFHQITGCLLLLECSQMTAAA BmOR-5 EERQEVDDTLKKCIQHHTLIIGFVRIMSETYGLAVLIYYAFQQVVGCLLLLQCSQMELKT BmOR-7 EERQEVDNTLKKCIQHHTLIIGFVRIMSETYGLAVLIYYAFQQVVGCLLLLQCSRLDLKT HR13 EELKEVFSKLREYIQHHNLILEFSSEMSNAFGPALLAYMVFHQVSGCILLLECSQLDTKT BmOR-4 EENQVVLLKLQECIRYHNFIISFTVMMSNVYDVVIIVYYLFHQVTGCLLLLQCSTLDWES BmOR-1 EESKEVFARLRECIKHYGTVDDFANDMSETFGVILLVYYGFHQVSLCMLLLECSDLSTKA EposOR1 EENKKVAAGLKEIIIHYIMIKKFVEKTSNTYSVTLCFYYGFHMVAECILLLQCSTLEVEA BmOR-6 SEKK---GYLVSILEYHAYIIRIFGEVQSAFGLNVASNYLQNLIEDGLFLYQIMNGEKEN * * . : :: : : ...:. : : ::* : MsOR1 LMRYGLLTAVLFQQLIQLSVVVESVGTVTGYLKDAVYNVPWEYMDTQDRKTVCIFLMNVQ BmOR-3 LVRYGVLTVVLYQQLIQLSVIVESVGTVTGRLKDAVYEVPWEYMDTSNRKTVAIFLMNVQ HR6 LMRYLPLTIIMLQQLIQLSVIFELVGTESEKLKDAVYGVPWDCMDTKNRKVVMFFLMNVQ HR16 LMRYVPLTIILTQQLIQLSVIFELVGSESDKLKHAVYGLPWECMDVKNRRVVLIFLANTQ HR14 LVCYLPLTIMLFQQLIQLSIIFELVGSVSDKLKDAVYSLPWEAMDIKNKKTVAIFLMNVQ HR15 LVRYLPLTIILFQQLIQMSIIFELVGSVTDKLRDAVYGLPWEAMDTKNRKTVAFFLMNVQ HR11 LTRYLPLTIIMFGELVLLSIIFETIGTMSEKLKDAVYKVPWEYMDTKNRRTLLIFLIKVQ BmOR-5 VTRFGFLTLVLNQQLIQISVIFELLGYMSDKLQDAVYCVPWEYMDTSHRKMVYMMFRQSQ BmOR-7 ITRFGFLTTMVNQQLIQISVIFELLGYMNDKLQEAVYCVPWEYMDTSHRKMVYMMFRQSQ HR13 LVRYGPLTIVIFQQLIQISVIFELLGSSNDKLIDGVYLVPWEYMDTKNRKLVFTMLRQSH BmOR-4 LSRYGPLTLIIFQQLIQVSMIFEILGFLSDKLPNAVYSIPWEAMNVTNRKLVQVLLQKSQ BmOR-1 MLRYGPLTLIMIQQLIQISIIFELLGSVADRIPDAVYQLPWECMDVKNRRVVYGFLRRTQ EposOR1 LAKYGFLTVAVYQELIQLSVVFELIYAKGTSLIDAVYGLPWECMDNSSRRTVLILLQIVQ BmOR-6 VLMYGLMIILYLGGLIFLSIVLEEIRRQNYDLCEYVYALPWEGMSLENQKIFVVFLQRTQ : : : *: :*::.* : : . ** :**: *. :: . :: : MsOR1 EPVHINALGLAKVGVQAMAGILKTSFSYFAFLRTVSN--- BmOR-3 EPLHVNALGLAKVGVQSMAAILKTSFSYFTFLRTVSE--- HR6 EPVHVKAMGLANVGVTTMASILKTSLSYFTFLLSQTKEE- HR16 EPVHVKAMGVANVGVTSMAAILKTSMSYFTFLRSM----- HR14 EPVHVKALGLAEVGVTSMTAILKTSMSYFTFLRSK----- HR15 EPVHVKALGLAEVGVTSMTAILKTSMSYFAFLRSM----- HR11 EPIHVKAGGLVDVGVTTMASILKTSFSYFAFLRTF----- BmOR-5 IPLQLKAMNMLSIGVKTMVSILKTSVTYYLILKTVTTD-- BmOR-7 IPLQLKAMNMLSIGVKTMASILKTSVTYYLMLKTITANEA HR13 RSINLTMMSMVTVGVQTMTAILKTSFSYFVMLKTVAEEE- BmOR-4 KPIQFKAMNMMSVGVQTMASIIKTSISYFIMLRTIARD-- BmOR-1 NPVRFKAMGMLDVGVQTMASILKTSISYFVMLRTVAT--- EposOR1 QPLSLKACGMVPVGIQTMQAILKGSFSYFLMLRTFANQ-- BmOR-6 PDLEFETVCGMKAGVKPAFSIVKSMFSYYVMINSRF---- : . *: . .*:* .:*: :: : Figure C.1: ClustalX multiple sequence alignment of the moth PR clade, including members from B. mori, M. sexta, E. postvittana and H. virescens (Krieger et al., 2002; Krieger et al., 2004; Nakagawa et al., 2005; Wanner et al., 2007; Jordan et al., 2009; Patch et al., 2009). Amino acids in red indicate region of degenerate primer design for identifying PR genes in E.postvittana.
Appendix D 139 D. Appendix D – Solutions for nuclear DNA isolation Table D.1: Reagents for making 1 L of Nuclei Extraction Buffer, pH 6. Reagent 1 L Final concentration Mannitol 91g 0.5 M PIPES-KOH 3.78 g 10 mM MgCl2 7H2O 2.03 10 mM PVP K30 20 g 2% L-lysine monohydrochloride 36.52 g 200 mM EGTA 2.28 g 6 mM Sodium meta bisulphite 1.9 g 10 mM 800 mL of deionized water was heated up in the microwave for 2-4 minutes and poured into a glass beaker with a magnetic stirrer. PVP K30 was added to the beaker with vigorous stirring. Mannitol was added and completely dissolved before addition of PIPES, magnesium, lysine, and EGTA. Volume was adjusted to 1 L, pH 6. The buffer was split into two 1 L flasks, autoclaved and cooled to room temperature. The buffer was kept at 4°C until required. Just before starting the nuclei isolation, 0.9 g sodium meta-bisulphite was added to each flask. To one flask 0.2 mL -mercaptoethanol was added and this flask labelled as NEB complete buffer, while the other flask is NEB– no -mercaptoethanol. Both the flasks were kept on ice until used. The lysis buffer in part II of nuclear DNA isolation consists of 0.5% SDS, 5 mM EDTA, 150 mM Tris-borate pH 7.4. To prepare this solution the Tris buffer was titrated with boric acid, instead of the usual HCl, autoclaved and kept at room temperature.
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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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4.1 Introduction 4 Identification o
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Identification of putative odorant
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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
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Page 177 and 178: References 161 Lopez-Gomez, R. and
Page 179 and 180: References 163 Miura, N., Nakagawa,
Page 181 and 182: References 165 Pell, J. K., Macaula
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Page 185 and 186: References 169 Stowers, L. and Loga
Page 187 and 188: References 171 Turner, C. T., Davy,
Page 189 and 190: References 173 Vogt, R. G., Riddifo
Page 191 and 192: References 175 Widmayer, P., Heifet
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