The development of specialized plants to either remove harmful metals from the environment or to maintain crop productivity and a safe food supply in agricultural plants growing in metal contaminated soils requires an understanding of the mechanisms of metal tolerance in plants. This understanding will provide traits which can be used to screen plants produced by conventional breeding strategies or which can be used to engineer plants with the desired traits. Before this work can be done, however, fundamental studies of metal-induced gene expression must be conducted to identify the genes and gene products involved in metal tolerance. I propose to systematically investigate gene expression in a metal-tolerant plant tissue culture and identify genes and gene products involved in metal tolerance. Differential display reverse transcriptase polymerase chain reaction will be used to study cadmium-induced gene expression in a metal-tolerant plant tissue culture. Total RNA isolated from metal tolerant Datura innoxia plant cell culture in the absence and presence of cadmium will be used for reverse transcriptase reactions followed by amplification by PCR. The products of the amplification will be resolved by polyacrylamide gel electrophoresis and side by side comparisons of the amplification products from the different samples will detect differentially expressed cDNA fragments. These cDNA fragments will be excised from the gels for amplification, cloned and sequenced. The sequences will be used to search nucleotide and peptide sequence libraries for possible identification of the genes or proteins represented by the sequences. Confirmation of differential expression will be done by Northern analysis. Full-length cDNAs for any unidentified cDNA fragments will be isolated, sequenced and identified. This work will provide information about differentially- expressed cadmium-induced genes or gene products in D. innoxia and position us to answer questions about the metal-specificity and plant species-specificity of the information obtained with this proposed research in the future.
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