稻瘟菌蛋白激发子MoHrip2结构与功能及互作蛋白研究

发布时间：2018-04-17 00:21

  本文选题：蛋白质结构 + 功能域　； 参考：《中国农业科学院》2016年博士论文

【摘要】：在长期的进化中,植物形成了一套有效的免疫系统应对逆境环境。植物免疫是基于对环境中诱导子的识别,蛋白质激发子是最主要的诱导子之一,已成为植物免疫领域的一个研究热点。蛋白质激发子MoHrip2是从稻瘟菌中分离获得的一种外分泌蛋白,能够诱导烟草免疫系统的激活,提高水稻对白叶枯病和稻瘟病的抗性。然而,MoHrip2作为激发子的作用机理尚不清楚。本研究从蛋白质结构、结构与功能的关系、互作蛋白的筛选与验证以及生物学功能等方面对MoHrip2进行研究,获得了以下研究成果:1、解析获得MoHrip2的三维结构通过原核表达系统获得了MoHrip2蛋白以及掺入硒原子的蛋白衍生物,对蛋白质结晶条件进行筛选并优化,最终获得MoHrip2蛋白母体及衍生物的高质量单晶;利用X-射线衍射技术获得了MoHrip2蛋白母体及衍生物的衍射数据,分辨率分别可达2?和1.8?;通过硒原子产生的反常散射数据,确定了MoHrip2蛋白晶体的相位,每个不对称单元含有两个单体;利用COOT软件进行模型搭建和精细修改,解析获得了MoHrip2的三维结构,每个单体形成一个β-桶状结构,包括11个β-折叠片和一个很短的α-螺旋,分子内形成3对二硫键;通过DALI软件进行结构比对,发现MoHrip2与TL-XI蛋白在结构上具有很高的相似度。2、确定了MoHrip2的功能域基于二级结构,对MoHrip2蛋白进行截短并利用原核表达系统表达和纯化,获得了8个截短突变体;对各截短突变体进行诱导烟草HR和抗病性分析,确定位于中间部位的第76-89位共14个氨基酸残基组成的短肽段,是MoHrip2发挥激发子活性的功能域。3、获得MoHrip2在拟南芥中的互作蛋白Mo2BP1以MoHrip2为诱饵蛋白,通过酵母双杂交技术筛选拟南芥cDNA文库,获得了4个候选互作蛋白;通过双分子荧光互补实验进行体内验证,其中一个蛋白能够在烟草细胞中与MoHrip2互作,命名为Mo2BP1;Mo2BP1为一个类奇异果甜-病程相关蛋白,在植物体内参与响应外源刺激;对Mo2BP1进行适应大肠杆菌表达的密码子优化,并重组表达、纯化获得GST-Mo2BP1融合蛋白;通过GST pull-down实验验证两个蛋白在体外能够相互作用。4、Mo2BP1缺失影响MoHrip2的功能在烟草叶片细胞中瞬时表达Mo2BP1-GFP融合蛋白,使用共聚焦激光扫描显微镜观察荧光,确定Mo2BP1在烟草细胞中定位于细胞膜上;购买获得Mo2BP1缺失突变杂合拟南芥,通过培育、筛选获得其纯合突变体;通过分析MoHrip2在野生型和突变体拟南芥中诱导HR和抗病性的不同效果,确定Mo2BP1在MoHrip2诱导拟南芥产生HR和提高抗病性中发挥重要作用。5、MoHrip2在拟南芥中过表达提高抗病性通过农杆菌介导法转化拟南芥,获得MoHrip2稳定遗传表达的转基因植株;分析转基因拟南芥对灰葡萄孢菌的抗性,表明MoHrip2过表达能够提高拟南芥的抗病性。
[Abstract]:Over the course of long-term evolution, plants have developed an effective immune system for coping with adverse environments.Plant immunity is based on the recognition of elicitors in the environment. Protein elicitor is one of the most important elicitors and has become a hotspot in the field of plant immunity.Protein elicitor MoHrip2 is an exocrine protein isolated from Magnaporthe grisea. It can induce the activation of tobacco immune system and increase the resistance of rice to bacterial blight and blast.However, the mechanism of MoHrip2 as excitator is unclear.In this study, MoHrip2 was studied from the aspects of protein structure, relationship between structure and function, screening and verification of interacting proteins and biological functions.The following research results were obtained: 1: 1. The three-dimensional structure of MoHrip2 was analyzed and obtained by prokaryotic expression system. MoHrip2 protein and protein derivatives doped with selenium atom were obtained by using prokaryotic expression system. The crystallization conditions of proteins were screened and optimized.The high quality single crystal of MoHrip2 protein parent and derivative was obtained, and the diffraction data of MoHrip2 protein parent and derivative were obtained by using X-ray diffraction technique, the resolution of MoHrip2 protein parent and derivative were up to 2?Using anomalous scattering data from selenium atoms, the phase of MoHrip2 protein crystal was determined, each asymmetric unit containing two monomers. The three-dimensional structure of MoHrip2 was obtained by modeling and fine modification by COOT software.Each monomer forms a 尾 -barrel structure, consisting of 11 尾 -folds and a very short 伪 -helix, in which three pairs of disulfide bonds are formed within the molecule.The structural similarity between MoHrip2 and TL-XI protein was found to be very high. The functional domain of MoHrip2 was determined based on secondary structure. The MoHrip2 protein was truncated and expressed and purified by prokaryotic expression system. Eight truncated mutants were obtained.HR and disease resistance of the truncated mutants were analyzed, and the short peptides composed of 14 amino acid residues at position 76-89 were identified.The interaction protein of MoHrip2 in Arabidopsis thaliana (Mo2BP1) was obtained by using MoHrip2 as bait protein. CDNA library of Arabidopsis thaliana was screened by yeast two-hybrid technique, and four candidate interaction proteins were obtained.In vivo, a protein was identified to interact with MoHrip2 in tobacco cells by bimolecular fluorescence complementary assay. It was named Mo2BP1Mo2BP1 as a kiwifruit like sweet disease-course related protein, which was involved in response to exogenous stimuli in plants.The codon of Mo2BP1 adapted to E. coli expression was optimized and recombined to obtain GST-Mo2BP1 fusion protein.GST pull-down assay showed that the function of MoHrip2 could be affected by the deletion of MoHrip2 in vitro. The Mo2BP1-GFP fusion protein was transiently expressed in tobacco leaf cells. The fluorescence was observed by confocal laser scanning microscope.The homozygous mutant Arabidopsis thaliana (Arabidopsis thaliana) with Mo2BP1 deletion mutation was obtained and its homozygous mutants were obtained by breeding.By analyzing the different effects of MoHrip2 on inducing HR and disease resistance in wild type and mutant Arabidopsis thaliana,It was confirmed that Mo2BP1 plays an important role in the production of HR and the improvement of disease resistance in Arabidopsis thaliana induced by MoHrip2. The overexpression of MoHrip2 in Arabidopsis thaliana and the improvement of resistance to disease were transformed into Arabidopsis thaliana by Agrobacterium tumefaciens. Transgenic plants with stable genetic expression of MoHrip2 were obtained.The analysis of resistance of transgenic Arabidopsis thaliana to Grapevine showed that overexpression of MoHrip2 could improve the resistance of Arabidopsis thaliana.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.11
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本文编号：1761237