选用品质相对不稳定的小麦品种济南17和品质较稳定的品种豫麦34，于开花后15~18 d（灌浆中期）及30~33 d（灌浆后期）分别进行连续3 d的高温胁迫处理（昼夜温度为38℃，25℃）。提取籽粒总RNA，通过cDNA-AFLP分析获得差异条带。回收差异条带，再经过克隆、测序、BLAST比对，济南17、豫麦34分别获得85个和99个高温胁迫下差异表达基因片段的序列。经过反向Northern杂交验证后，济南17获得25个信号明显的序列，其中22个来自热诱导表达的基因，主要与小麦的胁迫响应基因、热激蛋白等同源；豫麦34获得31个信号明显的序列，其中25个来自热抑制表达的基因，主要与乙烯合成酶、吡咯啉-5-羧酸合成酶等同源。说明高温胁迫总体上诱导品质不稳定品种的基因表达，而抑制品质稳定品种的基因表达。济南17的序列有15个来自灌浆中期样本、10个来自灌浆后期样本，豫麦34的序列则分别有29个来自灌浆中期样本、2个来自灌浆后期样本，说明高温胁迫对灌浆中期基因表达的影响比灌浆后期显著，在品质稳定品种中则比品质不稳定品种中更为明显。2个品种在高温胁迫下的基因表达模式存在显著差异，济南17诱导表达胁迫响应基因，豫麦34抑制表达乙烯合成酶和吡咯啉-5-羧酸合成酶及胁迫响应基因，这可能是二者品质稳定性不同的重要原因。
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.
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