Project Number: 6054-43440-052-03-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Sep 1, 2016
End Date: Aug 31, 2021
Evaluate genetic diversity of pecan cultivars to highlight potential hypoallergenic cultivars.
Samples of genetically diverse pecan cultivars will be processed to isolate and sequence genomic DNA or messenger RNA. Pecan allergen gene gene sequences including the 2S albumin Car i 1, the 11S legumin Car i 4, and the 7S vicilin will be evaluated using bioinformatics software analysis to identify conserved and heterogeneous segments of allergen protein sequences. Several alignment programs will be used to assess regions of consensus, conservation, or heterogeneity for the 2S (Car i 1), 7S (vicilin), and 11S (Car i 4) allergen proteins. Segments of gene/protein sequences of high conservation or those of high heterogeneity will be highlighted for closer inspection and comparison with published and predicted IgE binding sites and protein modeling. Published and predicted allergen IgE binding sites will be scrutinized to identify protein sequences or structures central to recognition by IgE. Protein modeling software will be used to generate protein structure homology-models and Chimera software will be used to visualize model proteins and highlight sequences of interest. Proteomic profiling of selected pecan nut cultivars based upon protein sequence will be analyzed by gel electrophoresis, immunoassay, and mass-spectrometry. Protein extractions will be performed using borate-based buffers and samples will be subjected to 1 or 2-D SDS-PAGE to compare protein accumulation. Liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS) of total extracts and specific spots from SDS-PAGE will be analyzed to identify/confirm differences in predicted protein sequence, expression level, and extraction efficiency. IgE binding assays will be used to correlate gene sequence and proteomic allergen data to differences in allergen gene sequence, gene expression, or protein level. Biochemical fractionation methods may be used to reduce nut extract complexity and allow immunoassay of a specifically targeted pecan nut allergen. The proteomic and immunological differences within pecan nuts from different cultivars can be correlated to other nut quality characteristics including polyphenol and lipid content among others.