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

More documents

Recommendations

Info

Identification of putative odorant receptors from Epiphyas postvittana 102 Table 4.5: Analysis of the 454 transcriptome sequence data from the raw data obtained (before contig assembly) through to the processed data, assembled contigs and singletons. Before contig assembly No. of ESTs Bases (bp) N50 Average length (bp) Median (bp) Longest (bp) 903121 188511228 208.7 192 594 Contigs 21627 6884597 369 318.3 311 1994 Singletons 94579 16837407 234 178.0 152 579 Contigs + singletons 116206 23722001 279 204.1 175 1994 4.3.3 Mitochondrial DNA contamination Before performing whole genome sequencing, an attempt was made at minimising the sequencing of mitochondrial DNA thereby increasing sequencing of nucleic DNA (as the OR genes are nuclear), the nucleic was isolated first, then gDNA extracted from it. E. postvittana DNA prepared from nuclei contained less mitochondrial DNA than nuclear DNA based on semi-quantitative PCR of the mitochondrial and nuclear genes (Figures 4.3). The DNA prepared from nuclei was then used as a template for Illumina sequencing. Just to be cautious that the gDNA sample has minimal mtDNA contamination, one lane of Illumina sequencing was initially done on the gDNA. Bowtie, which is an ultrafast short read aligner (Langmead et al., 2009) was used for mapping the 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. The test parameters for bowtie allows up to only 3 mismatches. Only 92 out of the 13 million paired end reads were predicted to be mitochondrial, suggesting that mtDNA contamination is minimal. Another 7 lanes of Illumina sequencing was then done in order to obtain higher coverage of the genome and an attempt at assembling longer scaffolds for use as a sequence set able to be searched using blast.
Identification of putative odorant receptors from Epiphyas postvittana 103 850bp 650bp 1kb+ ladder Figure 4.3: Semi-quantitative PCR on nuclear (Takeout 3) and mitochondrial (cytochrome oxidase I) genes using total DNA extracted from purified nuclei as template. Lanes A to G show PCR performed on a 10 fold serial dilution of the DNA template from 1 in 100 to 1 in 100 million with E. postvittana Takeout 3 primers. Lanes H to N are PCR performed on the same template serial dilution with cytochrome oxidase I primers. 4.3.4 Illumina Sequencing The assembly of the 8 lanes of Illumina sequencing resulted in 299,968,860 bases being assembled into 843,963 scaffolds with the longest scaffold being 19,645 bp, with an N50 of 511 bp. The sequences were deposited into a privately held genome server at Plant and Food Research Ltd for blast querying and data mining. 4.3.5 Data Mining Nucleic DNA products Mitochondrial DNA products A B C D E F G H I J K L M N Tblastn of the E. postvittana 454 transcripts and genomic sequences with known B. mori OR sequences resulted in the identification of a further 49 putative ORs, bringing the total number of ORs identified from E. postvittana to 52. The ORs with shortest and longest predicted coding region lengths are EpOR49 (40 amino acids) and EpOR21 (475 amino acids) respectively. The full length of only one putative OR was identified (EpOR34). The highest level of amino acid identity shared between E. postvittana and B. mori ORs is 88% between EpOR2 and BmOR2. Interestingly, EpOR8 shares 71% amino acid identity with BmOR8, a larval-specific OR in B. mori.
Page 1 and 2:
http://researchspace.auckland.ac.nz
Page 3 and 4:
Abstract Abstract Olfaction or the
Page 5 and 6:
Acknowledgements Acknowledgements I
Page 7 and 8:
Contents v Contents Abstract……
Page 9 and 10:
Contents 4.2.5 Degenerate PCR .....
Page 11 and 12:
List of Figures List of Figures Fig
Page 13 and 14:
List of Tables List of Tables Table
Page 15 and 16:
List of Abbreviations gDNA Genomic
Page 17 and 18:
1.1 General overview 1 General Intr
Page 19 and 20:
General Introduction 3 moth in loca
Page 21 and 22:
General Introduction 5 Figure 1.1:
Page 23 and 24:
General Introduction 7 1.5 Componen
Page 25 and 26:
General Introduction 9 OBPs have al
Page 27 and 28:
General Introduction 11 A model has
Page 29 and 30:
Page 31 and 32:
General Introduction 15 Tanaka et a
Page 33 and 34:
General Introduction 17 Table 1.1:
Page 35 and 36:
Page 37 and 38:
General Introduction 21 et al., 200
Page 39 and 40:
General Introduction 23 the attract
Page 41 and 42:
General Introduction 25 leaves and
Page 43 and 44:
Page 45 and 46:
General Introduction 29 Expression
Page 47 and 48:
General Introduction 31 BmOr2 HvOr2
Page 49 and 50:
General Introduction 33 for lepidop
Page 51 and 52:
Functional characterisation of Epip
Page 53 and 54:
Page 55 and 56:
Page 57 and 58:
Page 59 and 60:
Page 61 and 62:
Page 63 and 64:
Page 65 and 66:
Page 67 and 68: 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
Page 95 and 96: 4.1 Introduction 4 Identification o
Page 97 and 98: Identification of putative odorant
Page 109 and 110: Microarray OR 454 Transcriptome OR
Page 117: Identification of putative odorant
Page 141 and 142: 5.1 Introduction 5 Concluding Discu
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
Page 159 and 160: Appendix E 143 V
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,
Page 181 and 182:
References 165 Pell, J. K., Macaula
Page 183 and 184:
References 167 Røstelien, T., Stra
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
show all

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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?