【作者】 王金子；

【导师】 陈保善；

【作者基本信息】 广西大学 ， 微生物学， 2012， 博士

【摘要】 低毒病毒是一类无衣壳的双链RNA病毒,感染低毒病毒CHV1的板栗疫病菌出现明显的低毒力现象和表型变化。目前对低毒病毒／板栗疫病菌系统在分子生物学水平上已经有了很深入的报道,但有关蛋白质组学方面的报道还较少。本论文主要通过以下几个方面的研究,在蛋白质组层面上明确病毒侵染对宿主产生的影响。根据丝状真菌自身的特点,优化蛋白质提取方法,使用二维液相系统对野生型菌株EP155和受低毒病毒感染菌株EP713进行了差异蛋白质组分析。ProteoVue和DeltaVue软件分析并建立了重复性较好的二维模拟凝胶图谱。以EP155为标准,共有296个蛋白质紫外吸收峰表现出2倍以上的变化：上调的209个,下调的87个。同样的蛋白质样品在双向电泳图谱中共找到71个差异表达蛋白质,变化趋势与之前的报道相一致。建立了一种优化的、适合于板栗疫病菌分泌蛋白质样品提取的方法。使用该方法可以很好的得到培养在EP液体完全培养基中1-7天的分子量和等电点分布均匀的分泌蛋白质样品,并用于聚丙烯酰胺凝胶和双向电泳的实验。在双向电泳凝胶上,质谱法共有137个分子量在10-150kDa,等电点在pH3-8之间的蛋白质得到有效鉴定。通过注释,这些蛋白主要包括细胞壁降解蛋白、毒力因子和植物与病原真菌相互作用的成分。其中20个蛋白质具有N-末端信号肽序列,123个与真菌分泌蛋白质组数据库预测结果相吻合。通过基因敲除高丰度分泌表达蛋白质,在受低毒病毒侵染的分泌蛋白质组中,找到2个受调控的蛋白质,分别是泛素蛋白和一个假设蛋白质。为了进一步明确板栗疫病菌与低毒病毒相互作用的关系,选择板栗疫病菌不同菌株进行了囊泡蛋白质分析。共有33个受低毒病毒调控的差异表达蛋白质在囊泡蛋白质组中被发现。主要涉及到蛋白质、核酸和碳水化合物代谢,信号传导,细胞内运输,压力应激和氧化还原反应。分析结果表明囊泡的基本功能未受低毒病毒的侵染而改变,但运输效率却随着病毒在宿主体内的转运而发生明显改变。其中4个仅在低毒病毒侵染株EP713中出现的蛋白质点被鉴定为病毒蛋白。通过质谱鉴定,确定4个病毒蛋白质点所含肽段在ORF B中均属于病毒蛋白p48。由此我们提出假设,在宿主体内病毒ORF B N端自剪切形成p48后,p48引导ORF B的复制和翻译,并通过翻译后的加工修饰形成具有不同功能的病毒蛋白异构体。更多还原

【Abstract】 Hypovirus is unencapsidated dsRNA virus. The hypovirulence and phenotype variation of the chestnut blight fungus, Cryphonectria parasitica happens by hypovirus CHV1infection. The reports about proteomics of C. parasitica are limited at the moment, although the molecular biology research of hypovirus/C. parasitica system are in-depth study. This thesis included several aspects which revealed the interaction between hyporirus and the pathogen fungus in proteomic level.According to the characteristics of filamentous fungi, the extraction of tots protein was improved. Differential proteomic analysis between wild-type strai EP155and hypovirulent strain EP713has been made by two dimensional liqu chromatographic fractionation. ProteoVue and DeltaVue software analyzed at build two-dimensional simulation of gel patterns with good reproducibility.2protein UV absorption peaks were detected which show more than2fold changes the base of EP155:209up-regulated and87down-regulated. The same prot samples were used on2D-gels and71differential expressed proteins were fou The variation tendency was consisitent with the previous report.An optimized protein extraction method suitable for the preparation of secre proteins of a phytopathogenic filamentous fungus, C. parasitica, was developed, evaluated by SDS-PAGE and two-dimensional gel electrophoresis (2-DE), method was both reliable and comprehensive in obtaining secreted proteins wide range of molecular weight and pI, when the fungus was cultured in the complete liquid medium from one to seven days. A total of137unique proteins MW ranging from10to150kDa and pI ranging from pH3to8on2-DE gels identified by tandem mass spectrometry. By annotation, these proteins are m cell-wall-degrading enzymes, virulence factors and components involve plant-pathogen interactions. Of which,20proteins got a signal peptide and proteins were predicted to be involved in known secretory pathways by Fungal Secretome Database (FSD). Through knockout of high abundant secreted gene, we found2differential expressed proteins in secretome which belong to ubiquitin and a hypothetical protein.To expand our understanding of virus-host interactions, the vesicle proteins of different strains were analyzed. Thirty-three differentially expressed protein spots of vesicle protein were found in response to the virus infection. These proteins were implied to have functions in metabolisms of protein, nucleic acid, and carbohydrate, signal transduction events, cellular transport, response to stress and oxidation/reduction. The result indicated that the virus infection did not affect the function of transport vesicle but greatly changed transport efficiency. Four virus protein spots displayed in virus-infect vesicle protein samples. The identified peptides all came from ORF B of hypovirus CHV1and located in the scope of p48by mass spectrum analysis. We supposed that after autoproteolysis of NH2-terminal portion of ORF B to produce p48, the viral protein, p48may lead the viral ORF B replication and translation, then went through post-translational modification to produce protein isoforms with different functions.更多还原