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为了解小麦响应水分胁迫后复水条件下的基因表达特征,以小麦(Triticum aestivum L.)主栽品种陕229的3叶1心期幼苗为材料,采用消减杂交技术,构建了干旱复水条件下的SSH-cDNA表达文库。从消减文库中随机挑选59个插入片段大于400bp的阳性克隆测序,去除冗余序列和嵌合序列后,获得高质量EST序列32条(GenBank登录号为ES466767~ES466798)。序列比对分析表明,小麦干旱后复水的基因表达与植物对其他非生物胁迫的响应具有交叉性;17条EST序列与已知编码蛋白的基因同源性较高,涉及植物的信号传导、能量代谢、转录调控等方面;其他序列为新的EST。以其中一个与乙烯受体基因(ERS)同源性较高的EST序列为基础,采用同源克隆及RACE技术从小麦中分离了4个乙烯受体基因的全长cDNA序列,长度分别为2090、2271、2216和1886bp。4个全长cDNA序列所编码氨基酸序列的同源性极高(99%以上),且均具有ERS典型的GAF、HisKA和HATPase-c跨膜结构,分别命名为TaERS1(HM347272),TaERS2(HM601437),TaERS3(HM601438)和TaERS4(HQ111523)。植物ERS氨基酸序列多重比较表明,小麦与水稻的相似性最高(93%);TaERS基因的全长cDNA序列间比较共发现SNP位点23个。定量PCR表达分析显示,小麦TaERS家族基因参与了小麦植株响应水分胁迫和复水的调控机制。研究结果有助于进一步认识小麦干旱后复水的基因表达谱和小麦水分高效利用机制,探索小麦乙烯受体在水分高效利用中的作用。
In order to understand the gene expression characteristics of wheat in response to water stress after rewatering, the seedlings of 3-leaf and 1-heart of a wheat (Triticum aestivum L.) cultivar Shaan 229 were used as materials to establish a drought rehydration condition Under the SSH-cDNA expression library. Fifty-nine positive clones with insert sizes greater than 400bp were randomly selected from subtractive libraries. After removing redundant and chimeric sequences, 32 high-quality ESTs were obtained (GenBank accession numbers ES466767 to ES466798). Sequence alignment analysis showed that the gene expression of rehydration after drought in wheat was cross-resistant to other abiotic stress responses. The 17 ESTs shared high homology with known proteins and involved in plant signal transduction, Energy metabolism, transcriptional regulation and other aspects; other sequences for the new EST. Based on the EST sequence which has high homology with ethylene receptor gene (ERS), the full-length cDNA sequence of four ethylene receptor genes was isolated from wheat by homologous cloning and RACE, the length of which was 2090 , 2271, 2216 and 1886 bp. The sequences of the four full-length cDNAs showed high homology (more than 99%) with the homology of GAF, HisKA and HATPase-C of ERS, named TaERS1 (HM347272), TaERS2 (HM601437 ), TaERS3 (HM601438) and TaERS4 (HQ111523). Multiple comparison of the amino acid sequences of plant ERS showed that the similarity between wheat and rice was the highest (93%). A total of 23 SNPs were found in the full-length TaERS cDNA sequences. Quantitative PCR analysis showed that the wheat TaERS family of genes is involved in the regulation of wheat plants in response to water stress and rehydration. The results will help to further understand the gene expression profile of rehydration wheat after drought and the mechanism of efficient use of water in wheat to explore the role of ethylene receptors in the efficient use of water in wheat.