莖瘤固氮根瘤菌（Azorhizobium caulinodans ORS571）可溶性趨化受體的功能研究
發(fā)布時間：2018-08-01 11:19
【摘要】：莖瘤固氮根瘤菌(Azorhizobium caulinodans ORS571)屬于α變形菌綱,作為根瘤菌,A.caulinodans不僅可以在宿主植物毛萼田菁的根部和莖部結(jié)瘤,而且在自生狀態(tài)下也具有固氮能力。趨化是指可運動的細菌感應(yīng)環(huán)境中的化學(xué)濃度梯度,從而向有利于自身生長的環(huán)境方向運動的應(yīng)激過程。在細菌的趨化過程中,趨化受體感應(yīng)外界環(huán)境中的變化,并將信號傳遞至趨化通路中,引導(dǎo)菌體向最適的環(huán)境方向運動。本文對A.caulinodans的趨化系統(tǒng)進行了系統(tǒng)進化分析,并對其中的可溶性趨化受體的功能進行了初步探討,針對可溶性趨化受體Icp B在細菌自生與共生時所發(fā)揮的功能進行了深入研究,為揭示其與宿主毛萼田菁的共生機制提供了重要的理論依據(jù)。主要的研究內(nèi)容與結(jié)果如下:(1)A.caulinodans趨化系統(tǒng)的比較基因組學(xué)分析利用生物信息學(xué)及比較基因組學(xué)的方法對A.caulinodans的趨化基因簇與趨化受體基因進行分析,發(fā)現(xiàn)A.caulinodans的基因組中只含有一條趨化基因簇,還存在一個CheY基因與Che Z基因游離于趨化基因簇之外。基因組中共含有43條受體基因,所有受體的胞內(nèi)部分序列保守,均屬于38H型,并存在保守的甲基化位點與核心區(qū)域。甲基化酶CheR是非五肽依賴型。啟動子預(yù)測顯示趨化基因簇的啟動子為σ54型。(2)A.caulinodans可溶性趨化受體功能的初步探討構(gòu)建了A.caulinodans中六個可溶性趨化受體的基因敲除菌株和回補菌株。利用半固體培養(yǎng)基檢測突變株趨化能力發(fā)現(xiàn)所有受體缺失菌株的趨化圈均小于野生型,并且在固氮條件下缺失表型更明顯。利用綠色熒光蛋白(GFP)對可溶性趨化受體進行亞細胞定位,結(jié)果顯示所有的受體均定位在細胞的極端,并且在固氮條件下的表達量增強。競爭性結(jié)瘤實驗表明除含有PiLZ結(jié)構(gòu)域的AZC-3349外,其他五個含有氮端PAS結(jié)構(gòu)域的受體所對應(yīng)的突變株的競爭性結(jié)瘤能力均小于野生型菌株。(3)可溶性趨化受體IcpB在A.caulinodans自生與共生中的功能研究利用測定蛋白光吸收峰的方法驗證了Icp B氮端的PAS結(jié)構(gòu)域是與血紅素相結(jié)合,并且位于154位點上的組氨酸(H154)為血紅素結(jié)合的關(guān)鍵位點。利用氨基酸定點突變證明,H154對IcpB趨化、趨氧功能的發(fā)揮起到關(guān)鍵作用。Icp B的缺失使細菌的生物膜及胞外多糖產(chǎn)量增加,并且在固氮條件下二者的增量更為顯著。A.caulinodans在自生狀態(tài)下的固氮酶活均有所下降,icp B突變株所形成的莖瘤豆血紅蛋白含量明顯減少,瘤的體積與固氮酶活也明顯小于野生型。
[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.
【學(xué)位授予單位】：中國科學(xué)院煙臺海岸帶研究所
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q939.114
本文編號：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.
【學(xué)位授予單位】：中國科學(xué)院煙臺海岸帶研究所
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q939.114
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相關(guān)期刊論文 前2條
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2 陳文新,汪恩濤,陳文峰;根瘤菌-豆科植物共生多樣性與地理環(huán)境的關(guān)系[J];中國農(nóng)業(yè)科學(xué);2004年01期
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