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摘要：大豆疫霉引起的大豆根茎腐病是大豆的毁灭性病害之一，严重威胁全世界的大豆生产。在侵染植物的过程中，大豆疫霉会分泌大量的效应分子进入寄主体内干扰植物免疫，促进自身侵染。因此，深入探究效应分子干扰植物抗病性的机理，明确大豆疫霉致病的途径以及作用在寄主植物的靶标位点，可以为有效的病害防控策略提供新的思路及方向，同时对设计和改造抗病性有重要意义。本文探究了大豆疫霉效应分子Avh241的功能，通过筛选其寄主靶标，并从中选择候选互作蛋白，利用Co-IP和GST Pull-Down两种技术对其进行了互作验证，成功筛选到Avh241的互作蛋白GmHin1。该研究为进一步理解植物与病原菌的互作机制提供了理论依据。34617
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毕业论文关键词：效应分子；Avh241；寄主靶标；蛋白互作；Co-IP；GST Pull-Down
Identification of Host target of Phytophthora sojae effector Avh241
Abstract： Phytophthora sojae causes the Phytophthora root and stem rot disease of soybean. P. sojae delivers varieties of effectors into host cell to reprogram host immunity during infecting host plant. Therefore, it’s significant to learn the mechanism of how effectors interfere plant immunity and the way that P. sojae infect the host plant, which could provide guidance about control methods and strategies of plant diseases. Avh241 is one of the RxLR effectors from P. sojae, which can promote P. sojae infection on its host plant. Our research studies analyzes the function of Avh241. In this study, we screen out the host target protein which may interact with Avh241 and select one candidate interacting protein of Avh241 to do the research. We also provide the interaction between Avh241 and candidate interacting protein by Co-IP and GST Pull-Down. Our result is that we find an interacting protein named hin1 and demonstrate that it can interact with Avh241.
Key words: effector；Avh241；Host target；Interacting protein；Co-IP；GST Pull-Down
目 录
摘要 1
关键词 1
Abstract 1
Key words 1
引言 1
1 材料与方法 3
1．1 供试植物和供试菌株的保存和培养 3
1．2 目标基因的克隆及重组质粒的构建 3
1．2．1 目的基因的克隆 3
1．2．2 重组质粒的构建 3
1．3 大肠杆菌JM109感受态细胞的制备和转化 4
1．3．1 大肠杆菌JM109感受态细胞的制备 4
1．3．2 热击转化大肠杆菌JM109感受态细胞 4
1．4 大肠杆菌质粒提取 5
1．5 农杆菌GV3101感受态细胞的制备和转化 5
1．5．1 农杆菌GV3101感受态细胞的制备 5
1．5．2 电击转化农杆菌GV3101感受态细胞 5
1．6 目标基因的植物表达 6
1．6．1 注射烟草用缓冲液的配制(50 mL) 6
1．6．2 注射烟草用缓冲液悬浮农杆菌 6
1．6．3 注射烟草 6
1．7 植物蛋白的提取 6
1．8 蛋白样品Western 验证 7
1．9 蛋白互作验证 7
1．9．1 植物体内免疫共沉淀技术（Co-IP） 7
1．9．2 蛋白质Pull-Down技术 8
2 结果与分析 9
2．1 重组质粒pBinGFP-Avh241的构建 9
2．2 候选互作蛋白筛选结果 10
2．3 Avh241候选互作蛋白的克隆及重组质粒的构建 11