Plant & Animal Genome IX Conference

Poster: Sequencing & EST
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IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES UNDER HEAT STRESS IN WHEAT

Plant Molecular Genetics Lab, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX 79409-2122, USA

Heat stress causes large production losses to the wheat worldwide. Characterization of environmental stress induced genes can provide a better knowledge about the underlying molecular mechanisms of plant responses to heat stress and lead to the development of strategies for addressing this problem. Our objective is to analyze functional genomics of heat stress and identify differentially expressed genes in wheat. Winter wheat cultivar Mustang was chosen for this purpose. Two-week old seedlings were heat shock (HS) treated at 38 centigrade for 2 hours followed by 2 days of diurnal 2-h treatments at 38 centigrade. Total RNA were isolated from samples of the first 2-h long treatment and the second day of diurnal treatments and pooled together. PolyA mRNA was purified using magnetic beads (Promega). Three heat-stressed cDNA libraries were made: (1) non-subtracted cDNA library made with the Uni-ZAP vector system (Stratagene); (2) subtracted cDNA library made with the method of library subtraction; and (3) suppression subtracted cDNA library made with suppression subtraction hybridization (SSH) system (Clonetech). Three thousand random cDNA clones from the above three libraries (1,000 each) were end-sequenced with CEQ 2000 sequencer in our lab, producing a database of expressed sequence tags (ESTs). Our results indicate that suppression subtraction hybridization (SSH) is a powerful technique for rapidly identifying differentially expressed genes. Results on the efficiency of library subtraction will be presented. The BLASTN homology search analysis using heat-stressed ESTs was conducted and many putative gene homologs as well as novel sequences were identified. Experiments for functional analysis of novel genes are in progress.