| Nitrogen-fixing microorganism provides biological creatures about three-quarters of the nitrogen source. Nitrogen-fixing microorganism is nitrogen-fixing microbes by using nitrogenase converts the atmospheric nitrogen into ammonia that can be absorption and utilization between biological of process, not to consume a large amount of energy, not to damage the ecological environment and pollution.That Is good for environmental protection and the sustainable development of agricultural ecology. One of the highest efficiency in the biological nitrogen fixation is symbiotic nitrogen fixation. Symbiotic nitrogen fixation is the result of evolution between host plants that is beneficial symbiosis, interaction with each othe and Cooperative coevolution. In the process of nitrogen fixation of nodule, host plant growth metabolism of the nitrogen source is provided by nitrogen-fixing microbes, and nitrogen metabolism of microbial life the carbon source are necessary to provide by host plant.Symbiotic interaction of the legume plants and the rhizobia presents narrow spectrum, i. e. different rhizbia strains are just able to infect their corresponding host plants and form nodules. Symbiosis is a complex biological process, its successful proceeding relies on the signaling recognization, transmission and exchange by the rhizobia and the host legume, which is determined by a series of genes regulation of the both sides. Flavonoid compound, a signaling molecule secreted by legume roots, activates the expression of nodulation regulator gene NodD. As a transcription factor, NodD protein binds to the promoter region of other Nod genes and regulates their expression and results in the formation of another signaling molecule (the nodulation factors (NFs)) derived from rhizobia. Then, the NFs act upon the host plant roots and induce a series of early host plant biochemical and physiological response and changes of form, opening the process of nodulation.A two-component system (two-component system, TCS) is a kind of signal transduction components, it is widespread in bacteria, it is the main control system for bacteria to adapt to changes in intracellular physiological and external environment. Typical of a two-component system is controlled by a Histidine kinase (Histidine kinase, HK) and a Response regulatory proteins (Response regulator, RR). The histine kinase feels internal and external stimulation, sutophosphorylate, then pass phosphate groups to regulatory proteins reaction, so as to control the downstream related gene expression. Histidine kinases is mainly composed by signal structure domain, the polymerization and histidine phosphate transfer structure domain (DHp) and ATP-binding and structure of the catalytic domain (CA), after two structure domain contains a conservative N, H, F, G1and G2box (box). Reaction effect of regulatory proteins is generally consists of an effector domain and an accept domain (REC).The acidic soil accounted for a quarter of the world’s land, soil acidification affected the survival of plants and soil microorganisms and the interaction between host plants and soil microorganisms. Symbiotic nitrogen fixation of rhizobia in the soil and its host legume plants have also been seriously affected, influenced the rhizobia in N2fixed, and then affected the N input in agricultural systems. In some cases, nodule bacteria represents acid sensitive components in the symbiotic relationship; Sinorhizobium meliloti is particularly sensitive to acidity. This creates a problem, what mechanism do rhizobia to adapt to acid environment, why are rhizobium that some is acid tolerance and another is sensitive to acid?[Objective]1.The role of the two-component system, actS/actR genes from Sinorhizobium meliloti played in symbiosis and acid regulation was investigated.2. The role of the two-component system, ActS/ActR proteins from Sinorhizobium melilotiregulated downstream transcription factors and signal transduction pathways was studied.[Methods] The DNA fragments of the target gene or its proximate regions were cloned into a suicide vector to construct a recombinant plasmid, which was transferred into S. meliloti1021by triparental mating. The null mutants screened on LB/MC plates with appropriate streptomycin or neomycin or8%sucrose, by using the method of point mutations will put the ActS/ActR phosphorylation site mutation of two-component system into other amino acids to obtain mutant strains, were used to analyze the acid tolerance and symbiotic nitrogen fixation.[Results] The deletion mutants of actS or actR were not screened, but a plasmid insertion mutant of actR was successfully constructed. This mutant was sensitive to low pH, and showed lower competitive nodulation on alfalfa (Medicago sativa).[Conclusions] The ActS/ActR two-component system is required for S. meliloti survival and competitive nodulation on alfalfa, including that it plays regulatory roles in acid adaptation of bacteria and the symbiotic nitrogen fixation process. |
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