The Study Of Exopolysaccharide Gene And Their Role In The Symbiosis In Bradyrhizobium Japonicum USDA110

In recent years,excessive using of chemical nitrogen fertilizer has caused a great negative impact: soil fertility declines,chemical nitrogen fertilizer 's effect is getting lower and lower,serious pollution to the environment and so on.The symbiotic system of rhizobia and leguminous plants has the strongest nitrogen fixation capacity,and the amount of nitrogen fixation is more than half of the total biological nitrogen fixation,therefore,the researchers of biological nitrogen fixation believe that biological nitrogen fixation should be fully utilized to decrease the amount of chemical nitrogen fertilizer.In the symbiotic nitrogen fixation system,it has known that the extracellular polysaccharide of rhizobium is important in the symbiotic nitrogen fixation,the research of rhizobia extracellular polysaccharide not only helps us to understand the mechanism beteween bacteria and the host,but also contributes to the expansion of host range,improves the conditions of symbiosis,provides the theory basis for improving the efficiency of nitrogen fixation,helps to decrease the negative impact of chemical nitrogen fertilizer.In addition,many rhizobium can secrete large amounts of high viscosity or special rheological polysaccharides,which have potential application in industry.This project chooses Bradyrhizobium japonicum USDA110 as object of study,whose specific host is soybean,and researches the function of extracellular polysaccharide of this rhizobium in this model symbiotic system,in order to improve the efficiency of nitrogen fixation and decrease the use of chemical nitrogen fertilizer.This study used Bradyrhizobium japonicum USDA110 as the original strain to construct the mutant library.By using pLG107,the Tn5 transposon insertion mutant library has been constructed.By diluting and coating about 50000 single colonies and comparing the size of colony,I obtained 5 mutants that were interesting,because they are obvious smaller than WT,named BJ25,BJ32,BJ33,BJ57,BJ60.This BJ25 mutant has the biggest distinguish,and the distinguish is always stable,so finally I selected this BJ25 as final object of this study.By HiTAIL-PCR,the flanking sequence of Tn5 transposon was amplified,moreover,the inserted gene was confirmed by resequencing and completely same with the insertion site confirmed by HiTAIL-PCR.Clearly these genes have glycosyl transferase?succinyl-CoA synthase,geranylgeranyl pyrophosphate synthase's function,especially the mutant BJ25's insertion site is blr2358 and only has one insertion site,this gene's forecast function is glycosyl transferase.A paper told us that a gene cluster blr2358~blr2381 is about extracellular polysaccharide,and this blr2358 is the first gene of this gene cluster,so this mutant BJ25 strain was elected as this study's key object.After confirmed mutant strains BJ25's insertion site,I built the complementary strain of this mutant,and then compared all kinds of biological phenotypes and functions of wild type,mutant strain,wild type with an empty vecter,the complementary strain of this mutant,in order to explore the purpose gene's probable function.Comparing these treatment group with the size of single colony,the number of nodules and height of plant in plant experiment,the results show that BJ25's colony is obvious smaller than WT,nodule is less and height is lower than WT,but their complementary strain is the same with WT.This preliminary results show blr2358 is likely to be involved in the regulation of extracellular polysaccharide and can affect the nitrogen-fixing nodules.These results contribute to deeper study,and provide theoretical basis of utilizing this bacteria.