【作者】 李江利；

【导师】 蒋士君；

【作者基本信息】 河南农业大学 ， 植物病理学， 2011， 硕士

【摘要】 植物病原细菌的三型分泌系统(TTSS)是丁香假单胞菌(Pseudomonas syringae)侵染过程中重要的蛋白分泌系统。TTSS的编码、蛋白分泌等受到hrp基因的调节。研究hrp基因在病菌侵染过程中的表达情况有助于进一步探索hrp基因的功能,揭示植物病原细菌的分子致病机制,而egfp标记突变体的构建是开展寄主-病原物的互作研究的重要策略。本试验拟以增强型绿色荧光蛋白基因(egfp)作为报告基因,以植物丁香假单胞菌烟草致病变种和番茄致病变种为材料,以其hrpA基因和hrpZ基因为靶标,通过体外重组和双亲结合技术,分别构建靶标基因与egfp融合表达的突变体,以及靶标基因缺失的egfp标记突变体,为今后研究丁香假单胞菌-植物的互作提供有用的材料工具。主要试验结果为:(1)以丁香假单胞菌番茄致病变种DC3000菌株和烟草致病变种4号菌株的基因组DNA为模板,扩增出了hrpA、hrpZ基因及其上下游片段;以pEGFP-C1为模板扩增出了egfp ORF全长。将相关的DNA片段进行体外重组后,得到了4个与预期大小一致的目的大片段CDE3、CTE1、CTE3、CTE4,它们分别为hrpZPto::egfp、hrpAPsta::egfp、hrpZPsta::egfp融合型片段和△hrpZPsta::egfp缺失型片段,为下一步构建融合型、缺失型突变体的构建奠定了基础。(2)试验将获得的目的大片段CDE3、CTE1、CTE3、CTE4分别连接至克隆载体pMD19-T,提取阳性克隆质粒pMD19-CDE3、pMD19-CTE1、pMD19-CTE3、pMD19-CTE4的DNA与自杀型质粒pKSM1的DNA同时进行BamHI和XbaI双酶切,切胶回收的酶切片段进行连接,得到了适于进行双亲接合所用的自杀型重组质粒pKSM-CDE3、pKSM-CTE1、pKSM-CTE3、pKSM-CTE4。(3)将含有质粒pKSM-CDE3的大肠杆菌S17-1与番茄致病变种DC3000野生型菌株进行双亲接合后获得了hrpZPto::egfp融合型突变体;将含有质粒pKSM-CTE1、pKSM-CTE3、pKSM-CTE4的大肠杆菌S17-1分别与烟草致病变种4号菌野生型菌株进行双亲结合获得了hrpAPsta::egfp、hrpZPsta::egfp融合型突变体和△hrpZPsta::egfp缺失型突变体。将获得的4个突变体菌株划线于KB培养基(30μg/ml Nalr)上,在紫外灯下观察发现hrpAPsta::egfp融合型突变体有明显的绿色荧光,而hrpZPto::egfp、hrpAPsta::egfp融合型突变体及△hrpZPsta::egfp缺失型突变体则无绿色荧光。(4)与野生型菌株相比,以上构建的4个egfp标记突变体人工接种烟草后,pv. tomato DC3000的hrpZPto::egfp融合型突变体的过敏性诱导能力明显降低;hrpAPsta::egfp、hrpZPsta::egfp融合型突变体及△hrpZPsta::egfp缺失型突变体的致病性也明显降低。更多还原

【Abstract】 Type III secretory system (TTSS) of plant pathogenic bacteria is an important protein secretion system for Pseudomonas syringae infection. TTSS is regulated by Hrp gene. It is helpful to further explore the the function of hrp gene and reveal the molecular pathogenicity of pathogens that to study the hrp gene expression during the infection. To construct egfp marked-mutants is an important strategy for studying the interaction between plants -bacteriaIn this study, we intended to use enhanced green fluorescent protein (egfp) gene as a reporter, and hrpA gene and hrpZ gene of Pseudomonas syringae pv.tomato and Pseudomonas syringae pv.tabaci as the targets, to construct egfp-marked fusion mutants and egfp-marked deletion mutants, respectively, with recombination in vitro and parental conjugation. The major results are as follows:(1)hrpA、hrpZ gene and its down-stream and up-stream were amplified from the genomic DNA of P.s. pv.tomato DC3000 and P.s.pv.tabaci 4. The egfp full-legth ORF was amplified from plasmid pEGFP-C1 DNA. After recombination in vitro by PCR, 4 expected fragments CDE3、CTE1、CTE3、CTE4 were obtained that laid the foundation for construction of fusion mutants and deletion mutants.(2)After the expected recombined-fragments were cloned to vector pMD19-T, the positive cloning vector pMD19-CDE3, pMD19-CTE1, pMD19-CTE3, pMD19-CTE4 and the suicide plasmid pKSM1 was double digested with BamHI and XbaI, respectively. And the digested fragments were collected and ligated. So the research got plasmids pKSM-CDE3, pKSM-CTE1, pKSM-CTE3, pKSM-CTE4 which were suitable for parental conjugation.(3)The research obtained fusion mutant hrpZPto::egfp through the parental conjugation between Ecoli S17-1 containing the plasmid pKSM-CDE3 and P.s.pv.tomato wild type strain DC3000. Meantime, the research obtained fusion mutants hrpAPsta::egfp、hrpZPsta::egfp and deletion mutant△hrpZPsta::egfp through the parental conjugation between Ecoli S17-1 containing the plasmids pKSM-CTE1, pKSM-CTE3, pKSM-CTE4 separately and P.s.pv.tabaci wild type 4. When the fusion mutants and deletion mutant were cultured on KB plate(30μg/ml Nalr),only the fusion mutant hrpZPsta::egfp had significant green fluorescent colonies, while fusion mutants hrpZPto::egfp、hrpAPsta::egfp and deletion mutant hrpZPsta::egfp not.(4)Compared with the wild type strains, the resultant 4 egfp-marked mutants artificially inoculated to tabaco lealves showed different HRP capacity. The fusion mutant hrpZPto::egfp revealed a significantly reduced hypersensitivity-inducing, while the other 3 egfp marked mutants hrpAPsta::egfp, hrpZPsta::egfp and△hrpZPsta::egfp showed significant reduced pathogenicity to tobacco.更多还原