Screening And Functional Verification Of Genes Related To Symbiotic Nitrogen Fixation In Mesorhizobium MAFF303099

The reciprocal symbiosis between legumes and rhizobia can transfer the N₂in the air to ammonia,which is a requisite part of the N cycling.In the process of plant growth,symbiotic nitrogen fixation can provide with nitrogen nutrients that can be absorbed and utilized by plants.This way of supplying nitrogen nutrition is environmentally friendly,economical and efficient,and has important application value in agriculture.The establishment of an effective symbiotic interaction system requires complex gene expression regulation by rhizobia and legumes at the same time.In this study,Mesorhizobium japonicum MAFF303099 strain was used as the research object.Through the bio-analysis data of its transcriptome,expression profile and other data,a batch of genes that were up-regulated during the symbiosis process were screened.The candidate gene mutant strain was constructed by homologous recombination method.After these rhizobia were inoculated with Lotus japonicus,the effects on the number of nodules and nitrogenase activity were observed.The aim is to select mutants with a clear phenotype to study their physiological functions and the mechanisms involved in regulating symbiotic nitrogen fixation.The main findings are as follows:1.The 14 genes up-regulated in symbiotic nitrogen fixation in Rhizobium MAFF303099 were screened,and corresponding gene knockout vectors were constructed based on the p K18-mob-Sac B-Genta vector.The knockout of the target gene is achieved through the principle of homologous recombination.The mutant of Rhizobium MAFF303099 was screened by sucrose and antibiotic pressure.The results showed that when the concentration of gentamicin in the culture medium was 50?g/m L,the concentration of fosfomycin was 100?g/m L,and the concentration of sucrose was 10%,the gene knockout mutant of MAFF303099 strain could be obtained efficiently.14Rhizobium mutants were successfully constructed by this method2.Inoculate 10 mutants of rhizobia to the roots of Lotus japonicus respectively.During the peak period of nitrogen fixation,observe the difference of the number of nodules and nitrogenase activity between mutants and the wild-type rhizobia.Statistical analysis of the difference between the activity of nitrogenase and the number of nodules and the wild type.In two repeated nodulation experiments of the?mll6127,the nodules number was remarkable increased against control.;?mll7572 nitrogenase activity was significantly increased twice and?mlr5883 nitrogenase activity decreased significantly and stably.3.Perform bioinformatics analysis for mlr5883,and found that it has a conserved domain of aspartate aminotransferase,and has homologous genes in many other species of rhizobia.There are two similar genes on the MAFF303099 plasmid PLMa that located in the same branch of the evolutionary tree.Knockout of the most similar mlr9118 did not differ significantly in enzyme activity.4.Construct a mlr5883 replenishing expression strain,and incubating with Lotus japonicus for 21 days,it was found that its nitrogenase activity of forming nodule returned to the wild-type level.It was verified that the phenotype of reduced nitrogenase activity was caused by the deletion of mlr5883.5.Prokaryotic expression and purification of GST-MLR5883,and tested its AAT activity is 16.67U/mg.6.The tissue sections of the root nodules found that the morphology of the cells infected by the?mlr5883 strain was not significantly different from that of the wild type,but the number and diameter of starch granules in the root nodules were reduced.It indicates that the mutation of this gene may affect the metabolism and utilization of the carbon source supplied by the plant by the bacteroid.