【作者】 张鑫；

【导师】 张修国；

【作者基本信息】 山东农业大学 ， 植物病理学， 2015， 硕士

【摘要】 辣椒疫霉(Photophthora capsici)是一种具有很大危害性的蔬菜病原菌,侵染寄主范围广,包括辣椒、番茄、茄子、瓜类等多种茄科及葫芦科植物。随着这种病害的发生率和严重程度在逐年提高,人们对它的关注程度也在提高。近年来,辣椒疫病发生严重导致辣椒的减产,人们开始利用新的途径来研究辣椒疫霉的致病机制,随之越来越多的致病基因被发现。寄主和病原物之间的互作符合基因对基因假说(gene-for-gene hypothesis)。当抗病植物的抗病基因编码的蛋白识质别病原物的无毒基因编码的蛋白质时,植物的免疫反应被激活。无毒基因也被称为是效应基因。随着辣椒疫霉的基因组由美国能源部基因研究所(JGI)公布,许多效应基因被预测,但相关基因结构生物学还没有研究透彻。近年,卵菌效应因子转运机制成为病原物-寄主互作的热点问题,由于大部分的效应蛋白是胞内蛋白,而这些蛋白如何进入到植物体内,仍然还存在问题,本文试图通过研究转运机制以及植物抗病机制,为病害生物防治和选育抗病品种奠定重要基础。本研究通过生物信息学分析,从辣椒疫霉基因组中克隆到多个效应基因,成功通过体外原核表达和纯化获得高纯度和高浓度的蛋白质,最终得到一个效应分子蛋白晶体结构,其分辨率为3.2?,具体总结如下:1.通过生物信息学预测和分析,从辣椒疫霉的cDNA中克隆到50多个无毒基因,能够在原核表达系统成功表达和纯化的有3个基因,其氨基酸序列同源性很低,N端RxLR-deer基序保守及C端功能区存在很大差异,本研究主要以其中的RLXR23作为主要研究对象。2.重组蛋白质RXLR23通过亲和层析,离子交换层析和分子排阻层析纯化,得到稳定和高纯度蛋白质,由于RXLR23结构同源性<30%,不能通过分子置换法解析结构,需要通过硒代蛋白来求解相位,硒代蛋白与native蛋白的表达和纯化方法一致。3.本研究通过蒸汽扩散法中的悬滴法和坐滴法得到初步蛋白晶体,利用悬滴法优化最终得到高质量的蛋白晶体用于X衍射,利用单波长反常散射法求解相位,最终解析RXLR23晶体结构,该蛋白以二聚化的形式行使其功能。4.目前报道的某些效应因子能够与寄主质膜上的3-磷酸磷脂酰肌醇(PI3P)结合,可使效应因子直接进入到寄主细胞内,本研究试图通过RXLR23与PI3P的结合,在三维空间结构中解释效应分子与之结合的位点,利用蛋白表达纯化技术和晶体学技术,最终得到复合物晶体。5.在晶体优化过程中,初始得到的晶体质量衍射分辨率只有8?左右,通过截短,脱水,更换防冻液等方法,成功将分辨率提高到3.2?。本研究初步得到RXLR23蛋白的晶体结构和与PI3P复合物的晶体,可以在蛋白质三维空间结构上观察效应分子如何行使其相关生物学功能,为病原物与寄主间的互作提供重要理论依据。更多还原

【Abstract】 Phytophthora capsici is a kind of highly dynamic and destructive pathogen and can infect peppers,tomatoes,cucumbers and other species in Cucurbitaceae and Solanaceae.With incidence and severity of pepper blight caused by Phytophthora capsici increasing in recent decades,more and more people have paid attention on the infected mechanism.As the impact of crop loss by P.capsici has increased in recent years,there are new ways and resources to study pathogenic mechanism while many pathogenic genes are found.The interaction between host and pathogen is explained by gene-for-gene hypothesis.The plant immunity system can be activated by the directly or indirectly recognition between the protein encoded by the host resistance genes(R genes)and the protein encoded by the pathogen avirulence genes(Avr genes).Avr genes are also known as effector genes.Thanks to Phytophthora capsici genome published by JGI,more and more effector genes have been predicted but structural biology about effectors is poorly understood.Recently,transport mechanisms of oomycete effectors become one of the hottest issues during the interaction between pathogen and host.Most of effectors are intracellular protein but how these effectors are transported into host is still unclear.We attempt to study the transport mechanism to reveal host resistance mechanism that will make great foundation for biological control and breeding resistant cultivar.In this study,we cloned several effector genes from Phytophthora capsici genome.We successfully obtain high purity and concentration protein through vitro prokaryotic expression and ultimately get an effector crystal structure.The crystal diffracts to 3.2 ? resolution.The main results are as follows:1.Via bioinformatics analysis and forecasting,we cloned more than 50 Avr genes from c DNA of Phytophthora capsici.Three genes can be successfully expressed in prokaryotic expression system and proteins can be easily purified.From results of amino acid sequences alignment,their N-terminal is conserved Rx LR-deer motif while C-terminal keeps the diversity of sequence.Our major work is focused on RXLR23.2.I have obtained stable and high purity RXLR23 recombinant protein by affinity chromatography,ion exchange chromatography and gel filtration chromatography.Because the structural homology identity of RXLR23 is <30%,we can’t get its structure through molecular replacement instead by using single-wavelength anomalous dispersion.The approach of expression and purification of selenomethionine protein is the same as the native protein.3.We have got the initially protein crystals by using hanging drop and sitting drop method.After optimizing with the hanging drop method,we finally get high quality protein crystals for X-ray diffraction.Solved by using single-wavelength anomalous dispersion(SAD),RXLR23 is a dimmer that functions in plants.4.The current report is that some effectors can interact with phosphatidylinositol-3-phosphate(PI3P)which is located in the host cell membrane and then directly enter into the host cell.We attempt to explain the binding sites between RXLR23 and PI3 P.Finally,we obtain complex crystal of RXLR23 binding with PI3 P by protein purification and X-ray crystallography.5.In the crystal optimization process,initial crystal resolution is only about 8 ?.After using truncation,dehydration,replacement of antifreeze and other methods,crystal diffracts to 3.2 ? and belongs to the trigonal space group P3221.In our study,we preliminarily obtained the crystal structure of RXLR23 and the complexes crystal of PI3 P and RXLR23.We can observe spatial structure of RXLR23 and try to explain how effectors play role in biological functions.Thereby,structure of RXLR23 will provide important theoretical basis for the interaction between pathogen and host.更多还原