目录
摘要 II
关键词 II
ABSTRACT III
引言
1 文献综述 1
1.1小麦广谱抗白粉病Pm21基因 1
1.2鉴定Pm21基因发挥作用的过程 2
1.3钙信号转导途径基因参与调控Pm21 2
1.3.1 植物中的钙信号途径 2
1.3.2 钙信号转导途径参与调控Pm21基因介导的白粉病抗性相关研究 3
1.4.筛选互作基因的方法 3
1.4.1双分子荧光互补法研究蛋白互作的分子机制 3
1.4.2 酵母双杂交法研究蛋白互作的分子机制 3
1.4.3免疫共沉淀法研究蛋白互作的分子机制 4
1.5 本研究的目的和意义 4
2 材料与方法 5
2.1 实验材料 5
2.2 实验方法 5
2.2.1 簇毛麦CBL基因的克隆 5
2.2.2 簇毛麦CIPK上游启动子克隆 6
2.2.3 琼脂糖回收克隆产物 7
2.2.4 CBL3、CBL7、CBL10、CIPK基因的载体构建 7
2.2.5序列分析所用生物信息学工具 8
3 结果与分析 9
3.1 CBL基因的克隆 9
3.2 基因序列的分析 9
3.3 CBL蛋白结构分析 13
3.4 CIPK上游启动子序列克隆和序列分析 14
3.5 酵母表达载体的构建 15
4 总结与讨论 16
4.1 研究结论 16
4.2 讨论 16
致谢 17
参考文献 18
簇毛麦抗白粉病相关CBL基因克隆及载体构建
摘要
白粉病是小麦生长过程中对其产量和品质具有较大影响的病害之一，几乎在我国所有麦区均有发生，可侵染小麦整个生长周期。簇毛麦是小麦的近缘物种，含有丰富的抗病、抗逆等基因,是小麦育种的优异基因源。将小麦抗白粉病基因转入普通小麦中,培育小麦抗病新品种是小麦抗白粉病基因研究的重要目的之一。通 *51今日免费论文网|www.51jrft.com +Q: ^351916072# 
过筛选、克隆和利用抗白粉病基因的互作基因并开展基因的功能研究，对于解析其抗性的分子机制非常重要，也将为抗病基因的持久利用提供策略。本实验室前期克隆了簇毛麦广谱抗白粉病基因Pm21，其具有广谱的白粉病抗性，在不同小麦遗传背景下抗病性均表现稳定, 其编码一个CCNBSLRR类的抗病基因NLR1V,本研究前期通过利用酵母双杂交技术筛选到与NLR1V保守的CC结构域互作的CIPK基因片段，进而开展了互作基因的全长基因上游启动子序列克隆，以及多个上游CBL的克隆和相关表达载体的构建，为分析CBLCIPK互作关系和酵母中的回补验证，启动子特征和活性分析奠定基础，为解析该基因调控NLR1V基因介导的抗白粉病分子机制提供依据。
CLONING AND VECTOR CONSTRUCTION OF CBL GENE RELATED TO POWDERY MILDEW RESISTANCE OF HAYNALDIA
ABSTRACT
Powdery mildew is one of the serious diseases that have a great influence on the yield and quality of wheat production.It occurred in almost all the wheat regions in China and could infect the whole growth cycle of wheat.Haynaldia villosa is a wild relative species of wheat, which is rich in disease resistance and stress resistance genes, and is an excellent gene source of wheat breeding. It is one of the important purposes of wheat powdery mildew resistance gene research to transfer it into common wheat and cultivate new resistant wheat varieties. It is very important to screen, clone and utilize the interaction gene of powdery mildew resistance gene and carry out the function research of the gene, so as to analyze the molecular mechanism of its resistance and provide the strategy for the sustainable utilization of the resistance gene. Pm21, a broadspectrum powdery mildew resistance gene, was cloned in our laboratory. It encodes a CCNBSLRR resistance gene NLR1V, which showing a broadspectrum powdery mildew resistance and is stable in different wheat genetic background. In this study, the CIPK gene fragment interacting with conservative CC domain of NLR1V was screened by yeasttwohybrid technology, and then the fulllength gene sequences and promoter cloning of the interacting gene CIPK and its multiple upstream CBLs were carried out. It can provide the basis for the analysis of relationship of CBLCIPK and Y2H verification, and the characteristic of the promoter, which is help to elucidate the molecular mechanism of resistance to powdery mildew mediated by NLR1V gene.

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