番茄丁香假单胞菌抗病基因Pto互作蛋白功能分析

发布时间：2018-04-13 16:42

本文选题：丁香假单胞菌 + 效应蛋白　；参考：《合肥工业大学》2017年硕士论文

【摘要】：病原微生物对自身生命活动的调控、进化集中体现在与宿主的相互作用、攻防博弈中。丁香假单胞菌番茄致病变种Pseudomonas syringae pv.tomato(Pst)是引起细菌性斑点病影响番茄产量和品质的病原微生物。P.syringae pv.tomato与其宿主植物番茄之间的相互作用也是研究病原微生物-植物相互作用的一个重要模式。Pto是植物识别、防御丁香假单胞菌的抗性蛋白。Pto可以识别结合病原菌注入植物细胞的效应蛋白AvrPtoB、AvrPto,并由此触发下游防御反应,最终在侵染部位产生超敏反应诱导组织坏死,防止病原菌的定殖和繁殖。因此,Pto成为病原微生物-植物相互作用的一个枢纽蛋白,但Pto介导的免疫途径的框架仍未完整阐明。本文通过克隆番茄Pti(Pto interaction protein)基因Pti4、Pti5和Pti6,分别构建植物瞬时表达载体,利用农杆菌介导方法在烟草叶片中进行表达,并进一步利用免疫共沉淀技术,在植物体系中检测它们与番茄Pto、病原菌效应蛋白AvrPtoB的相互作用。同时,通过Pti4、Pti5和Pti6基因载体构建、番茄遗传转化,获得过量表达转基因植株。通过病原菌Pst DC3000接种,检测Pti基因在番茄防御丁香假单胞菌侵袭中的作用。实验结果表明,利用所构建Pti4、Pti5和Pti6基因植物表达载体,在烟草中获得了预期大小Pti4、Pti5和Pti6蛋白,并在植物体系中验证了它们与Pto蛋白,及其活性突变体Pto~(Y207D)的相互作用,表明Pti蛋白参与Pto介导的植物免疫应答途径,但它们相互作用强弱的差异,则提示Pti4、Pti5和Pti6蛋白可能在具体功能作用中有所不同。通过免疫共沉淀进一步分析发现,Pti4、Pti5和Pti6蛋白与病原菌效应蛋白AvrPtoB有相互作用。AvrPtoB与Pti蛋白的互作是否使其成为病原菌毒性效应蛋白的靶标,还是促进Pto蛋白对AvrPtoB的识别,仍需进一步实验探讨。此外,通过农杆菌介导遗传转化,获得了Pti4、Pti5基因过量表达转基因番茄植株。利用真空渗透法接种病原菌Pst DC3000后,与野生型相比,转基因植株叶片、茎秆病斑,叶片菌落计数均明显减少,表明Pti4和Pti5基因可提高番茄对P.syringae pv.tomato的抗性。本工作为进一步探讨与Pto互作的这些Pti转录因子的功能、深入了解它们在病原微生物-植物相互作用关系中的角色,以及应用于植物抗病遗传改良都提供了良好基础。
[Abstract]:The regulation of pathogenic microorganisms on their own life activities is mainly reflected in the interaction with the host and the game of attack and defense.The interaction between P. syringae pv.tomato and its host plant tomato, Pseudomonas syringae pv.tomatoPstis, which causes bacterial spot disease, affects the yield and quality of tomato, is also a study of pathogenetic microorganism and plant interaction.An important pattern of action. Pto is plant recognition,The resistance protein. Pto of Pseudomonas clove can recognize the effector protein AvrPtoBn, which is injected into plant cells by pathogenic bacteria, and trigger the downstream defense reaction, and finally produce hypersensitive reaction to induce tissue necrosis at the site of infection.To prevent colonization and reproduction of pathogenic bacteria.Therefore, Pto is a pivotal protein in the pathogenetic microorganism-plant interaction, but the framework of the immune pathway mediated by Pto has not been fully elucidated.In this paper, plant transient expression vectors were constructed by cloning tomato Pti(Pto interaction protein gene Pti4, Pti5 and Pti6, respectively. Agrobacterium tumefaciens mediated expression was used in tobacco leaves, and immunoprecipitation technique was further used.Their interaction with tomato, pathogen effector protein (AvrPtoB) was detected in plant system.At the same time, transgenic plants were obtained from transgenic tomato by construction of Pti4 Pti5 and Pti6 gene vectors.The role of Pti gene in tomato defense against Pseudomonas cloves was detected by inoculation of pathogen Pst DC3000.The results showed that the plant expression vectors of Pti4, Pti5 and Pti6 genes were used to obtain the expected size of Pti4Pti5 and Pti6 proteins in tobacco, and their interaction with Pto protein and its active mutant Ptochy207D was verified in plant system.The results indicated that Pti protein was involved in the Pto mediated plant immune response pathway, but the differences of their interactions suggested that Pti4Pti5 and Pti6 proteins might be different in specific function.By immunoprecipitation, it was found that the interaction between Pti4Pti5 and Pti6 protein and pathogenic effector protein (AvrPtoB). The interaction of AvrPtoB and Pti protein made it a target of virulence effector protein of pathogenic bacteria, or promoted the recognition of AvrPtoB by Pto protein.Further experiments are needed.In addition, transgenic tomato plants were obtained by Agrobacterium tumefaciens mediated genetic transformation.Compared with wild-type transgenic plants inoculated with Pst DC3000 by vacuum osmotic method, the number of leaf, stem spot and colony of transgenic plants decreased significantly, indicating that Pti4 and Pti5 genes could increase the resistance of tomato to P.syringae pv.tomato.This work provides a good basis for further exploring the functions of these Pti transcription factors interacting with Pto, understanding their roles in the relationship between pathogenic microorganisms and plants, and applying them to genetic improvement of plant disease resistance.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.412

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