目录
摘要Ⅰ
关键词Ⅰ
AbstractⅡ
引言
引言
1材料与方法1
1. 1实验材料 1
1.1.1 植物材料1
1.1.2 菌株材料1
1.2实验方法 1
1.2.1大肠杆菌转化1
1.2.2农杆菌感受态的制备2
1.2.3植物表达载体的构建与烟草中蛋白瞬时表达3
1.2.4 Avr3b的亚细胞定位及Western验证4
1.2.5辣椒疫霉侵染5
2结果与分析5
2.1 Avr3b植物中候选互作蛋白的筛选6
2.2 Avr3b与植物MAPK13蛋白互作6
2.2.1 Avr3b与植物MAPK13蛋白体内CoIP互作6
2.2.2 Avr3b与植物MAPK13蛋白体外Pulldown实验6
2.2.3 Avr3b与植物MAPK13蛋白在植物中的共定位情况7
2.3 大豆MAPK13蛋白初步功能研究8
2.3.1 大豆MAPK13蛋白引起烟草组织的坏死8
2.3.2 大豆MAPK13蛋白正调控植物免疫反应9
2.4 Avr3b抑制植物MAPK13蛋白在烟草上引起的细胞坏死9
3讨论10
致谢10
参考文献12
附录1 Buffer Inoue及BufferA、B、C的配置体系16
附录2 菌落PCR反应体系16
附录3 农杆菌悬浮缓冲液配置体系16
大豆疫霉RxLR效应分子Avr3b毒性靶标的筛选与研究
摘要
大豆疫霉是典型的土传病原菌，其引起的根腐病每年都会导致全球大豆生产损失严重。在侵染寄主的过程中，大豆疫霉分泌效应分子进入寄主体内，影响植物的免疫反应并促进疫霉菌侵染。在大豆疫霉基因组中发现了300多个RxLR效应分子，其中有9个RxLR基因是大豆疫霉的无毒基因。Avr3b是其中一个无毒效应分子。Avr3b的羧基端含有一个Nudix结构域，具有Nudix活性，能够抑制寄主体 *51今日免费论文网|www.51jrft.com +Q: ^351916072^ 
内活性氧的积累。已有的研究表明Nudix结构对大豆疫霉能否发挥自身的全部毒力是必须的。Nudix结构域的重要氨基酸发生突变，不仅会影响Avr3b的Nudix活性，甚至会导致大豆疫霉丧失侵染寄主的能力。此前的研究虽已知效应分子Avr3b的部分功能及作用机制，但其在植物中的毒性靶标仍未找到。本实验在植物体内筛选出Avr3b的靶标蛋白植物MAPK13蛋白，并对其功能以及Avr3b和蛋白间的关系进行了研究，进一步加深对大豆疫霉与寄主的互作机理的理解，揭示效应分子Avr3b抑制植物免疫的机制，以寻找更加高效的防治策略, 更有助于大豆疫霉引起的植物根腐病的防控，为生产提供指导。
SCREENING THE HOST TARGETS OF PHYTOPHTHORA SOJAE RXLR EFFECTOR AVR3B
ABSTARCT
Phytophthora sojae causes the soybean root rot diseases, which lead to serve damages on soybean production all over the world. It can influence soybean’s immunity system and enhance its pathogenicity by secreting effectors into host cells. There are over 300 RxLR effectors in the genome of P. sojae. Among these effectors, nine have been proved to be avirulent effectors. Avr3b is one of the avirulent effectors. Moreover, Avr3b is one of the few effectors that have enzyme activity domain. Avr3b’s carboxyl terminal includes a Nudix motif which makes it has hydrolytic enzyme active and can inhibit the accumulation of reactive oxygen in hosts. This Nudix motif is necessary for the virulence function of P. sojae. When Nudix motif mutation occurs to some important amino acids, it not only influences the activity of Avr3b, but also leads to Phytophthora sojae losing infestation ability. From the existing researches, we have known part of the function and mechanism of Avr3b. However, Avr3b’s virulence targets in plants remain unknow. In our experiment, we identified the target protein, GmMAPK13, and studied its function and the relationship between Avr3b and GmMAPK13 to understand the mechanism between the host and Phytophthora sojae so that we can reveal how Avr3b suppresses plant’s immune system. We hope that we could find out more efficient prevention strategies to control the plant root rot diseases which are caused by Phytophthora sojae and provide guidance for production.

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