To reveal the molecular mechanism of cold resistance of winter wheat variety Dongnongdongmai 1, under natural conditions in the north region of China, we collected tillering nodes before (5℃) and in winter season (-25℃) respectively, then constructed cDNA library of Dongnongdongmai 1 containing low temperature stress related genes by suppression subtractive hybridization. A total of 300 positive clones selected from cDNA library randomly were sequenced to obtain 230 high quality expressed sequence tags (EST). After BLAST and functional annotation, we found complex and diverse types of genes in the library, including stress response genes (such as heat shock protein, CS66, Wcor8, and other related genes) and genes encoding 40S and 60S ribosomal subunit. These genes with high frequency may play an important role in protecting Dongnongdongmai 1 from injuring under low temperature. Two unknown genes and Clp selected from the cDNA library were analyzed through quantitative PCR. The expression levels of these genes in Dongnongdongmal 1 were increased in various degrees with the decrease of temperature; however, their expression patterns were different in Jimai 22 (a winter wheat cultivar with low resistance to low temperature). This result suggests that these genes may play an important role in resistance to low temperature stress.%为揭示高抗寒冬小麦品种东农冬麦1号的抗寒分子机制,在北方寒地自然条件下,于越冬前(5℃)和越冬期(-25℃)分别对分蘖节取样,利用抑制消减杂交技术构建该品种低温胁迫相关基因的cDNA文库.在cDNA文库中随机挑选300个阳性克隆测序,获得230条高质量的表达序列标签(EST).对该序列进行BLAST比对及功能注释,发现文库中基因组成类型复杂多样,其中应对胁迫的基因(如热激蛋白、CS66、Wcor8等)以及参与编码60S和40S核糖体亚基的基因出现的频率较高,这些基因可能与东农冬麦1号的安全越冬有密切关系.对文库中筛选到的2种未知基因和Clp基因进行荧光定量PCR分析,发现其表达水平随着温度降低而不同程度升高;在弱抗寒性小麦品种济麦22中,这些基因则有不同的表达模式.推测这些基因在抗低温胁迫过程中发挥重要作用.
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