title: Identification of cold-responsive genes in a New Zealand alpine stick insect using RNA-Seq.
abstract: The endemic New Zealand alpine stick insect Micrarchus nov. sp. 2 regularly experiences sub-zero temperatures in the wild. 454-based RNA-Seq was used to generate a de novo transcriptome and differentiate between treatments to investigate the genetic basis of cold tolerance. Non cold-treated individuals were compared to those exposed to 0°C for 1 h followed by a 1 h recovery period at 20°C. We aligned 607,410 Roche 454 reads, generating a transcriptome of 5235 contigs. Differential expression analysis ranked candidate cold responsive genes for qPCR validation by P-value. The top nine up-regulated candidates, together with eight a priori targets identified from previous studies, had their relative expression quantified using qPCR. Three candidate cold responsive genes from the RNA-Seq data were verified as significantly up-regulated, annotated as: prolyl 4-hydroxylase subunit alpha-1 (P4HA1), staphylococcal nuclease domain-containing protein 1 (snd1) and cuticular protein analogous to peritrophins 3-D2 (Cpap3-d2). All three are novel candidate genes, illustrating the varied response to low temperature across insects.
authors.affiliation: Landcare Research, Auckland, New Zealand. Dunningl@landcareresearch.co.nz
authors.forename: Luke T
authors.initials: LT
authors.lastname: Dunning
authors.forename: Alice B
authors.initials: AB
authors.lastname: Dennis
authors.forename: Duckchul
authors.initials: D
authors.lastname: Park
authors.forename: Brent J
authors.initials: BJ
authors.lastname: Sinclair
authors.forename: Richard D
authors.initials: RD
authors.lastname: Newcomb
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Buckley
mesh_terms: D000818:Animals; D057773:Cold Shock Proteins and Peptides / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D019476:Insect Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D007313:Insecta / Q000235:genetics* / Q000378:metabolism; D015233:Models, Statistical; D009520:New Zealand; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome / Q000235:genetics*
chemical_list: D057773:Cold Shock Proteins and Peptides; D019476:Insect Proteins; D012333:RNA, Messenger