Monday, December 10, 2007 - 9:53 AM

Characterization of seven new proteins from type-I lepidopteran peritrophic matrix

Umut Toprak, umt968@mail.usask.ca1, Martin Erlandson, erlandsonm@agr.gc.ca1, Cedric Gillott, cedric.gillott@usask.ca2, and Dwayne Hegedus, hegedusd@agr.gc.ca1. (1) Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK, Canada, (2) University of Saskatchewan, Department of Biology, 112 Science Place, Saskatoon, SK, Canada

The peritrophic matrix (PM) performs several functions including the compartmentalization of digestion, the regulation of nutrient uptake and as a barrier to pathogen infection; however, many aspects of its structure and function remain poorly defined. To date, only 5 PM proteins have been identified from Lepidoptera; insect intestinal mucins and peritrophins with either multiple chitin binding domains or a chitin deacetylase-like domain. We employed a genomics and proteomics approach to survey the proteins expressed by the midgut epithelium that associate with the Mamestra configurata (bertha armyworm) PM. Our approach has been to couple 1 and 2-dimensional gel separation of PM proteins with MALDI-mass spectrometry to generate de novo sequence information for specific PM proteins. In parallel, ca. 3900 ESTs were generated from a midgut cDNA library yielding a database of genes expressed in the midgut. 5` RACE (Rapid Amplification of cDNA Ends) was performed to obtain the full-length sequences. Transcription size for each protein was confirmed by northen blot analysis and each target protein was examined in PM, larval carcas and midgut by western blot analysis with antibodies developed from conjugated peptides specific to the amino acid sequences of each protein. Three new insect intestinal mucins (IIM), three small peritrophins with small chitin binding domains and a protein with a chitin deacetylase-like domain were identified. This research will ultimately lead to the identification of proteins that can serve as targets for “designer” insect control molecules, as well as to further our understanding of insect midgut physiology and digestive biochemistry.

Species 1: Lepidoptera Noctuidae Mamestra configurata (bertha armyworm)