Construction And Characters Of Sinorhizobium Fredii Mutant In NifR3-ntrBC

Biological nitrogen fixation is of tremendous importance to the enviroment and to world agriculture. And root-nodule symbioses with legumes are the most important kind of all kinds of nitrogen fixations. S.fredii . HN01 is a fast-growing Chinese isolate that is able to form nitrogen-fixing nodules on soybean. It is better suited for commercial inoculation production because it is a fast grower and produces fewer.The aim of this study was to clone nifR3-ntrBC genes, construct mutants, and study the effects of mutations of these genes on nodulation, nitrogen fixation and growth, so as to elucidate the possible functions to symbiosis.In this study, a recombinant plasmid pGXHN300 was first mutated by using of transposon Tn5gusA5 mutagenesis, and a recombinant plasmid pGXHN305 in which the Tn5gusA5 being inserted in ntrB was detected by analysis of the insert site. Through DNA sequencing the whole nifR3-ntrBC sequence was obtained. 3 recombinant plasmid were constructed via pK18mob and 3 mutants were gained by transported the 3 recombinants into HN01 by triconjugation respectively, via homologous recombination caused by a single crossover event。 A plasmid pGXHN306 containing nifR3-ntrBC genes was constructed by subcloning pGXHN300 and 3 complement strains were constructed by pGXHN306 transported into the 3 mutants.The tests of growth status were performed with HN01, mutants and complement strains. The results showed that the growth status were normal when growing on medium with ammonium chlorite as the only N sources but the mutants defective when on medium with nitrate as the solo N sources.the nodulation assay revealed that GXHNR lost the ability of nodulation while the other two mutants a day delay in nodulation time and the nodulation efficiency ability was decreased compared with HN01. Nitrogenase activity assay showed that nitrogenase activity of the mutants was also decreased compared with HN01 . We conclude that the nifR3-ntrBC genes are required for efficient nitrogen fixation and nitrogenase activity in HN01.