Title: Embryonic transcriptome analysis of the Caribbean fruit fly, Anastrepha suspensa Authors
|Schetelig, Marc Florian|
|Xavier, Nirmala -|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 10, 2010
Publication Date: N/A
Technical Abstract: The embryonic transcriptome of the Caribbean fruit fly, Anastrepha suspensa, was sequenced by 454 pyrosequencing in an effort to isolate embryonic promoters and genes involved in programmed cell death. A cDNA library was constructed from total RNA pooled from various time points in embryogenesis using the SMART cDNA synthesis kit (BD Biosciences), normalized using the Kamchatka crab duplex-specific nuclease (Evrogen), titrated and sequenced on a 454-FLX platform. To specifically isolate genes promoting programmed cell death, a second transcriptome was constructed from total RNA isolated from embryos treated with gamma-irradiation known to promote premature cell death. In total, 58 million bases were obtained from 342,052 reads with an average length of 200 bp that formed 16,288 contigs ranging from 91 to 1,019 nt in length and 313,668 singlets. In vitro functional assays were performed on genes selected from the cell death-induced library. Putative A. suspensa cognates to the Drosophila hid and reaper pro-apoptotic genes were isolated and tested. These genes were also modified to include or delete binding and regulatory regions known to be important to Drosophila gene function, and were comparatively tested with their Drosophila homologs to verify function. Sequence information from the ongoing Medfly whole genome sequencing project also will be used to identify and compare cell death genes between different tephritid species. Those shown to be most effective may then be tested for use as lethal-effectors in pest species to develop conditional lethality strains for use in new biologically-based control strategies.