Isolation Of Differentially Expressed Genes Under Heat Stress During Grain Filling Period In Winter Wheat

Wheat quality traits consistency improvement, breeding high yield and qualitycultivars with good adaptability are important breeding objectives in China. Heat stressduring grain filling period is one of enviroment factors affecting quality consistency; itcan badly affect wheat yield and quality and has come to be primary physiologicalobstacle to improving wheat yield and quality in Chinese north area. For the momentdominating studies of heat stress during grain filling period in wheat were about yield,quality, physiology and biochemistry, but studies of gene expression, especially of thepatterns of wheat seeds gene expression under heat stress were rarely reported. Thestudying actuality of heat stress during grain filling period restricts quality consistencygenetical improvement. Studies of gene expression patterns under heat stress during grainfilling stage will provide important information for breeding wheat cultivars with highquality.In the present study, two wheat cultivars, Jinan 17 and Yumai 34 with differentquality consistency under various environments, were used to study the influence of hightemperature on gene expression. They were exposed to high temperature (38/25℃day/night) for three days in middle stage of grain filling (from 15 to 18 days post-anthesis)and late stage (from 30 to 33 days post-anthesis) in a climate chamber. Spikelets inmiddle of heads were harvested, and RNA of kernels was isolated with a combinedtechnique of cold phenolic and Trizol single-step methods, cDNA was obtained by thereverse transcription of total RNA, and differential bands were detected in subsequentcDNA-AFLP analysis. In total, 410 and 316 differential bands were detected from Jinan17 and Yumai 34, respectively. The differential fragments were cloned, sequenced andblasted in NCBI, and 85 and 99 positive fragments of differentially expressed genesunder heat stress were obtained from Jinan 17 and Yumai 34, respectively. After the positive fragments were validated by reverse Northern blotting, 25 positive fragmentsisolated from Jinan 17 showed intense signal, and 22 of them were induced under heatstress, which were notablely homologous to stress response genes and heat shock proteinof wheat. Meanwhile, 31 positive fragments showed intense signal were observed fromYumai 34, and 25 of them were suppressed, which were notablely homologous to stressresponse genes, ethylene forming enzyme, pyrroline-5-carboxylate synthetase. Theresults indicated that gene expression was more induced under heat stress in Jinan17,whereas suppressed more in Yumai 34, which might lead to differences in heat toleranceand quality consistency. Five fragments from Jinan 17 and two fragments from Yumai 34did not have any homology sequences in the BLAST analysis, while other fragmentshave homology protein or nucleic acid sequences in wheat or other crops. Two fragmentsinduced in response to heat stress were notablely homologous to the storage protein genes,which might induce the expression of transcripts related to storage protein under heatstress. Fifteen differential fragments were detected from medium stage of grain filling inJinan 17, whereas those from late stage were 10, while 29 and 2 differential fragments inYumai 34 were observed in medium and late stages, respectively. It indicated that geneexpression was more significantly affected by heat stress in medium stage of grain fillingthan late stage, especially in Yumai 34.The difference of gene expression patterns between two wheat cultivars wereobserved, stress response genes were induced in Jinan 17, ethylene forming enzyme andpyrroline-5-carboxylate synthetase as well as stress response genes were suppressed inYumai 34, which may result in the different responses in heat tolerance and qualityconsistency.The identification and characterization of heat stress responsive genes in wheat mayprovide a molecular biological understanding of gene expression patterns and regulationinvolved in the heat stress in wheat. This study can provide useful information about heattolerance and quality consistency of wheat.