【作者】 郜刚；

【导师】 屈冬玉；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2008， 博士

【摘要】 马铃薯(Solanum tuberosum)是重要的粮菜兼用作物,其适应性强、丰产性好、营养丰富、经济效益高、适于加工,在食物安全结构中具有举足轻重的地位。马铃薯种植和生产常受青枯病(Ralstonia solanacearum)的严重威胁。因此加速病害控制和抗病育种研究显得非常重要,这就迫切要求阐明马铃薯与青枯菌互作的分子机理,挖掘和利用优良抗性基因用于马铃薯抗病遗传结构的改造。目前关于马铃薯应对青枯菌的防卫或抗病相关基因的种类、数量及其表达调控等知之甚少。本论文针对马铃薯与青枯菌互作早期受病菌诱导的防卫相关基因及其表达模式进行初步研究,目的在于为揭示马铃薯与青枯菌互作的分子机制提供一些有用信息。本研究以伤根灌菌法接种马铃薯,应用抑制差减杂交技术构建病原诱导后差异表达EST文库;应用SMART技术构建富集青枯菌诱导的防卫相关基因cDNA文库;从差异表达EST文库中筛选与防卫相关的增强表达基因片段,采用基因本体论(gene ontology,GO)对其进行功能注释和分类;结合快速扩增cDNA末端技术获得部分防卫相关基因的全长cDNA,然后对其诱导表达模式进行qRT-PCR和Northern杂交验证。主要结果如下:1.通过构建马铃薯与青枯菌互作早期受病菌诱导表达基因的差减EST文库,筛选鉴定出上调表达EST共计189条。利用同源检索结合GO术语(GO term)初步确定了防卫相关基因122个,它们分别属于生物过程(25.42%)、分子功能(40.98%)、细胞组分(15.57%)。随机抽取5条EST做RT-PCR分析,证明其在病程早期明显受到青枯菌的诱导上调表达。2.结合GO分类和先验基因功能分析,展示了自根系接种青枯菌诱导感病马铃薯基因型防卫相关基因的EST内容;通过与同类病害的相关EST的纵向比较以及与另外一种维管束系统病害葡萄皮尔斯病病原苛养木杆菌诱导的葡萄EST转录谱的横向比较分析,找出了它们与本研究中防卫相关基因EST的异同;初步鉴别出一些可用于辨识抗性的关键防卫基因,这些基因可用来作为开发抗病品种可能的分子靶标。3.利用富集青枯菌诱导的防卫相关基因cDNA文库与RACE结合的方法,克隆了11条关键防卫基因的全长cDNA,包括非特异性脂质转移蛋白(StLTPa1,StLTPa7,StLTPb1,StLTPb3, StLTPf10)、印膜蛋白(StREMa4)、植物铁蛋白(StFb8)、开心蛋白(StYPa5)、蛋白酶抑制子(StPIa3)、MBF转录因子(StMBFb7)、NAC转录因子(StNACb4)等。4.结合青枯菌诱导、化学处理、环境扫描电子显微镜和EDAX能谱分析,发现非特异性脂质转移蛋白基因StLTPa7在马铃薯与青枯菌互作早期表现与钙相关、受病菌诱导的系统表达模式,非生物激发子水杨酸、茉莉酸、脱落酸以及一定浓度的氯化钙能够诱导该基因增强表达,钙离子通道抑制剂钌红可以抑制其表达;原位杂交显示其转录物主要在马铃薯茎、叶维管束系统的韧皮部细胞中积累,暗示其在青枯菌入侵和定殖的微管束系统中发挥作用。StLPTa7基因可能参与马铃薯系统获得抗性中的长距离信号转导。对这种钙离子相关的基因表达模式在抗病反应中的作用机制提出了一个可能的工作模型。5.对另一个非特异性脂质转移蛋白基因StLTPa1的表达分析表明,该基因在马铃薯的抗病基因型和感病基因型中差异诱导表达:在抗病基因型中的诱导表现得更快、更高、更强。在远离根部接种位点的组织和靠近接种位点的组织中的表达差异明显。该基因同样受水杨酸、茉莉酸和脱落酸的诱导并产生一定时间的持续效应。原位杂交结果显示StLTPa1主要在马铃薯茎、叶维管束系统的韧皮部细胞中表达。其表达特性与植物的抗病类型有一定的因果关系。6.从马铃薯根际土壤和块茎中分离到两株低温适应型青枯菌菌株,对其进行微生物学、病理学特征分析和分子鉴定,发现其属于青枯菌生理小种3号、生化变种2号,具有小菌落、小细胞、胞壁加厚、生长缓慢、细胞自聚、对固体培养基具强粘附性、潜伏感染等诸多小菌落变异株(small colony variants,SCVs)特征。检测其潜伏感染马铃薯后,非特异性脂质转移蛋白基因StLTPa7和StLTPa1的表达,结果表明SCV不能诱导这两个基因的上调表达,说明该防卫基因的表达与病菌的致病性相关。通过文库构建、基因克隆及其表达模式研究,本论文确定了自根系接种的感病马铃薯基因型早期防卫相关基因的种类、数量;初步确定了非特异性脂质转移蛋白的表达模式;发现青枯菌诱导的马铃薯防卫相关基因与苛养木杆菌诱导的葡萄防卫相关基因在生物过程和分子功能方面具有很大的相似性,而在细胞组分上具有较大的差别,而且不同的接种方法诱导的马铃薯青枯病防卫相关基因有很大的差异;青枯菌小菌落变异株不能诱导nsLTP基因的上调表达表明了该基因的表达与青枯菌的致病性有关。这些实验结果为进一步大范围基因表达分析、深入研究青枯病防卫机制奠定了基础。更多还原

【Abstract】 On a global scale, potato (Solanum tuberosum) is an important crop species for the purpose of grain and vegetable consumption. Potato has wide adaptability, well fertility, high economic efficiency and rich nutritional value to be suitable for industrial processes. It plays a significant role in the food safety architectures. The plantation of potato and the production of potato are seriously threatened by bacterial wilt disease (Ralstonia solanacearum). It’s very important to improve the genetic resistance of potato cultivars for disease control. This presses for clarification of the molecular mechanism of potato-Ralstonia interactions and for discovery and utilization of the good resistance genes to improve the hereditary constitution for disease resistance. At the present time, very little is known about the amount and type of defense related genes or resistance genes expressed during the stages of potato-Ralstonia interaction, without mentioning the regulations. In order to acquire some useful information about the molecular mechanism, here we presented the screening and identification of potato defense related genes and their expression patterns.The SSH (suppression subtractive hybridization) technique was used to construct an EST library. SMART (switching mechanism at 5’ end of RNA transcript) and LD-PCR (long distance PCR) techniques were used to construct a full length cDNA library. Gene ontology method was used for classification and functional annotation of the related genes. For validation of the expression patterns of the defense related genes, qRT-PCR and Northern blotting were used. The main results obtained are listed bellow:To identify potato genes differentially expressed during the early stages of infection, two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) approach. Subtraction hybridization was conducted between cDNAs from stems and leaves of the susceptible genotype‘zhongshu 3’inoculated with R. solanacearum and the mock inoculated with water, in both directions. A total of 189 unique sequences were obtained from the library enriched for the inoculated susceptible‘zhongshu 3’sequences. By homology search and gene ontology analyses, the identified ESTs were mainly putatively categorized as belonging to: biology process (25.42%), cellular component (15.57%), and molecular function (40.98%). 22 ESTs (18.03%) were distributed among the unknown GO principles. RT-PCR analyses of 5 transcripts identified as differentially expressed between the inoculated and mock-inoculated plants revealed distinct kinetics of gene up-regulation at the early stage of the disease.Using gene ontology hierarchy and prior known genes, this is the first study to display the extent of EST transcript diversity in susceptible genotype of potato after infection by R. solanacearum which were poured into soil surrounding wounded roots. By the comparative analyses with other two similar research works including ESTs from potato inoculated with R. solanacearum by so called‘stem culture in Bacterial solution method’and ESTs from grapes inoculated with Xylella fastidiosa, some similarities and differences were identified among the defense-related genes. This study has identified likely molecular targets for developing bacterial wilt resistant varieties and for characterizing their key defense genes.A full length cDNA library was constructed, with which RACE (rapid amplification of cDNA ends) method was combined to clone a dozen of key defense genes involved in the early stages of potato-Ralstonia interaction. These genes included non-specific lipid transfer protein (StLTPa1, StLTPa7, StLTPb1, StLTPb3, and StLTPf10), remorin (StREMa4), plant ferritin (StFb8), yippee-like protein (StYPa5), proteinase inhibitor (StPIa3), multiprotein bridging factor (StMBFb7), NAC transcription factor (StNACb4) and so on.The non-specific lipid transfer proteins (nsLTPs) gene StLTPa7’s expression pattern was investigated by using combined methods of inoculation with the pathogen, chemical treatments with abscisic acid, salicylate and methyl jasmonate, and ESEM-EDAX (environmental scanning electron microscopy-electron dispersive x-ray spectrometer) analysis. The results showed that StLTPa7 gene displayed a complex Ca2+-associated pattern of expression during the early stage of potato-Ralstonia interaction. Transcripts of the StLTPa7 gene were systemically up-regulated by infection with R. solanacearum. It was stimulated by salicylic acid, methyl-jasmonate, abscisic acid and CaCl2. The Ca2+ channel blocker, ruthenium red, partially blocked pathogen-induced expression of StLTPa7. In situ hybridization results showed that StLTPb7 mRNA was localized in inner phloem cells of vascular tissues of potato leaves and stems. These results suggested that the gene was involved in long distance signaling and played a key role in plant defense mechanisms. A putative working model on the Ca2+-associated expression pattern of StLTPa7 was proposed to explain the mechanism involved in the defense response to bacterial wilt.The transcripts of another nsLTP gene StLTPa1 accumulated in potato leave and stem tissues infected by R. solanacearum and showed significant differences between resistant and susceptible genotypes of potato. StLTPa1 transcription was induced faster, higher and stronger in resistant genotypes compared with susceptible genotypes by the pathogen. Dominant differences in the pathogen-induced gene expression pattern between the upper and lower leaves and stems were observed within the same genotypes. In situ hybridization localized the StLTPa1 mRNA in inner phloem cells of vascular tissues. Salicylic acid, methyl jasmonate and abscisic acid induced StLTPa1 gene expression in different manners. These three signal molecules could produce long-lasting effects on the expression of StLTPa1. These results showed that StLTPa1 be pathogen-responsive gene in potato defenses and involved in a complex gene regulation network. These results suggested the causality between the gene expression and genotypes with different resistance.Two R. solanacearum cold-adapted strains isolated from the potato rhizosphere soil and potato tuber were identified as race 3 biovar 2 by microbiological, pathological and molecular methods. The strains had the classical characters of small colony variants (SCVs): pinpoint colonies that were 1/10 smaller than those of the wild-type strain, smaller cells with thicker extracellular substance, rough and strongly cohesive colony on solid media, cell-cell aggregating behavior, reduced growth rate and symptomless latent infection. To our knowledge this is the first isolation of SCVs in plant pathogen of R. solanacearum. The SCVs could not induce the up-regulated expression of StLTPa1 or StLTPa7 gene, showing that the expressions of these genes were related to the pathogenity of R. solanacearum.By construction of SSH-EST and cDNA library, gene cloning and expression analysis, the content of R.solanacearum-induced ESTs and the amount and type of defense related genes expressed during the early stages of potato-Ralstonia interaction was preliminarily determined and the biological processes in which the defense related genes involved in came to understand. This study has identified some key defence gene as likely molecular targets for developing bacterial wilt resistant varieties and for characterizing their resistance genes. Based on the comparative EST analyses, the similarities and differences were of defense-related genes involved in two types of vascular disease, and it is clear that different inoculation methods could induce different defence-related genes. The expression patterns of two defense genes of StLTPa1 and StLTPa7 were displayed. It was deduced that there must exist a complicated network composed with multiple genes, multiple pathways and multiple biological proceeds in the early stages of potato-Ralstonia interaction.Therefore the SSH library that we have created provides a basis for the identification of potato defense genes that are up-regulated during the early interaction with the pathogen, which could be important for next intensive study on gene expressions and for the molecular mechanism of potato defense to R. solanacearum.更多还原