【作者】 彭丹辉；

【导师】 彭惠茹；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2005， 硕士

【摘要】 小麦是我国重要的粮食作物。但随着温室效应的加剧,全球平均气温的不断升高,高温已经成为危害我国小麦生产的重要因素,每年造成巨大的经济损失。因此,开展小麦的耐热性研究对我国的农业生产具有重要的理论和现实意义。本研究的目的是通过对小麦苗期热胁迫前后基因表达谱的分析来揭示小麦热胁迫响应的分子机理,并分离相关基因,为通过基因工程手段提高小麦耐热性奠定基础。 以38℃热胁迫的小麦幼苗为处理组(HS),同期未经处理的小麦幼苗为对照组(CK),分别与Affymetrix Barley1基因芯片杂交。杂交结果显示在总共22840个探针中,对照组(CK)和热胁迫组(HS)共检测到8486个探针,占总探针数的37.2%。采用半定量RT-PCR和RealTimeRT-PCR的方法对12个基因进行验证,其中11个与芯片的结果一致,表明杂交结果真实可信。 对杂交结果的分析表明,共有892个探针发生了差异表达,其中469个表现为热胁迫后上调,423个表现为热胁迫后下调。差异表达的基因涉及到逆境胁迫、信号转导、物质运输、光合作用、蛋白代谢、脂肪代谢、碳水化合物代谢等多个方面。其中有大量热激蛋白和分子伴侣基因上调表达。上调表达的热激蛋白涉及到热激蛋白各个家族,且上调表达的平均倍数明显高于其它家族基因的上调倍数。另外泛素-蛋白酶体通路中的多个关键酶的基因也发生了差异表达,预示着负责蛋白质选择性降解的泛素-蛋白酶体通路也参与到了植物对热胁迫的应答机制。 选取芯片中的两个热胁迫后上调表达的基因,利用电子克隆方法,获得了它们具有完整ORF的序列,分别命名为TaCDPK和TaPK,TaCDPK推测的氨基酸序列具有钙依赖蛋白激酶(CDPK)典型的EF手性结构和丝氨酸/苏氨酸蛋白激酶催化域,N端有一段豆蔻酰化位点;TaPK推测的蛋白具有典型的丝氨酸/苏氨酸蛋白激酶催化域,N末端具有两段核定位信号。更多还原

【Abstract】 Wheat is one of the most important cereal crops in China. In recent years, the global average temperature has increased due to the enhanced "Greenhouse Effect", heat stress has come to be one of the serious climatic threats for wheat production and causes severe economic losses. However, the molecular basis of crops adaptation to high temperature is not completely characterized. The aim of this study is to analyze the gene expression profile of wheat in response to elevated temperature and isolate the genes induced by heat stress.To define wheat molecular response to elevated growth temperatures, gene expression profiles were examined subjected, to heat stress by using Affymetrix 22k Barley 1 Genechip. cRNA derived from heat treated wheat seedling (38℃ for 1 hour,HS) and untreated wheat seedling(CK) were used to hybridized two chips respectively. As a result, 8486 probe sets produced present calls in those two chips, covered 37.2% of total 22840 probe sets. Among them approximately 10.5% (n=892) probe sets were significantly up or down regulated (ratal >=2 and p-value < 0.05).Semi-quantitative RT-PCR and Realtime RT-PCR were used to verify the hybridization result. Among the 12 genes we have examined, 11 genes exhibited the similar results with the chips. So we believe that the chips result is credible.Analysis showed the differentially expressed genes involved in stress response, signal transduction, transport, photosynthesis, protein metabolism, lipid metabolism and carbohydrate metabolism etc. As expected, a large number of the genes homologous to known chaperones and heat shock proteins in other organisms were highly induced. In addition, several predicted genes including those encoding key enzymes in Ubiquitin-Proteasome pathway had differentially expressed.The complete ORF of two heat-induced genes were cloned by in silico cloning method. Bioinformatic analysis showed that one sequence is encoding a calcium depended protein kinase (CDPK) and another is encoding a Ser/Thr protein kinase. In addition one myristoylation site was detected in the N-terminal of the predicted CDPK and two nuclear targeting signal sequences was found in the N-terminal of the predicted Ser/Thr protein kinase. This result indicate those CDPK and Ser/Thr protein kinase would be anchored to membrane and sorted to nuclear after translation respectively.更多还原