茎瘤固氮根瘤菌（Azorhizobium caulinodans ORS571）可溶性趋化受体的功能研究
发布时间：2018-08-01 11:19
【摘要】：茎瘤固氮根瘤菌(Azorhizobium caulinodans ORS571)属于α变形菌纲,作为根瘤菌,A.caulinodans不仅可以在宿主植物毛萼田菁的根部和茎部结瘤,而且在自生状态下也具有固氮能力。趋化是指可运动的细菌感应环境中的化学浓度梯度,从而向有利于自身生长的环境方向运动的应激过程。在细菌的趋化过程中,趋化受体感应外界环境中的变化,并将信号传递至趋化通路中,引导菌体向最适的环境方向运动。本文对A.caulinodans的趋化系统进行了系统进化分析,并对其中的可溶性趋化受体的功能进行了初步探讨,针对可溶性趋化受体Icp B在细菌自生与共生时所发挥的功能进行了深入研究,为揭示其与宿主毛萼田菁的共生机制提供了重要的理论依据。主要的研究内容与结果如下:(1)A.caulinodans趋化系统的比较基因组学分析利用生物信息学及比较基因组学的方法对A.caulinodans的趋化基因簇与趋化受体基因进行分析,发现A.caulinodans的基因组中只含有一条趋化基因簇,还存在一个CheY基因与Che Z基因游离于趋化基因簇之外。基因组中共含有43条受体基因,所有受体的胞内部分序列保守,均属于38H型,并存在保守的甲基化位点与核心区域。甲基化酶CheR是非五肽依赖型。启动子预测显示趋化基因簇的启动子为σ54型。(2)A.caulinodans可溶性趋化受体功能的初步探讨构建了A.caulinodans中六个可溶性趋化受体的基因敲除菌株和回补菌株。利用半固体培养基检测突变株趋化能力发现所有受体缺失菌株的趋化圈均小于野生型,并且在固氮条件下缺失表型更明显。利用绿色荧光蛋白(GFP)对可溶性趋化受体进行亚细胞定位,结果显示所有的受体均定位在细胞的极端,并且在固氮条件下的表达量增强。竞争性结瘤实验表明除含有PiLZ结构域的AZC-3349外,其他五个含有氮端PAS结构域的受体所对应的突变株的竞争性结瘤能力均小于野生型菌株。(3)可溶性趋化受体IcpB在A.caulinodans自生与共生中的功能研究利用测定蛋白光吸收峰的方法验证了Icp B氮端的PAS结构域是与血红素相结合,并且位于154位点上的组氨酸(H154)为血红素结合的关键位点。利用氨基酸定点突变证明,H154对IcpB趋化、趋氧功能的发挥起到关键作用。Icp B的缺失使细菌的生物膜及胞外多糖产量增加,并且在固氮条件下二者的增量更为显著。A.caulinodans在自生状态下的固氮酶活均有所下降,icp B突变株所形成的茎瘤豆血红蛋白含量明显减少,瘤的体积与固氮酶活也明显小于野生型。
[Abstract]:As a rhizobia, A. caulinodans can not only nodule in the root and stem of the host plant, but also have the ability of nitrogen fixation in autogenous state. Chemotaxis refers to the stress process in which the chemical concentration gradient in the mobile bacteria induces the environment to move in favor of their own growth. In the process of bacterial chemotaxis, chemoattractant receptors induce changes in the external environment, and transmit signals to the chemotaxis pathway, leading the bacteria to move towards the optimal environment. In this paper, the phylogenetic analysis of the chemotactic system of A.caulinodans was carried out, and the function of soluble chemokine receptor (Icp B) was preliminarily discussed. The function of soluble chemotactic receptor Icp B in bacterial autogenesis and symbiosis was studied. It provides an important theoretical basis for revealing the symbiotic mechanism between Sesbania and its host. The main contents and results are as follows: (1) the comparative genomics analysis of A.caulinodans chemotaxis system uses bioinformatics and comparative genomics methods to analyze the chemotaxis gene cluster and chemokine receptor genes of A.caulinodans. It was found that there was only one chemoattractant gene cluster in the genome of A.caulinodans, and a CheY gene and Che Z gene were isolated from the chemoattractant gene cluster. There are 43 receptor genes in the genome, all of them are conserved, all of them belong to 38H type, and there are conserved methylation sites and core regions. Methylase CheR is non-pentapeptide dependent. Promoter prediction showed that the promoter of chemoattractant gene cluster was 蟽 54 type. (2) A preliminary study on the function of A.caulinodans soluble chemoattractant receptor was carried out to construct gene knockout and complement strains of six soluble chemoattractant receptors in A.caulinodans. The chemotaxis ability of mutant was detected by semisolid medium, and the chemotaxis circle of all receptor-deficient strains was smaller than that of wild type, and the deletion phenotype was more obvious under nitrogen fixation condition. The subcellular localization of soluble chemoattractant receptors by green fluorescent protein (GFP) showed that all the receptors were located at the extreme of the cells and the expression of these receptors was enhanced under nitrogen fixation. Competitive nodulation test showed that, except for AZC-3349 with PiLZ domain, (3) the function of soluble chemoattractant receptor IcpB in A.caulinodans autogenesis and symbiosis; The PAS domain at the nitrogen end of Icp B is combined with heme. And histidine (H 154), located at 154 site, is a key site for heme binding. It was proved by amino acid site-directed mutation that H154 played a key role in IcpB chemotaxis and oxygen-inducing function. The absence of Icp B increased the production of biofilm and extracellular polysaccharides of bacteria. In addition, the increment of A. caulinodans under nitrogen fixation condition was more significant. The activity of nitrogenase in autogenetic condition decreased significantly. The hemoglobin content of stem tumour bean formed by mutant strain B was obviously decreased, and the volume of tumor and activity of nitrogenase were also significantly lower than that of wild type.
【学位授予单位】：中国科学院烟台海岸带研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q939.114
本文编号：2157411
[Abstract]:As a rhizobia, A. caulinodans can not only nodule in the root and stem of the host plant, but also have the ability of nitrogen fixation in autogenous state. Chemotaxis refers to the stress process in which the chemical concentration gradient in the mobile bacteria induces the environment to move in favor of their own growth. In the process of bacterial chemotaxis, chemoattractant receptors induce changes in the external environment, and transmit signals to the chemotaxis pathway, leading the bacteria to move towards the optimal environment. In this paper, the phylogenetic analysis of the chemotactic system of A.caulinodans was carried out, and the function of soluble chemokine receptor (Icp B) was preliminarily discussed. The function of soluble chemotactic receptor Icp B in bacterial autogenesis and symbiosis was studied. It provides an important theoretical basis for revealing the symbiotic mechanism between Sesbania and its host. The main contents and results are as follows: (1) the comparative genomics analysis of A.caulinodans chemotaxis system uses bioinformatics and comparative genomics methods to analyze the chemotaxis gene cluster and chemokine receptor genes of A.caulinodans. It was found that there was only one chemoattractant gene cluster in the genome of A.caulinodans, and a CheY gene and Che Z gene were isolated from the chemoattractant gene cluster. There are 43 receptor genes in the genome, all of them are conserved, all of them belong to 38H type, and there are conserved methylation sites and core regions. Methylase CheR is non-pentapeptide dependent. Promoter prediction showed that the promoter of chemoattractant gene cluster was 蟽 54 type. (2) A preliminary study on the function of A.caulinodans soluble chemoattractant receptor was carried out to construct gene knockout and complement strains of six soluble chemoattractant receptors in A.caulinodans. The chemotaxis ability of mutant was detected by semisolid medium, and the chemotaxis circle of all receptor-deficient strains was smaller than that of wild type, and the deletion phenotype was more obvious under nitrogen fixation condition. The subcellular localization of soluble chemoattractant receptors by green fluorescent protein (GFP) showed that all the receptors were located at the extreme of the cells and the expression of these receptors was enhanced under nitrogen fixation. Competitive nodulation test showed that, except for AZC-3349 with PiLZ domain, (3) the function of soluble chemoattractant receptor IcpB in A.caulinodans autogenesis and symbiosis; The PAS domain at the nitrogen end of Icp B is combined with heme. And histidine (H 154), located at 154 site, is a key site for heme binding. It was proved by amino acid site-directed mutation that H154 played a key role in IcpB chemotaxis and oxygen-inducing function. The absence of Icp B increased the production of biofilm and extracellular polysaccharides of bacteria. In addition, the increment of A. caulinodans under nitrogen fixation condition was more significant. The activity of nitrogenase in autogenetic condition decreased significantly. The hemoglobin content of stem tumour bean formed by mutant strain B was obviously decreased, and the volume of tumor and activity of nitrogenase were also significantly lower than that of wild type.
【学位授予单位】：中国科学院烟台海岸带研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q939.114
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