| Bradyrhizobium japonicum USDA110 is a symbiotic bacterium that nodulates soybean.It is very important for agricultural production to study the mechanism of slow rhizobium participating in symbiotic nitrogen fixation and find high efficient nitrogen fixing strains.C-di-GMP(Cyclic guanosine diphosphate),as the second messenger in bacteria,is involved in the regulation of bacterial motility,extracellular polysaccharides and biofilm formation.the GGDEF domain is fully capable of DGC activity and either an EAL or HD-GYP domain is capable of c-di-GMP hydrolysis.Both regulate the intracellular c-di-GMP content.With the rapid development of genome sequencing,these potential protein resources in bacteria have been excavated rapidly.However,both the mechanism of c-di-GMP synthesis and degradation and the role of c-di-GMP in symbiotic process have not been identified in USDA110.In this study,the metabolic gene activity of c-di-GMP in USDA110 and its role in symbiosis with plants were studied.The results are as follows:1.42 genes encoding GGDEF,EAL or HD-GYP protein were identified by bioinformatics method in B.japonicum USDA110.Then 16 genes about c-di-GMP metabolic protein of the USDA110 strain were overexpressed into the plasmid vector and transformed into PAO1 and ? wsp F,respectively.If the protein contains DGC activity,it can produce the shrinking phenotype.If the protein contains PDE activity,it can restore? wsp F to the wild type colony phenotype.The results showed that 6 genes were detected DGC activity in the gene containing the GGDEF domain,In 4 potential PDE activity genes,only the gene bll2340 containing the HD-GYP domain can transform the ? wsp F colony phenotype from collapse to smooth.2.The effects of 16 genes on the EPS,motility and biofilms in PAO1 and ?wsp F were tested.It was found the overexpression GGDEF domain gene bll4347,bll5123,bll1362,bll7151 and blr2401 could increase the production of EPS and biofilm in PAO1,andinhibite the swarmming motility.However,overexpression HD-GYP domain gene bll2340 could decrease the EPS content of ?wsp F,its swarmming ability was not affected,and the twitching motility ability was restored to the wild type level.3.The activity of acetylene reductase in nodulation was determined by gas chromatography.The activity of nitrogenase in the nodulation of the strain containing GGDEF domain gene bll4347,bll5123,blr0624,bll2326 was higher than that of the strain containing EAL and HD-GYP.It was suggested that c-di-GMP was involved in nodule formation and affected the activity of nitrogenase.4.The symbiosis mechanism of c-di-GMP between rhizobium and legume was studied by test of exopolysaccharide,motility and adhesion to legume plant roots.More viscous substances were produced on the surface of the colonies when overexpressed bll4347,bll5123,bll7151,blr0624 gene,and those strain's mobility and adhesion to the root hairs were also enhanced.Overexpression bll2326 gene strain produced viscous substances on the surface,but there was no significant change in the motile and adhesion ability of the strain.The overexpression of blr2401,bll7113,bll3041,bll1500,bll6704,bll2340 gene showed that the colony surface of the strain was desiccated.The overexpression of bll2326,bll3041,blr0402,blr1644,bll2340 could weaken the adhesion ability of root hair.5.In this study,4 strains of USDA110 with high mobility,EPS production and adhesion ability of root hairs were obtained by over-expression of USDA110 c-di-GMP related metabolic genes.In this study,USDA110 c-di-GMP related genes were selected as the research object to explore the effect of GGDEF signaling pathway on intracellular c-di-GMP level,and constructed a high-yield EPS strain.Studying the changes of EPS from the point of view of GGDEF protein signaling pathway can deeply reveal the horizontal factors that affect the production of EPS and participate in nitrogen fixation symbiosis of host plants.It provides theoretical support for artificial improvement of symbiotic efficiency of rhizobium and plant in the future. |
PDF Full Text Request