【作者】 贾永健；

【导师】 张修国；

【作者基本信息】 山东农业大学 ， 植物病理学， 2008， 博士

【摘要】 辣椒疫病是一种世界性土传病害,严重影响我国及世界各国的辣椒生产,造成巨大的经济损失,该病由辣椒疫霉菌(Phytophthora capsici Leonian)引起。辣椒疫霉菌是一种土传卵菌,以卵孢子或厚垣孢子在土壤及病残体中越冬,可以存活数月甚至更长时间,感病品种的重复种植能够导致卵孢子在土壤中大量积累。该菌可以在任何生长季节侵染寄主植物引起种苗死亡、茎腐、枯萎、果实腐烂;寄主范围广,可以侵染辣椒、西葫芦、黄瓜、茄子番茄等9科20多种作物。目前,对于辣椒疫病的防治主要采用轮作、化学防治和培育抗病品种。但是农药残留容易对环境造成污染;辣椒疫霉生理小种的变化造成抗病品种效果不稳定。因此,寻找探索辣椒疫霉菌重要的致病因素来控制病害已经成为科学家研究的热点。研究表明植物病原菌与寄主互作过程中分泌的果胶酶是重要的致病酶,这些酶包括多聚半乳糖醛酸酶、果胶甲基酯酶、果胶裂解酶,它们产生于病原菌识别、定殖及与寄主建立寄生关系的过程中,降解或软化细胞壁的果胶、中胶层乃至破坏寄主的防御体系,增强病原菌对寄主的亲合力。因此,植物病原菌在侵染寄主过程中所分泌的细胞壁降解酶是一类重要的致病因子,其活性高低是界定植物病原菌侵染与否或致病力强弱的重要技术指标之一。其中,果胶甲基酯酶(PME)是许多植物病原菌的致病因子,在植物发病过程中起重要作用。本研究以辣椒疫霉为研究对象,筛选多聚半乳糖醛酸酶、果胶甲基酯酶、果胶裂解酶活性较高的辣椒疫霉菌株,并构建了其基因组DNA文库,应用Pool PCR方法分离和克隆了5个pme基因。构建了pcpme1基因真核表达载体,通过诱导表达获得重组蛋白,用Anti-His抗体通过Western Blot检测了其表达情况,用Ni-NTA树脂纯化了表达的PCPME1,并制备了特异性抗体,用Western Blot检测了pcpme1在辣椒体内的表达情况;用PNGase F对PCPME1进行去糖基化处理,研究了糖基化位点对其活性的影响;找到pcpme1的活性中心位点,对其进行定点突变,然后进行真核表达,获得纯蛋白;应用RT-PCR和Northern Blot方法分析了其在辣椒体内的表达情况;用表达的原始PCPME1、去糖基化的蛋白,定点突变后的蛋白分别接种辣椒叶片,透射电镜观察其对寄主细胞壁的破坏作用。主要研究内容如下:采用分光光度法测定辣椒疫霉菌SDPH-33的多聚半乳糖醛酸酶(PG)、果胶裂解酶的活性;滴定法测定了果胶甲基酯酶活性;结果发现,辣椒疫霉的致病性与其PGs、PELs、PMEs的活性密切相关。利用Pool PCR法筛选SDPH-33的基因组文库,从文库中共分离克隆了5个pme基因,并对其结构特征进行分析。对其序列和蛋白结构进行分析发现, 5个pme基因具有很高的同源性,所编码的蛋白具果胶甲基酯酶的保守序列和活性位点(GxYxE、QDTL、QAVAL、DFIFG和LGRPW),并具有不同数目的糖基化位点,其蛋白结构也具明显的相似性。根据比对不同来源的PME的氨基酸系列构建的进化树表明,辣椒疫霉的pme基因与卵菌的pme基因聚在一起,亲缘关系最近,明确了卵菌在自然界中的分类地位。用SDPH-33的游动孢子悬浮液接种4-6叶期辣椒叶片,提取发病叶片的总RNA,合成cDNA。根据pcpme的序列设计特异性引物,RT-PCR检测接种叶片内pcpme的表达情况。结果发现,接种后pcpme在发病辣椒体内均能表达,其表达量随着时间延长而加强,第7天表达量最高,而在健康辣椒组织中则没有发现任何pcpme的表达。结合RT-PCR的结果及基因的糖基化位点数目确定pcpme1为关键的致病基因,根据pcpme1的序列设计引物,PCR扩增片段以制备探针,Northern Blot结果表明,pcpme1基因在接种辣椒叶片内得到表达,结果于RT-PCR相似。根据已经克隆的pcpme1基因的序列,设计特异性引物,PCR扩增其成熟肽片断。重组至酵母分泌型表达载体pPIC9K,构建成重组表达载体pPIC9K-pcpme1,转化至大肠杆菌JM109中,筛选得到阳性克隆。重组质粒线性化后转化毕赤酵母GS115感受态细胞,经过G418筛选和PCR扩增转化子基因组筛选,得到数株基因工程酵母菌。SDS-PAGE分析发现,目的蛋白分子量约为60KDa,比预期蛋白的分子量大,用anti-his抗体检测到pcpme1已经在毕赤酵母中获得了高效表达。通过Ni-NTA树胶纯化系统并透析后获得了表达的PCPME1融合蛋白,发现其对提取的辣椒细胞壁具有降解活性,通过将纯化的PCPME1蛋白免疫家兔,制备特异性抗体。Western Blot分析结果表明,pcpme1基因在发病的辣椒组织中获得了充分的表达,且随着发病程度的提高表达量逐渐增高。将纯化的PCPME1蛋白用PNGase F处理,获得了去糖基化的蛋白,分子量约为37Kda与预期蛋白的分子量相当,根据去糖基化前后蛋白的活性测定确定了糖基化对PMEs的活性不起决定性作用。根据前人报道的结果预测PCPME1的活性位点,并对其进行定点突变,将突变后的基因转化毕赤酵母,并诱导其表达,用Ni-NTA树胶纯化系统并通过透析获得了突变后的蛋白,突变前后蛋白的活性测定结果表明突变的氨基酸为该蛋白的活性位点。用纯化的野蛋白PCPME1、去糖基化的蛋白、突变后的蛋白分别接种4-6叶期的辣椒幼苗叶片,发现野蛋白和去糖基化的蛋白能够在叶片上引起病斑,且病斑的大小随接种时间的延长而增大;突变后的蛋白接种后则不能形成病斑;接种无菌水的对照叶片也没有表现症状。将接种后的辣椒用透射电镜观察其细胞壁的结构发现接种野蛋白和去糖基化的蛋白细胞壁被不同程度地降解,而接种突变蛋白和无菌水的辣椒其细胞壁基本保持完整。本实验的结果表明pcpme1是编码辣椒疫霉果胶甲基酯酶基因家族中起致病作用的关键基因之一。更多还原

【Abstract】 Phytophthora blight of peppers is a worldwide soil-borne disease, and it has become a serious threat to the agricultural production of China and other countries in the world, and result in enormous economic loss. The disease was caused by phytophthora capsici Leonian. P. capsici is a soilborn oomycete, and it can survive in the soil as oospores or chlamydospore for several months even longer, repeated cultivation of susceptible hosts results in a high density of oospores in siol. P. capsici can attack the host plant at any growth stage and causes seedling death, crown rot, foliar blight, ang fruit rot; the pathogen can also cause several crop lossed in pepper, other cucurbits, eggplant, and tomato and so on.At present, the phytophthora blight of peppers was controlled mainly by crop rotation, application of chemicals and breeding resisitant host. However, phytotoxicity and chemical residues may pose a serious threat to the environment, strong variability of physiological strains of P. capsici caused the instability of resistant host. So search the important pathogenic factors of P. capsici and tocontrol the phytophthora blight of peppers have become the focus of the phytopathologist. Many previous studies have shown that plant pathogens must breach cell walls before they can infect the hosts, in which plant pathogens can secrete an array of pectinases during plant-pathogen interactions. These enzymes, such as polygalacturonase, pectate lyase, and pectin methylesterase, appear to play a key role in pathogenicity in most pathogens for the decomposition of pectin, which is one of the main components in the plant cell wall, and make the host more susceptible to the pathogen. The pathogenicity of many plant pathogens is dependent upon the synthesis and secretion of numerous pectinases, and the maceration of the plant parenchymatous tissues result primarily from the enzymatic degradation of the pectic polysaccharides by pectinases in plant cell walls, which indicated that the pectinase activities were important factors in the maceration of the plant tissues, and pectinase activity is an important factor to determine the pathogenicity of microorganisms.Among these pectinases, pectin methylesterase is an important pathogenitic factor, and plays a major role in the pathogenisis of phytophthora blight. The study took the P. capsici as subject, analysis the polygalacturonase, pectate lyase, and pectin methylesterase activities of high pathogenic P. capsici SDPH-33. Five pme genes were obtained by screening genomic library of SDPH-33 through Pool PCR. Then PCPME1 fusion protein was acquired according to heterologous eukaryotic expression and purified by Ni-NTA resin, western blot was used to test whether the pcpme1 expressed in Pichia pastoris. purified PCPME1 was used to prepare antibodies in New Zealand white rabbits, then the antibody was used to tested the expression of pcpme1 in pepper. The effect of potential glycosylation site to the PCPME1 activity was analysised by deglycosylation through PNGase F. we predict the activity site of pcpme1 and analysis the function by site-directed mutagenesis, and got the purified mutational protein by eukaryotic expression and affinity chromatograph. At last, the expression level of pcpme1 in pepper tissues was investigated by RT-PCR and Northern Blot. The purified fusion protein, deglycosylated protein, and mutational protein were used to inoculate pepper leaves, PMEs activity varied in PMEC treated pepper leaves was consistent with symptom development in pepper leaves. transmission electron microscope was used to analysis the enzymatic degradation of the plant cell wall. Main researchs were as follows:The activities of PGs and PELs were determined with spectrophotometer, and PMEs activities were analysized by neutralization titration; the results indicated that there was a high correlation between activities of PGs, PMEs, and PELs and the pathogenicity of P. capsici. The genomic library of SDPH-33 was screened by Pool PCR, and 5 pcpmes were isolated from the library, and structural features of the 5 genes were analysized. Alignment of these amino acid sequences indicated that there were high similarities between the 5 pme genes, and the pcpmes contain 5 characteristic sequence segments and active sites(GxYxE、QDTL、QAVAL、DFIFG and LGRPW). All the amino acid sequences of the 5 pcpmes possess 3-8 potential N-linked glycosylation sites, and have a similar tertiary structure. The evolution tree based on the alignment of PME amino acid sequences with different sources showed that PMEs from oomycetes formed their own cluster.The leaves of peppers with 4-6 fully expanded leaves were inoculated with the zoospores suspension of SDPH-33 and then the total RNA of infected leaves was isolated and cDNA was synthesized. Specific primers were designed based on the sequences of 5 pcpmes and the expression of these pcpmes in pepper leaves was detected by RT-PCR. The results indicated that all these genes can express in infected peppers respectively and the expressed level became stronger with the time prolong after inoculation, and then reached the peak on 7th day, however, there was no expression of pcpmes in the healthy peppers. Based on the result of RT-PCR, the pcpme1 was determined to be the most important pathogenetic gene. The non-conservative fragment of pcpme1 was amplified with specific primers and then was used to synthesize probe and then the probe was used to determine the expression of pcpme1 in pepper leaves, the similar result to RT-PCR was obtained.Gene-specific primers were designed and synthesized to amplify the mature peptide of pcpme1according to known sequence. The amplified fragment was inserted into pPIC9K to construct expression plasmid pPIC9K/pcpme1, and then it was transformed into E. coli JM109 to obtain recombinated plasmid. The plasmid pPIC9K/pcpme1 was transformed into Pichia pastoris GS115 competent cell after linearized with restriction enzyme SacI. After screening by G418and PCR amplification of these transformants, several recombinant strains with pcpme1 inserted were obtained. The result of SDS-PAGE indicated that Heterologous expression of pcpme1 in Pichia pastoris produced a protein of 60 kDa that was not corresponded to the predicted mass of this protein. Western Blot with anti-His antibody showed that pcpme1 can express efficiently in Pichia pastoris. Expressed PCPME1 was purified through Ni-NTA system, and was used to prepare antibodies in New Zealand white rabbits, then the antibody was used to tested the expression of pcpme1 in pepper, the result indicated that pcpme1 can express in infected peppers and the expressed level became stronger with the time prolong after inoculation, and then reached the peak on 7th day, however, there was no expression of pcpmes in the healthy peppers. The purified N-linked wild type protein PCPME1 was treated with PNGase F, and the molecular mass of the deglycosylated PCPME1 was 37KDa, which was similar to the predicted mass of PCPME1. The activity of deglycosylated PCPME1 was similar to that of N-linked wild type PCPME1, which suggested that glycosylation does not play a major role in the activity of PCPME1. Site-directed mutagenesis of the activity sites of pcpme1 was carried out by over lap PCR amplification. Comparison of the activities between wild type and mutational PCPME1 showed our hypothesis was correct. The wild type, deglycosylated and mutational PCPME1 were used to inoculate the leaves of peppers with 4-6 fully expanded leaves, lesions appeared in wild type and deglycosylated PCPME1, and the lesions became larger with the time prolong after inoculation, while mutational PCPME1 and sterile water could not cause any lesion on the surface of pepper leaves. The cell walls of the leaves treated with wild type and deglycosylated PCPME1 were found degradated with different levels through transmission electron microscope, while those treated with mutational PCPME1 and sterile water kept almost integrity.The results of this study indicate that pcpme1 was one of the key pathogenetic genes that encode pectin methylesterase in the pathogenesis of P. capsici.更多还原