Submitted to: Plant Molecular Biology International Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 7/15/2003
Publication Date: 9/1/2003
Citation: Cary, J.W., Rajasekaran, K., Bennett, C. 2003. A proteomics approach for the isolation of seed coat-specific genes from cotton. Plant Molecular Biology International Conference Proceedings. S5-90:93.
Technical Abstract: Aflatoxins are toxic and carcinogenic polyketide secondary metabolites produced by the filamentous fungus Aspergillus flavus during growth on crops such as corn, cottonseed, peanuts, and treenuts. Efforts are underway in our lab to develop transgenic cotton expressing genes for antifungal proteins and peptides that inhibit the ability of A. flavus to infect cottonseed and produce aflatoxins. We are interested in identifying novel promoter sequences of cotton genes that will control expression of these antifungal genes in a tissue-specific manner. We have shown that during invasion of cottonseed, A. flavus most often enters the seed at the chalazal end and ramifies along the inner seed coat prior to colonizing the lipid acid-rich cotyledons. Based on this observation we were interested in identifying cottonseed coat-specific genes and characterizing their promoters. Cotton bolls were harvested at 22, 29, and 36 dpa and the seed coat (SC) tissue separated from the cotyledonary (COT) tissues. Total protein was extracted from these tissues and subjected to 2-dimensional electrophoresis. Three proteins that were observed to be either unique to SC or expressed at significantly higher levels than observed in COT were chosen for protein sequence analysis. The three proteins were digested with trypsin and the peptide sequences determined by mass spectroscopy. A search of the protein databases with the peptide sequence data showed that the three proteins shared homology with deduced amino acid sequences from Gossypium arboreum genes encoding glutathione-S-transferase (GST), 2-nitropropane dioxygenase (2-npd), and a fruit-ripening factor. Utilizing PCR primers based on Gossypium arboreum and Arabidopsis thaliana DNA sequences the genes encoding the GST and 2-npd were amplified from cotton genomic DNA. Results of Southern hybridization for gene copy number and Northern hybridization analysis of RNA from various cotton tissues for expression patterns of GST and 2-npd will be discussed.