Confirmation of Differential Expression of Selected Genes Expressed Under High Temperature Stress in Wheat by Real-Time PCR.

Real-time PCR allows quantification of starting amount of messenger RNA in real-time.  Real-time PCR can compare mRNA levels of a gene of interest between different experimental conditions, and thus can confirm expression level of that gene. A microarray gene expression study was carried out in wheat under high temperature stress conditions to find genes related to long-term heat stress tolerance in wheat. The objectives of the present study were confirmation of the expression of selected genes potentially related to heat tolerance. A subset of 14 ESTs was selected from microarray experiments based on the significant expression level of those genes under heat-stress conditions in two sampling dates. Leaf tissues were collected at 4, 7, and 10 days from both heat-treated and control plants. A total of 48 cDNA samples (24 from offspring and 24 from parents) were used in the study. House keeping gene   β-actin was used to adjust the concentration of cDNA, and a dilution series was used to estimate primer efficiency. Six different genes, namely Rubisco activase, IP3 kinase B, a Zinc finger protein, a Leucine Rich Repeat protein, a RNA binding protein, an auxin efflux carrier protein, showed consistently higher expression in tolerant lines. On the other hand, thaumatin-like protein, Abscisic acid-inducible protein kinase, Pheophorbide a oxygenase, Clp protease, and Papain family cysteine protease were consistently higher expressed in sensitive lines. Results from Real-Time PCR confirmed their differential expression. The differential expression of those genes may be attributed to genotypic variations in response to heat stress. The up-regulated genes in the tolerant lines might have potential to be related to the heat tolerance, while the genes up-regulated in sensitive lines could be potentially related to rate limiting factor of senescence under heat stress conditions.