【作者】 常佳丽；

【导师】 韦革宏；

【作者基本信息】 西北农林科技大学 ， 微生物， 2010， 硕士

【摘要】 本论文采用来自中国西北地区白花三叶草的两株根瘤分离菌hbh54.2和hbh3.1作为研究对象,对二者的关系进行了初步的研究。经过对两株菌的16S rDNA全序列进行测定,构建了系统发育树状图,确定了根瘤分离菌hbh54.2位于土壤杆菌属Agrobaterium,hbh3.1位于快生根瘤菌属Rhizobium。在根瘤菌多样性研究领域,从根瘤中分离得到土壤杆菌的现象极为常见,但是对于这种现象还没有一个确切的解释,本研究希望通过设计一系列实验,能够初步揭示在三叶草根瘤中土壤杆菌与根瘤菌的关系问题,证明土壤杆菌存在于根瘤中的意义所在,为进一步的深入研究提供理论基础。本研究通过对两株供试菌的功能基因进行PCR扩增,检验其是否具有共生性以及致病性,结果表明,土壤杆菌hbh54.2在常规条件下无法扩增产生相应的结瘤基因nodA和固氮基因nifH,与植物回接结瘤预实验中其不能与三叶草共生结瘤的结果一致,此外,二者均不能扩增出致病基因virC的目的条带,间接证明二者均不携带Ti质粒,不具有致病性。本研究通过设计盆栽法植物回接结瘤实验,检验两株供试菌单独回接以及共同回接时对寄主植物白花三叶草各项生长指标的影响,结果表明,与不接菌对照相比,单独回接以及共同回接所导致的植株整体生长量的变化有一定的差异,此外,共同回接还能够促进根瘤菌hbh3.1与寄主植物的结瘤情况,体现在根瘤数量的增加,该结果从植物的角度证明了土壤杆菌hbh54.2与根瘤菌hbh3.1相互作用的意义可以体现在促进原寄主植物的生长发育上。经过一系列促植物生长生理特性测定实验,结果发现,与根瘤菌hbh3.1相比,土壤杆菌hbh54.2具有较强的分泌铁载体和生长素吲哚乙酸IAA的能力,从生理的角度出发,可以推断土壤杆菌hbh54.2能够弥补根瘤菌hbh3.1的一些缺陷与不足,当前者与后者共存于根瘤中时,前者通过分泌IAA,为植物提供外源生长激素,控制植物体内IAA的浓度,调节植物的生长发育;通过分泌铁载体,螯合细胞周围有限的铁元素,除供自身所需之外,也有利于后者对铁元素的吸收,进而提高其固氮效率。本研究还利用增强型绿色荧光蛋白EGFP基因标记土壤杆菌hbh54.2,将标记的土壤杆菌单独接入白花三叶草根部,并设计同步实验让其与未标记的根瘤菌hbh3.1共同侵染宿主植物,以及待植物与hbh3.1共生结瘤后再引入共生体系的根际附近,结果表明,只有当二者共同存在的情况下,标记土壤杆菌才能进入根瘤组织内部,单独侵染以及结瘤后再侵染,标记土壤杆菌均无法进入植物体内,说明土壤杆菌hbh54.2不具有单独入侵白花三叶草的能力,只有在相应根瘤菌hbh3.1的携带之下才有可能进入宿主植物体内并定殖于根瘤组织中。本论文首次将促植物生长生理特性实验结果与标记菌回接实验结果结合起来,初步推断土壤杆菌能够在相应根瘤菌的携带下进入根瘤组织,在根瘤内,土壤杆菌通过分泌IAA调节植物生长,使植物为根瘤菌提供充足的碳源,同时土壤杆菌还能分泌铁载体,为维持根瘤菌固氮酶(钼铁蛋白)正常的生理活性提供铁元素。这个推断初步揭示了土壤杆菌与根瘤菌共存于根瘤中的意义所在,为今后进一步深入研究二者的关系开辟了一个新的方向。更多还原

【Abstract】 Hbh54.2 and hbh3.1, two strains isolated from the same nodule of white clover in China’s northwest region , are used in this thesis for the preliminary study of the relationship of Rhizobium and Agrobacterium. A phylogenetic tree is constructed on the basis of the 16S rDNA sequences of the two strains, which locates hbh54.2 to the genus of Agrobaterium and hbh3.1 to the genus of Rhizobium. As we know, in the study of rhizobia diversity, it is very common to isolate Agrobacterium-related strains from root nodules. However, up to now, there is no precise explanation to account for this phenomenon. In our study, a series of experiments are designed under the aim of revealing the relationship of Rhizobium and Agrobacterium that can exist in the same nodules of the host plant, which can provide theoretical basis for the further research.In our study, through the PCR amplification of the functional genes of the two tested strains, we can verify whether the tested strain is symbiotic or pathogenic. As is shown from the results, tested strain hbh54.2 habours no nodulation gene(nodA) and nitrogen-fixation gene(nifH), which is consistent with the results of the plant nodulation pre-experiment as is demonstrated that it cannot induce root nodules in the white clover. In addition, virulence gene(virC) can not be amplified when using the template of the two tested strains, indicating that both of the tested strains do not carry Ti plasmid, which is representative of pathogenecity.Through pot plant-nodulation experiments, it can be seen from the consequence that there is difference on the mass growth of the host plant among different kinds of disposal condition, one is designed to inoculate with only one kind of strain, another is co-inoculating with two tested strains simutaneously or unsimutaneously. Additionally, it also shows that co-inoculation can promote the nodulation process. In a word, the results indicate that the significance of the interaction of hbh54.2 with hbh3.1 can be reflected in aspects of promoting the growth and development of the original host plant.After a series of experiments testing the physiological characteristics of plant-growth promoting bacteria, we find an interesting phenomenon. Compared with hbh3.1, hbh54.2 has a strong ability of producing siderophores and secreting plant-growth hormone (indole acetic acid, IAA). Therefore, it may be inferred that hbh54.2 can make up some of the defects and shortcomings of hbh3.1, when the two strains co-exist in the same nodules, the former can provide exogenous growth hormones to host plants through the secretion of IAA, controling the concentration of IAA of the plants in vivo and regulating plant growth condition, what’s more, through the secretion of siderophore, it can also chelate Fe element which is limited around the cells, not only for its own requirements, but also better for the latter in enhancing the efficiency of nitrogen fixation.This study also used the EGFP(enhanced green fluorescent protein) gene as the marker gene of hbh54.2, expecting to find out how hbh54.2 could locate in the nodule. As the results demonstrate, only under the circumstance of inoculating with hbh54.2(marked with egfp) and hbh3.1 together, can hbh54.2 show its existence in the nodules, while there is no fluorescence detected in the nodules in other situation, which indicates that hbh54.2 do not have the ability to invade the white clover alone, only with the help of the appropriate rhizobia(hbh3.1) can it be carried into the host plants and colonize in the root nodule tissues.This paper integrated the results of experiments testing the physiological characteristics of plant-growth promoting bacteria and plant nodulation tests with egfp marked strain for the first time. After analyzing the whole results, we conclude that Agrobaterium can enter the root nodules under the carrying of the appropriate Rhizobium; in the nodules, in order to make host-plant provide sufficient carbon source for Rhizobium, Agrobaterium can regulate plant-growth condition through the secretion of IAA, and also it can secrete siderophores to help Rhizobium absorb Fe for the maintenance of the normal physiological activity of nitrogenase (MoFe protein). The inferred conclusion revealed the significance of the coexistence of Agrobacterium with Rhizobium in the nodules, opening a new direction for further study of the relationship of Agrobacterium and Rhizobium in the future.更多还原