目录
摘要I
关键词I
AbstractII
引言
引言
青枯菌与植物互作的研究1
1 植物先天免疫系统1
病原相关分子模式触发的免疫（PTI）1
效应因子触发的免疫（ETI）1
2 青枯菌致病因子2
2.1 III型分泌系统2
2.2 II型分泌系统2
2.3胞外多糖I2
3 青枯菌效应因子作用机制3
3.1激发HR3
3.2抑制植物的防御反应3
3.3破坏与植物防御相关的蛋白酶4
第二章 青枯菌效应因子Rip46功能和作用机制初探5
1 材料与方法5
1.1材料5
供试菌株5
供试植物5
载体5
常用培养基及其配制5
1.2克隆目的基因7
1.3构建表达载体8
1.3.1入门载体的构建8
1.3.2转化大肠杆菌DH5α8
1.3.3质粒提取9
1.3.4植物表达载体的构建9
1.4本氏烟瞬时表达效应因子10
1.5效应因子亚细胞定位11
1.6疫霉接种实验11
1.7敲除突变体致病性分析11
1.7.1构建敲除载体11
1.7.2青枯菌感受态制备12
1.7.3电转青枯菌13
1.7.4交换验证13
1.7.5敲除突变体接种番茄13
1.8酵母双杂交13
1.8.1检测DNABD融合毒性13
1.8.2文库与诱饵菌株交配14
1.8.3筛选表达互作蛋白的酵母二倍体14
2 结果与分析15
2.1效应因子Rip46克隆和表达载体构建15
2.2瞬时表达Rip46引起细胞死亡15
2.3效应因子Rip46的亚细胞定位16
2.4表达Rip46的烟草对寄生疫霉的抗性17
2.5青枯 *51今日免费论文网|www.51jrft.com +Q: ￥351916072$ 
菌Rip46敲除突变体致病性测定18
2.6鉴定青枯菌效应因子Rip46互作蛋白20
3 讨论21
致谢23
参考文献24青枯菌效应因子Rip46功能和作用机制的初步研究
摘要
茄科劳尔氏菌（Ralstonia solanacearum）是世界上分布最广、危害最严重的十大植物病原细菌之一，寄主范围广泛，可以侵染50多个科的400多种植物，导致多种重要作物的毁灭性病害。注射器状的III型分泌系统（typeIII secretion system, T3SS）分泌的III型效应因子（typeIII effectors, T3Es）是青枯菌致病的关键因子。青枯菌T3Es数目众多，它们可通过与植物蛋白互作干扰植物的免疫反应。目前仅有少数效应因子的功能被揭示，绝大多数效应因子的功能和作用机制尚不明确。本研究对青枯菌效应因子Rip46的功能和作用机制进行了初步探究。以青枯菌GMI1000菌株70个III型效应因子作为材料，通过农杆菌介导的瞬时表达在本氏烟上筛选到诱导细胞死亡的效应因子Rip46。亚细胞定位实验结果表明该效应因子定位于植物细胞质。在本氏烟中瞬时表达Rip46基因未见其促进寄生疫霉对植物的侵染。在青枯菌中敲除效应因子Rip46基因并评估突变体在番茄上的致病性，发现青枯菌效应因子Rip46敲除突变体在番茄上的致病性有所减弱，表明Rip46是一个毒性效应因子。最后通过酵母双杂交法筛选该效应因子在植物中的潜在互作蛋白，为今后进一步揭示Rip46的作用机制提供了基础，并为揭示青枯菌的致病机制提供线索。
A PRElIMINARY STUDY ON THE FUNCTION AND MECHANISM OF RAlSTONIA SOlANACEARUM EFFECTOR RIP46
ABSTRACT
Ralstonia solanacearum is one of the top ten most widely distributed and most seriously harmful plant pathogenic bacteria in the world. It has a very broad host range, and can infect more than 400 plants in more than 50 families, leading to the devastating diseases of many important crops. The type III effectors (T3Es)secreted by the syringeshaped type III secretion system(T3SS)are the key virulence factors in R. solanacearum. There are a large number of T3Es in R. solanacearum, and they can interfere with plant immune responses by associating with plant proteins. So far, only a few of T3Es’ functions have been revealed, and the function and mechanism of most effectors are still unclear. In this study, the function and mechanism of the R. solanacearum effector Rip46 were preliminarily investigated. Using 70 T3Es of R. solanacearum strain GMI1000 as materials, through the agrobacterium mediated instantaneous expression in Nicotiana benthamiana we screened the effector Rip46, which can cause cell death. Subcellular localization experiments indicated that the effector Rip46 was located in the cytoplasm of plants. The instantaneous expression of Rip46 gene in N. benthamiana did not promote the infection of Phytophthora parasitica on plants. Knocking out of Rip46 gene in R. solanacearum and evaluating the pathogenicity of the mutant in tomato, it was found that the pathogenicity of the mutant in tomato was weakened, suggesting that Rip46 was a virulent effector. Finally, the potential interaction proteins of this effector in plants were screened by yeast twohybrid method, which provided a basis for further revealing the mechanism of Rip46 and a clue for revealing the pathogenic mechanism of

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