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<UL> <LI>Clone gene from Tex6 coding for a chitinase that is inhibitory to growth of A. flavus. <LI>Produce recombinant chitinase in a bacterial expression system to confirm that the corresponding gene encodes the inhibitory activity. Use the recombinant chitinase to determine the organ specificity and abundance of the protein in corn.<LI> Develop a specific probe from the sequence of the gene that can be used by researchers working on resistance to aflatoxin contamination for mapping resistance in corn populations.
Approach: A partial amino acid sequence for the purified chitinase will be used to design a series of nested degenerate primers to amplify a DNA fragment unique to the Tex6 chitinase. Positive clones will be characterized by sequencing. The cloned gene will be over-expressed in bacteria, purified, and tested for activity. The organ specificity and abundance of the protein in corn will be determined by co-migration of extracted protein with the recombinant protein. To develop a specific probe from the sequence of the gene that can be used to follow resistance in corn to aflatoxin accumulation, we will design highly specific primer pairs of at least 20 bp each to target the unique sequences of the coding region and 5' and 3' untranslated regions of the gene. The primer pairs will be used to amplify the chitinase gene sequence with PCR, and the parents of several mapping populations will be screened with these primers to identify amplification product length polymorphism that can be mapped. In a new initiative, A. flavus microarrays containing the 5,200 expressed gene sequences will be analyzed for identification of specific genes involved in plant-fungal interactions, as well as those expressed under conditions conducive to aflatoxin production in the fungus.