| 其他摘要 | Sebania cannabina is an excellent legume crop for improving coastal saline soils, which could from root nodules with Rhizobium and Ensifer related groups. Ensifer alkalisoli YIC4027 was isolated from root nodules of S. cannabina in the Yellow River Delta. It had good growth-promoting ability, nitrogen-fixing ability and strict host specificity, and it occupied a dominant position. Rhizobium can symbiotically fix nitrogen with S. cannabina, which can increase the tolerance of S. cannabina to soil nitrogen deficiency and promote its growth and development. Therefore, the S. cannabina-Rhizobium symbiosis improvement system is of great significance to help the transformation of coastal saline land. The establishment of the symbiosis system of rhizobia and host S. cannabina is first that the rhizobia reach the roots of the plant through chemotaxis, and then through a series of interactions, finally form an infection cavity and symbiotic nodules. Therefore, chemotaxis plays an important role in the process of symbiotic nodulation and bacterial recognition. In the chemotactic pathway of bacteria, chemoreceptors are responsible for sensing external chemical stimuli and realizing signal amplification. They are divided into transmembrane chemoreceptors and soluble chemoreceptors. YIC4027 exhibited obvious host specificity in symbiosis with S. cannabina, and had a great advantage in the abundance of Rhizobium sibiricum, but its chemotactic signal transduction pathway and symbiosis-specific mechanism have not been reported yet. There is an urgent need to carry out relevant research for discussion.In this study, the comparison of genes and protein sequence analyses of the chemoreceptors in E. alkalisoli YIC4027, and functions of the transmembrane chemoreceptors CDS_1340, CDS_2131, CDS_4153 and the soluble chemoreceptor CDS_2666 were discussed. It laid the foundation for revealing the symbiosis mechanism of YIC4027 and host S. cannabina. The main research contents were as follows: chemoreceptor genes were analyzed via bioinformatic and comparative genomic methods. It found specific chemoreceptor proteins and predicted their related functions; Mutants of four chemoreceptors were constructed by the method of triparental conjugative transfer and their related functions were identified. The main results were as follows:(1) Chemoreceptors analysis of E. alkalisoli YIC4027. The results of biosynthesis analysis showed that there were two che clusters in E. alkalisoli YIC4027. We identified 13 methyl-accepting chemotaxis protein homologs containing conserved methylated domain and contained different signal sensing structures, of which 10 were transmembrane chemoreceptors and 3 were soluble chemoreceptors, only one of them had a PAS domain, and the PAS domain combined with the FAD prosthetic group to sense the redox state. In addition, the cytoplasmic domains were composed of 36 heptad repeats, except CDS_5173, which was composed of 36 heptad repeats. They had a conserved core region and methylation sites. The methylase CheR was pentapeptide-independent. The che cluster was predicted to regulate by σ54 promoter.(2) The four chemoreceptor mutants had no significant effect on the basic growth characteristics of E. alkalisoli YIC4027. Firstly, the four chemoreceptor-encoding genes CDS_1340, CDS_2131, CDS_4153 and CDS_2666 in strain YIC4027 were knocked out to obtain mutants Δ1340, Δ2131, Δ4153 and Δ2666, respectively. The basic physiological parameters of the mutants were determined, including the growth ability of the strains and the salt tolerance test of the strains. The results showed that the growth abilities of the mutant strains were consistent with that of the wild type. The results of the salt tolerance of the strains showed that the growth abilities of the 4 mutants were basically the same as that of the wild type, and they could grow normally under the concentration of 1-4% NaCl.(3) Chemoreceptors CDS_2131 and CDS_2666 played a role in the chemotaxis of E. alkalisoli YIC4027. Chemotaxis detection mainly included the use of capillary method to detect the chemotaxis of the root exudates of S. cannabina, and the use of semi-solid plate method to detect the chemotaxis of chemical signal substances and oxygenation. The results of chemotactic ability of the root exudates of S. cannabina showed that the chemotactic ability of the mutants Δ2131 and Δ2666 was lower than that of the wild type, which was 34.7% and 83.4%, respectively. This study showed that the mutant Δ2666 had a more obvious impaired ability to induce secretions. The mutants Δ2131 and Δ2666 had different degrees of missing responses to different chemical signal substances. Oxygenation experiments showed that the mutant Δ2666 had a weakened ability to sense oxygen. (4) Chemoreceptors CDS_2131 and CDS_2666 affected the autogenous state of E. alkalisoli YIC4027. Autogenous state ability detection mainly included cell coagulation, biofilm content, extracellular polysaccharide production, and hydrogen peroxide stress. The results of the coagulation experiment under oligotrophic conditions showed that the flocculation abilities of Δ2131 and Δ2666 were enhanced, which increased by 57.5% and 60.0% in the early stage of culture, but there were no significant difference with the wild type YIC4027 in the later stage. The biofilm production was detected by the method of crystal violet staining. The results showed that the biofilm yields of mutants Δ2131 and Δ2666 were 68.2% and 87.2% higher than those of the wild type, respectively. The production of extracellular polysaccharides (EPS) was tested, and the results showed that the colony morphologies were different on the medium of different carbon sources, and the production of extracellular polysaccharides of the mutants Δ2131 and Δ2666 were higher than that of the wild type. The hydrogen peroxide stress experiment was used to detect the resistance to reactive oxygen species, and the results were consistent with the results of extracellular polysaccharides. The survival rate of wild type was 26%, and the survival rates of mutants Δ2131 and Δ2666 were 41% and 45%, respectively. (5) Chemoreceptors CDS_2131 and CDS_2666 affected the establishment of a symbiosis system with S. cannabina. The symbiosis state ability detection showed that the colonization ability and competitive nodulation ability of the mutants Δ2131 and Δ2666 were weaker than the wild type and complemented mutant strains, indicating that CDS_2131 and CDS_2666 participated in the interaction with the host S. cannabina. Inoculation of wild type and mutants to cultivate S. cannabina, the results showed that there was no significant difference in the growth of S. cannabina. The colonization abilities and the nodulation ability of mutants Δ2131 and Δ2666 were weaker than that of the wild type. The results showed that CDS_2131 and CDS_2666 were involved in the interaction with the host S. cannabina.In summary, this study found that YIC4027 encoded a large number of chemoreceptor genes and had different types of ligand domains, indicating that it might have a complex chemotactic signal transduction network; The functions of two chemoreceptors CDS_2131 and CDS_2666 had been identified: affecting the movement of bacteria, which in turn affected the ability of root colonization and nodulation. On the one hand, this study not only enriches the cognition of chemoreceptor functions, but also initially reveals that the chemoreceptors affect the symbiosis process of YIC4027 and the host S. cannabina. On the other hand, it has improved the understanding of the establishment mechanism of the symbiosis improvement system of rhizobia and S. cannabina, and provided a theoretical basis for the biological improvement model of coastal saline land. |
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