【作者】 付丽；

【导师】 张修国；

【作者基本信息】 山东农业大学 ， 植物病理学， 2012， 博士

【摘要】 辣椒疫病(Pepper Phytophthora Blight)是世界范围内普遍发生的一种土传病害,能侵染包括辣椒、西红柿、茄子、黄瓜、南瓜和甜瓜等在内的多种茄科及葫芦科植物。1922年,Leon Leonian首次报道了其在美国新墨西哥州辣椒作物上的危害,并将其病原命名为Phytophthora capsici。辣椒疫霉菌以卵孢子或厚垣孢子在土壤的病残体中越冬,可以存活数月至更长时间,在适宜条件下引起茎腐、根腐和枯萎,寄主范围广,给我国及世界各国农业生产造成巨大的经济损失。由于辣椒疫霉菌株具有较强的变异性,培育抗病品种效果不理想,一直以来辣椒疫病的防治以化学防治为主,带来的后果就是农药残留和环境污染。因此,探索辣椒疫霉菌重要致病因子,研究病原菌抗病性生理生化机制,结合抗病性遗传、抗病基因定位与基因工程,进行抗病性鉴定和抗病育种方面研究成为病害防治的前景。植物病原菌与寄主互作过程中,植物自身形成了一系列复杂的防御系统,病原物要想侵染寄主必须克服寄主的防卫系统,从寄主中获取养分才能进行繁殖并扩大侵染。因此在侵染过程中病原菌分泌多种细胞壁降解酶或致病酶类,破坏植物的防御系统,包括：角质酶(cutinase),果胶酶(pectinase),纤维素酶(cellulase),半纤维素酶(hemicellulase)和蛋白酶(protease)等。PEL(Pectate Lyase),是一种典型的果胶降解酶,又称pectate ranseliminases,由β消去反应裂解α-1,4糖苷键,在反应产物的非还原端残基C-4和O-5之间产生一个双键。可裂解高度酯化的果胶,能迅速降低粘度。许多真菌、细菌及卵菌的侵染过程都可以产生果胶裂解酶,卵菌中的疫霉属基因组存在大量的果胶裂解酶基因,对于大量的基因信息区分主要的致病靶基因,进而解释重要致病靶基因的功能及其在寄主侵染过程中的作用,显得极为重要。本研究对高致病辣椒疫霉菌株SD33内Pcpel基因家族的致病遗传机制及其功能进行了分析。具体内容如下：(1)辣椒疫霉菌高致病菌株SD33内Pcpel基因克隆及生物信息学分析。首先对辣椒疫霉基因组序列进行生物信息学分析,特别分析了基因组内Pcpel基因家族信息。以高致病辣椒疫霉菌株SD33为材料,结合已得到的13个Pcpel序列,利用同源序列法克隆了该菌株内其他9个Pcpel基因并进行BLAST分析；利用DNAman软件对克隆的序列进行比对,发现辣椒疫霉的PEL蛋白基因序列在总长上显示了相对高的差异,序列分为两种结构域,同属于果胶裂解酶家族；但仍有保守的区域存在,该部分的保守残基于催化功能有关；大部分序列都含有信号肽和糖基化位点。最后利用构建进化树的方法选取了12个基因进行了功能分析。(2)辣椒疫霉Pcpel基因在病原-寄主互作过程中的表达模式分析。利用游动孢子悬浮液接种离体辣椒叶片,在接种后1d、3d、5d、7d取样,然后提取发病叶片总RNA。反转录后用qRT-PCR分析12个候选基因在辣椒疫霉侵染寄主过程中的表达模式。实验结果显示12个PEL基因的表达模式基本一致,其表达量都是逐渐上升的趋势,都在第7d的表达量最高,也就是后期达到峰值。这说明PEL蛋白在病原侵染的后期阶段强烈表达,病原与植物防御系统后期反应Pcpel的作用力较大。辣椒疫霉果胶裂解酶基因家族的成员在侵染后期参与克服植物防御反应,在致病过程中起到重要作用。(3)辣椒疫霉Pcpel基因在植物体内的功能分析。将12个Pcpel基因,分别构建了PVX表达载体,然后转化农杆菌进行瞬时表达。利用农杆菌侵染法接种本氏烟和辣椒叶片。记录每个基因表达后所引起两种植物症状变化。结果显示本氏烟和辣椒的叶片症状不同,并且各个基因成员在同一种植物上表达后的症状也有较大差异；其中接种本氏烟的叶片大多出现褪绿症状,黄化症状不明显,个别基因接种没有引起植物防卫反应；而接种的辣椒叶片症状比较明显,除了产生褪绿黄化症状外,个别基因(Pcpel1、Pcpel16和Pcpel20)的表达还出现了大面积连片的黑褐色病斑症状。这说明了Pcpel家族成员基因拥有果胶裂解酶的作用,但是不同Pcpel基因差异性较大(4)沉默突变体的构建及基因沉默现象的分析。借助PEG介导的原生质体转化方法,得到了12个沉默突变体菌株,实现了12个候选基因的沉默。根据载体pHAM34酶切位点及各基因序列设计基因特异性引物,将基因序列反向插入载体,构建了12个候选基因的沉默载体。将重组质粒及标记质粒pHspNpt共同转入辣椒疫霉SD33原生质体,利用抗生素G418筛选转化子。得到的转化子在V8培养基上生长并记录生物性状,包括菌丝生长速率、菌丝形态、游动孢子产率等。同时提取转化子菌体总RNA,反转录后,荧光定量PCR分析各候选基因在各转化子内的沉默效率。结果显示,12个转化子中除了Pcpel15、Pcpel18、和Pcpel20外,其余9个基因的沉默效率都在70%以上,没有出现多个基因在同一个转化子内共沉默的现象,这可能是相关基因同源性较低,没有发生染色体异染色质化的原因。此外,基因沉默并没有引起疫霉菌株生物性状的变化。(5)过量表达突变体的构建及过量表达的影响。借助PEG介导的原生质体转化方法,得到了12个过量表达突变体菌株,实现了12个候选基因的过量表达。引物设计及载体同沉默方法相同,将基因正向插入载体,构建了12个基因的过量表达载体。得到的转化子在V8培养基上生长并记录生物性状,包括菌丝生长速率、菌丝形态、游动孢子产率等。同时提取转化子菌体总RNA,反转录后,荧光定量PCR分析各候选基因在各转化子内的过量表达效率。结果显示,12个转化子中除了Pcpel17和Pcpel19外,其余10个基因的相对过量表达效率都在25倍以上,没有出现多个基因在同一个转化子内共过量的现象。此外,基因过量表达并没有引起疫霉菌株生物性状的变化。(6)转化子的致病性检测。利用游动孢子接种方法处理辣椒幼苗叶片,以野生型菌株为对照,记录叶片发病情况及病斑大小。分析结果发现沉默突变体的致病力发生变化,Pcpel1、Pcpel16、Pcpel20病斑明显减小,其他基因的变化较小,Pcpell1的接种基本没有变化。过量表达的结果表明,大部分的基因过量表达转化子接种病斑面积没有明显加大,只有Pcpel16、Pcpel20的病斑面积加大,病斑出现时间提前,比沉默转化子接种发病面积明显增加。Pcpel1过量表达的接种面积反而变小,说明这个基因的过量表达影响了菌体的致病性,可能是致病过程中的关键基因。综合实验说明部分PEL基因沉默或过量表达导致菌株致病力发生变化,大部分基因没有影响菌体的致病性,而且Pcpel1、 Pcpel16、Pcpel20是辣椒疫霉中在病原侵染过程中起到重要作用的PEL。综合所有实验结果,我们认为辣椒疫霉果胶裂解酶基因是以基因家族形式存在的,在寄主与病原互作过程中扮演着重要角色。辣椒疫霉果胶裂解酶不仅能使植物产生常见的褪绿、黄化,还会引起寄主叶片细胞壁完全失去防御能力,导致细胞死亡,组织坏死的能力。基因家族各成员功能存在较大差异,即使功能域相同的基因之间也可能存在巨大的功能差异,对基因家族功能的研究不能局限于个别基因功能的分析。Pcpel基因沉默不会引起菌株表型变化但会导致转化子致病力明显降低。此外很难实现基因家族单一成员或者全部成员基因沉默,但是并不影响对该类基因功能的分析。将该家族12个基因进行稳定沉默以及致病性分析,发现该家族部分成员沉默会导致菌株致病性降低。利用稳定基因沉默对于基因家族成员功能的研究是个很好的选择。更多还原

【Abstract】 Phytophthora blight of peppers is a worldwide disease, which impacted on agriculture almost in every country, and caused enormous losses on economy. It can infect pepper, tomato, eggplant, cucumber, pumpkin, melon and so on. In1922, Leon Leonian described Phytophthora capsici after observing a blight of peppers at the New Mexico Station. The oospores of P. capsici are capable of surviving in the soil for several years, germinating in favorable environmental conditions. After germinating, it can cause stem rot, stem wilt and root rot in a wild host range. This has caused huge economic losses to our country and the worldwide agricultural production. At present, the pepper blight was controlled mainly by chemical methods and breeding resisitant cultivars. However, strong variability in pathogenicity of P. capsici caused the instability of resistant breeds. For a very long time, the chemical methods were the most effective and rapid method to prevent and congtrol P. capsici with the consequence of pesticide residues and enviromental pollution. So it is very important to study the disease control in phytophthora blight of peppers by carrying out resistance identification and disease-resistance breeding via investigating the infection mechanism, resistance biochemistry mechanism, resistance genetics, and quantitative trait loci and genetic engineering as well, so as to the resistance identification and application in breeding for disease resistance.The success infection of pathogens depend on their ability to overcome the extensive defences of their plant hosts and this ability in turn depends on specific physiological functions in the pathogens. During the infection the pathogens could secret a series of cell wall degrading enzymes (CWDE) or pathogenicity-related enzymes, including cutinase, pectinase, cellulase, hemicellulase, and protease. Pectate lyase, also known as pectate transeliminases, exhibits a P-elimination mechanism in the cleavage of α-1,4-glycosidic bonds of polygalacturonic acid which results in the formation of a double bond between C4and C5at the non-reducing end via E2elimination mechanism and an elimination of CO2It is one of the most important CWDEs, it can be secreted by many fungi, bacteria and oomycete. There are many PEL genes in the genome sequence of Phytophthora spp, however, the reason why Phytophthora species have a large PELs gene family and the roles these genes play during infection process are still unclear.In this work, we carried out the pathogenesis and functional analysis of PELs gene family from P. casici. The focus of the present study was to elucidate in vitro and in vivo the function of PELs gene family in P. casici by stable gene silencing, over-expression and agroinfection for the first time. The details are as follows:(1) We analyzed the presence of PELs in a high-virulent P. capsici strain SD33isolated from China and carried out the bioinformatics analysis. Genome-wide identification of PELs was performed in the released P. capsici genome sequence. A series of primers were designed according to the released P. capsici genome sequence (JGI). The PCR products of expected length were cloned and then sequenced in company. There are9sequences accepted with the primers. And a database search in BLAST confirmed that they were PEL genes. Sequences alignment was performed using DNAMAN software. The results showed that all these22genes were classfied into two clades, but remain had conserved motif and a relative conserved bexakis-residue. Most genes have signal peptide and N-glycosylation sites. The tree was constructed on the basis of alignment of the selected PEL genes predicted protein sequences. Phylogenetic trees were generated by neighbor-joining, as implemented in PAUP’4.0Beta. At last we chosed twelve PELs for the founction research.(2)The expression pattern analysis of Pcpel genes in the process of pathogen-host interaction. Twelve Pcpel genes expression pattern analysis in infection. The leaves of peppers with4-6weeks fully expanded were inoculated with zoospores of SD33and then the total RNA of infected leaves were isolated from the1th,3th,5th and7th day, and cDNA was synthesized. Specific primers were designed based on the sequence of twelve Pcpel genes and the expression of Pcpel genes in different pepper leaves was detected by Quantitative Polymerase Chain Reaction (QRT-PCR). The results indicated that Pcpel genes could express in infected peppers respectively and the expressed level became stronger with the time prolong after inoculation, and then reached the peak on the7th day. But different genes have different levels. The results indicated that Pcpel genes played important roles in infection process.(3) The Pcpel genes’function in plants. Agrobacterium mediated transient expression of twelve Pcpel genes in tobacco and pepper plants showed function diversity of different genes. We observed that diverse changes when Pcpel genes were expressed in pepper and tobacco leaves. These symptoms including necrosis, chlorisis in varying degrees, wrinkle and roll, appeared on different plants corresponding to individual genes at different time points. In pepper, all the genes have more visible symptoms in varying degrees then in tobacco. Three genes (Pcpell, Pcpel16and Pcpel20)have heavier symptoms than other genes. These results indicated that paralogous in Pcpel gene family had distinct biological functions in different plant species.(4) Silenced transformants construct and analysis. We got stable transformation lines for Pcpel genes using PEG-mediated protoplast gene-silencing mathod. We assessed the expression levels of other members in the gene family by Real-time RT-PCR. Twelve transformants were selected. Individual genes expression levels didn’t affect other genes expression level. All the selected genes except Pcpel15, Pcpel18and Pcpel20were silenced at a degree above70%. Phenotypes of the twelve transformants’colonies were examined and no differences were observed between controls and the transformants.(5) Over-expressing transformants and analysis. We also got stable over-expressing lines for Pcpel genes using PEG-mediated protoplast gene-silencing method. We assessed the expression levels of other members in the gene family by Real-time RT-PCR. Twelve transformants were selected. Individual genes expression levels didn’t affect other genes expression level. All the selected genes except Pcpel17and Pcpel19were over-expressing at a degree above25fold. Phenotypes of the twelve transformants’colonies were examined and no differences were observed between controls and the transformants.(6) Transformants pathogenicity testing. The virulence of silenced and over-expressing lines is tested with pepper leaves. To determine the effect of Pcpel gene silencing and over-expression on virulence, we used the induced zoospores inoculation assay to measure virulence in the susceptible pepper cultivar. Most leaves inoculated with the silenced lines showed significantly different spots at the same time point. Most genes have the same or smaller spots with the wild type and the CK, only3genes have obviously small spots. Generally, silenced strains showed obviously different reduction of virulence or pathogenicity. The same to the over-expressing lines, most lines didn’t showed significantly biger spots at the same time point, only Pcpel16and Pcpel20over-expressing lines indicated the bigger spots. The over-expressing line of Pcpel1inoculated area but smaller, indicating that it may be the key gene in the pathogenic process. Generally, not all the over-expressing strains showed obviously increase of virulence or pathogenicity. These results indicated that paralogous in Pcpel gene family had distinct biological functions with different genes.We evaluated Pcpel gene family roles for inducing hypersensitive response in different plant species, which documented the avirulence function and distinct biological functions. Pcpel genes expression deficiency could decrease virulence, genes expression increase could enhance virulence which suggested the probable roles in the process of plant pathogen interaction. This is the first report that directly demonstrates virulence functions of PELs from P. capsici. These results provide further information in comprehending the PELs pathgenicity.更多还原