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The research objectives of this project include the characterization of RAPs to assess their role in resistance. Genes corresponding to RAPs will be cloned, and gene expression studied at different stages of kernel development and under normal, stress, and infected conditions. The identity of regulatory genes will be sought, as will be the association of specific alleles with resistance. Completion of these objectives should enhance the development of resistant corn lines through marker-assisted breeding and/or genetic engineering. This would save growers millions of dollars per year, and improve the safety of feeds and food.
Clone the corresponding genes encoding resistance-associated proteins (RAPs). Study biochemical and physiological role(s) of RAPs in kernel resistance. Examine the expression of RAP genes during kernel development. Study the polymorphism of these genes to see whether there is a particular allele associated with host resistance. Perform promoter analysis of RAP genes and identify possible regulatory genes using the yeast one-hybrid system.
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Resistance-associated genes will be cloned using PCR and RACE methods with degenerate primers corresponding to the peptide sequences. The expression of these genes in resistant and susceptible genotypes during kernel development under normal and stress conditions will be examined using northern or real time PCR. The corresponding genes will be overexpressed in E. coil for antibody production, in vitro biochemical and antifungal studies, and also will be expressed in tobacco for testing their biological functions relating to antifungal and stress resistance. The promoters of these genes will be analyzed to identify a common motif, which will then be used in the yeast one-hybrid system to identify the master regulatory progein/gene(s).
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The contamination of corn with aflatoxins, toxic and highly carcinogenic metabolites produced by the fungus Aspergillus flavus after infection, has caused a loss of millions of dollars to U.S. growers, and has been linked to health problems in humans and domestic animals. In the past two decades, several corn lines have been identified as resistant to aflatoxin contamination. However, it has been difficult to transfer this resistance into commercial corn lines, because no specific traits related to resistance have been identified. Recently, however, kernel proteins have been discovered using proteomics technology, that are either unique, or expressed at higher levels in resistant compared to susceptible lines. That some of these resistance-associated proteins (RAPs) are stress-related, adds to the potential importance of these discoveries, since drought-stress dramatically increases aflatoxin levels.