关键词: 普通小麦, 灌浆期, 高温胁迫, cDNA-AFLP, 基因表达, 品质稳定性

Abstract:

Studies of gene expression patterns under heat stress during grain filling stage will provide important information for breeding wheat cultivars with high quality. In the present study, two wheat cultivars, Jinan 17 and Yumai 34 with different quality stability under various environments, were used to in the influence of high temperature on gene expression. The wheat plants were exposed to high temperature (38℃/25℃ day/night) for three days in the middle (from 15 to 18 days post-anthesis) and late stage (from 30 to 33 days post-anthesis) of grain filling in a climate chamber. Spikelets in middle of heads were harvested, and RNA of kernels was extracted with a combined technique of cold phenolic and Trizol single-step methods. cDNA was obtained by the reverse transcription of total RNA, and differential bands were detected subsequently in cDNA-AFLP analysis. In total, 410 and 316 differential bands were detected from Jinan 17 and Yumai 34, respectively. The differential fragments were cloned, sequenced and blasted in NCBI, and 85 and 99 positive fragments of differentially expressed genes under heat stress were obtained from Jinan 17 and Yumai 34, respectively. After the positive fragments were validated by reverse Northern blotting, 25 positive fragments isolated from Jinan 17 showed intense signal, and 22 of them were induced under heat stress, which were notablely homologous to stress response genes and heat shock protein of wheat. Meanwhile, 31 positive fragments showed intense signal were observed from Yumai 34, and 25 of them were suppressed, which were notablely homologous to stress response genes, ethylene forming enzyme, pyrroline-5-carboxylate synthetase. The rusults 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 tolerance and quality stability. Five fragments from Jinan 17, and two fragments from Yumai 34 did not have any homology sequences in the BLAST analysis, while other fragments have homology protein or nucleic acid sequences in wheat or other crops. Two fragments induced 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 heat stress. Fifteen differential fragments were detected from medium stage of grain filling in Jinan 17, whereas those from late stage were 10, while 29 and 2 differential fragments in Yumai 34 were observed in medium and late stages, respectively. This indicated that gene expression was more significantly affected by heat stress in medium stage than in late stage of grain filling, especially in Yumai 34. The difference of gene expression patterns between two wheat cultivars were observed, stress response genes were induced in Jinan 17, ethylene forming enzyme and pyrroline-5-carboxylate synthetase as well as stress response genes were suppressed in Yumai 34, which may result in the different responses in heat tolerance and quality stability. The identification and characterization of heat stress responsive genes in wheat may provide a molecular biological understanding of gene expression patterns and regulation involved in the heat stress in wheat.

Key words: Common wheat (Triticum aestivum L.), Grain filling period, Heat stress, cDNA-AFLP, Gene expression, Quality stability