Efficiency Of Different Horsebean Rhizobial Nitrogen Fixation And Lipopolysaccharide Transporter

In recent years,due to the irrational application of chemical fertilizers in agriculture,many environmental and ecological problems such as soil compaction,fertility degradation,and eutrophication of water bodies has been increasingly appeared.The research of legume-rhizobium symbiotic nitrogen fixation system,which can increase soil fertility,grain yield,and protect the ecological environment,is of great significance.Rhizobia are obligate aerobic Gram-negative bacteria that exist in the soil and they can make the symbiotic association with leguminous plants to form a legume-rhizobium symbiotic nitrogen-fixing system,which converts atmospheric nitrogen into a plant-available compound.They provide nutrition for leguminous plants and have the highest nitrogen fixation efficiency in biological nitrogen fixation.Therefore,it is of great value to study the rhizobium-legume symbiotic nitrogen fixation process,and then further improve its nitrogen fixation efficiency.The symbiotic nitrogen fixation by rhizobium and leguminous plants is a complex biochemical process.The lipopolysaccharide,which located on the outer membrane of the cell,serves as a signal recognition factor and plays an important role in maintaining cell membrane stability,protecting against plant antibiotic damage,and inducing immune response process.The change of the structure of lipopolysaccharide can cause the loss of the symbiotic nodulation process,which indicates that lipopolysaccharide also plays an important role in the process of rhizobium-legume symbiotic fixation of nitrogen.To study the mechanism of nitrogen fixation will provide important evidence to improve fixation efficiency.In this study,five strains of rhizobium(Bnc3-4,Blgs21-3,9Z-3,Blgs19-2,and Bdz5-3)with different nitrogen fixation efficiency were used as the research objects.The effects of bean seed exudate on the growth of strains,lipopolysaccharide production,lipopolysaccharide band pattern,and the transcriptional levels of lipopolysaccharide transporter gene of rhizobium were investigated,in the aim to provide support for elucidating the roles of lipopolysaccharide in the process of rhizobium-signal recognition.The main research contents and results are as follows:(1)The bean exudate can promote the growth of rhizobium,and this promotion effect increases with the proliferation of the concentration of exudate;when the cell grows to logarithmic growth phase,the strain can grow faster after a short period of induction time.When the culture exposed to exudate for about 4 h,the growth of Bnc3-4,Blgs21-3 and 9Z-3 change a lot,but the significant changes for strains Blgs19-2 and Bdz5-3 happened latter(8 h).(2)Bean exudate has different effects on the yield of lipopolysaccharide and SDS-PAGE band patterns of rhizobium with different nitrogen fixation efficiency.Lipopolysaccharide content was measured by spectrophotometry(204 nm and 258 nm)and phenol sulfuric-acid methods.It was found that the content of lipopolysaccharide under the exudate treatment samples was slightly higher than that of the control.BN-PAGE electrophoresis results showed that the lipopolysaccharide bands were different between strains,between treatment and control sample.Additionally,the cluster analysis of electrophoretic bands showed that small similarity was found between the strains Blgs19-2,9Z-3 and Bdz5-3.(3)Based on the effect of exudate on the lipopolysaccharide and the cluster analysis,two strains,Bdz5-3 and Bnc3-4,were selected for further study.After the strain grows to the exponential growth phase,the exudate was added and samples were taken at different time points.The results of real-time quantitative PCR showed that the transcriptional expression levels of Lpt gene were different at different time points.The effects of exudate on the expression level of Lpt in rhizobium were different,but the relative expression levels of Lpt genes in the same strain were basically the same.Among them,the Lpt genes of Bdz5-3 were up-regulated immediately after the addition of exudate(15 min),while the transcriptional expression levels of Bnc3-4 Lpt genes reached the highest level when exposed to the exudate for 8 h.