【作者】 范慧艳；

【导师】 韩成贵；

【作者基本信息】 中国农业大学 ， 植物病理学， 2014， 博士

【摘要】 甜菜坏死黄脉病毒(Beet necrotic yellow vein virus, BNYVV)基因组包含4-5条正单链RNA,是甜菜丛根病的病原物,对甜菜生产造成严重危害,在全球各个甜菜主产区均有分布。该病毒在不同寄主上的系统侵染能力和致病性不尽相同,本论文在已有的研究基础上,主要对BNYVV侵染系统寄主本生烟和大果甜菜的生物学、转录组学和蛋白组学特点进行比较分析。本实验室前期研究表明,BNYVV RNA5在系统侵染本生烟时会发生高频的自然丢失。为了探究导致RNA5不稳定的原因,先对RNA5在不同系统寄主本生烟和大果甜菜上的稳定性和复制积累量进行了比较。RT-PCR和Northern blot分析表明,RNA5在大果甜菜上可以稳定地系统侵染,RNA复制积累量也明显高于本生烟。利用RNA4、RNA5以及重组RNA分子转染本生烟原生质体,证明RNA5复制效率低是导致其在本生烟上不稳定的主要原因,且该复制能力主要与其非翻译区序列密切相关。BNYVV系统侵染本生烟,当含有RNA4时可以造成叶片下卷、植株矮化的严重型症状,但不含RNA4时则表现为温和型症状。激光共聚焦扫描显微镜观察发现,RNA4编码的致病因子p31与RFP的融合蛋白主要定位在本生烟表皮细胞的细胞核内。由此推测,RNA4导致的严重型症状的原因可能是p31的核定位影响了植物的转录谱表达进而改变了植物的正常生理代谢。因此,利用新一代测序技术对BNYVV不同分离物侵染本生烟后叶片组织的转录组表达进行了分析。将BN3(RNA1+2+3), BN34(RNA1+2+3+4)侵染的样品与健康样品比较,共得到3,016条差异表达的转录本,其中包含大量由病毒侵染激活的寄主抗病候选基因。数据分析表明,由RNA4导致的严重症状表型可能与基因沉默、泛素化途径、细胞壁合成以及赤霉素代谢等相关基因有关。为了比较BNYW在本生烟和大果甜菜上致病机制的差异,利用Illumina测序平台对大果甜菜也进行了转录组测序,并对BNYW侵染后整个转录谱变化进行了比较。通过De novo拼接,共获得75,917条unigene,261个基因在BNYVV侵染后表达发生明显变化,其中128个基因上调表达,133个基因下调表达。GO功能分析表明这些基因主要涉及生物性刺激反应和初级代谢过程。蛋白质是生理功能的具体执行者,对蛋白质的研究将直接阐明发病的生理和病理条件下的变化机制。从mRNA水平进行研究,只包括了转录水平的调控,不能反映蛋白质的表达水平。蛋白质组学是从整体角度分析细胞蛋白的动态变化,可以获取样品完整的蛋白表达图谱。为此,利用双向电泳(Two-dimensional electrophoresis,2-DE)技术结合质谱鉴定和生物信息学方法,初步分析了BN3、BN34侵染本生烟后叶片组织的蛋白质组变化,共鉴定出13个差异表达蛋白质点,功能分析表明这些蛋白主要与植物抗病性、细胞骨架、应激反应、光合作用及氧化还原等功能相关。本实验第一次提供了BNYVV与本生烟相互作用的蛋白质表达全貌,为今后更好地理解BNYVV在寄主植物上的致病机理提供蛋白质组学方面的信息。更多还原

【Abstract】 Beet necrotic yellow vein virus (BNYVV), contains either four or five plus-sense single stranded RNAs and is the causal agent of rhizomania disease in sugar beet, which is a widely distributed virus in most regions of the world. The virus exhibits various systemic infectivity and pathogenicity in different hosts. Based on the previous researches, this dissertation was mainly focus on biological, transcriptomic, proteomic analysis of Nicotiana benthamiana and Beta macrocarpa infected by BNYVV with different RNA components.Our previous studies have demonstrated that RNA5was usually eliminated spontaneously in N. benthamiana. To understand the cause of RNA5instability, the maintenance and accumulation of BNYVV RNA5were investigated during systemic infections of N. benthamiana and B. macrocarpa. RT-PCR and Northern blot analysis showed that RNA5had a higher level of accumulation and was maintained more consistently in B. macrocarpa than in N. benthamiana. Comparative analysis of β-glucuroidase (GUS) expression from RNA4and RNA5replicons and recombinants revealed that untranslated regions (UTR) sequence in RNA5had a positive effect on replication in protoplasts and systemic infection of N. benthamiana. These data demonstrate that the low replication efficiency of RNA5depends on the untranslated sequence and regards to systemic infectivity in N. benthamiana.BNYVV can infect N. benthamiana systemically, which causes severe curling and stunting symptoms in the presence of RNA4or mild symptoms in the absence of RNA4. Confocal laser scanning microscopy (CLSM) analysis showed that the RNA4-encoded pathogenicity determinant p31protein fused to the red fluorescent protein (RFP) was localized mainly in the nuclei of N. benthamiana epidermal cells. This suggested that the severe RNA4-induced symptoms might result from p31-dependent modifications of the transcriptome. Therefore, we used next-generation sequencing technologies to analyze the transcriptome profiling of N. benthamiana in the response to infection with different isolates of BNYVV. Comparisons of the transcriptomes of mock, BN3(RNA1+2+3), and BN34(RNA1+2+3+4) infected plants identified3,016differentially expressed transcripts, which provided a list of candidate genes that potentially are elicited as a response to viral infection. Our data indicate that modifications in the expression of genes involved in RNA silencing, ubiquitin-proteasome pathway, cellulose synthesis, and metabolism of the plant hormone gibberellin may contribute to the severe symptoms induced by RNA4from BNYVV. To compare the disease developing mechanism between N. benthamiana and B. macrocarpa, we also characterized the transcriptome of B. macrocarpa and analyzed global gene expression of B. macrocarpa in response to BNYVV infection using Illumina sequencing platform. The overall de novo assembly of cDNA sequence data generated75,917unigenes. Furthermore, comparative analysis of the two transcriptomes revealed that261genes differentially expressed in the infected plants compared to the control, including128up-and133down-regulated genes. GO analysis showed that the changes of the differently expressed genes mainly enriched in response to biotic stimulus and primary metabolic process. Proteins are specific physiological functions executor, the study of protein exrpession changes will directly elucidate the mechanism under physiological and pathological conditions. The research at mRNA level can only reflect the regulation of transcription, but can not reflect the expression of the protein. Proteomics is the large-scale study of proteins, with which we can gain a global understanding of protein expression pattern of samples. Using two-dimesional electrophoresis (2-DE) followed by mass spectrometry and bioinformatics method to analyze the protein expression changes in BN3-and BN34-infected N. benthamiana leaves. The significant expressional changes in N. benthamiana proteins were mostly related to pathogenesis-related proteins, cytoskeletal proteins, stress reponse proteins and proteins involved in photosynthsis, oxidation reduction and metabolism. Thirteen differentially expressed proteins were successfully identified. The study is the first attempt to comapre the complex picture of Nbenthamiana protein expression during BNYVV infection, hence provides large scale valuable protein-related information for better understanding the host immune response to BNYW and potentially mlecular pathogenesis.更多还原