The Construction Of Nh4+-excerting Mutant Strains And Research Of NH4+-Excerting Mechanism Of Kosakonia Radicincitans GXGL-4A

Kosakonia radicincitans GXGL-4A,a nitrogen-fixing bacterial strain that isolated from maize root was firstly subjected to evaluate promotional effect on the growth of maize and rice.Different densities of GXGL-4A were used in the pot experiment,and the results showed that GXGL-4A had significant influence on the growth of maize and rice within a certain range of application concentrations.Prokaryotic expression vector containing an enhanced green fluorescent protein gene,egfp was constructed and then applied for tracing colonization of GXGL-4A in seedling of maize using fluorescence microscopy.The tagged bacterial cells of GXGL-4A were successfully detected in different parts of plant including root,stem and leaf of maize.It should be noted that root surface was considered to be the most important colonization site.An enclosed system(with sterile soils)and an open system(with natural soils)were used for the dynamic monitoring of GXGL-4A population,respectively.The gfp-marked GXGL-4A bacterial cells were released to rhizosphere soil of maize,and then the population dynamic trend within half-a-month treatment was determined through plate dilution method.The population of GXGL-4A decreased whether in open system or in enclosed system.The population of GXGL-4A reduced sharply one day after inoculation,and subsequently the dynamic curves declined slowly.In conclusion,strain GXGL-4A showed apparent colonization capacity in rhizosphere of maize.Complete genome sequencing was performed using the third generation sequencing platform,PacBio System and genome features within Kosakonia species were compared with each other.Two gene clusters associated with nitrogen fixation were eventually identified in GXGL-4A and the number of nitrogen-fixing genes decreased significantly compared with Pseudomonas stutzeri A1501.The ammonium vector,AmtB of GXGL-4A was predicted to be a membrane protein with eleven transmembrane helixes by bioinformatic analysis.In the past decade,AmtB protein had been confirmed to be responsible for the transportation of extracellular ammonium to maintain dynamic balance of nitrogen pool of bacterial cells.Construction and optimization of CRISPR/Cas9 editing system of GXGL-4A was carried out.Mutants with modified ammonium secretion abilities were identified and further characterized with respcect to growth curve and nitrogenase activity.