Studies On NifR3 Gene Of Sinorhizobium Fredii HN01

Sinorhizobium.fredii HN01 is a fast-growing Rhizobium strain that is able to form nitrogen-fixing nodules on soybean.It is suited for commercial production because its fast grower and fewer extracelluar polysaccher characteristics.The nitrogen fixation genes(nif genes)are essential for nitrogen-fixing.In this study,we detected the activity of a potential promoter region P1 upstream of the nifR3 and researched the function of nifR3 of S.fredii HN01 in symbiotic nitrogen fixation.A 1.5 kb DNA fragment include the whole nifR3 gene and a putative promoter gerion P1 were firstly cloned by PCR method.Sequence analysis showed that nifR3 gene shares 80%and 83%identities with that of Sinorhizobiurn meliloti 1021 on nucleotide and amino level,respectively.The putative promoter fragement P1 was fused to the gus reporter gene to determine its promoter activity.A 280-bp of the putative promoter region P1 fragment showed promoter activity by measurement ofβ-Glucuronidase activity. The suicide plasmid pK18mob was for construction of the mutant of HN01 disrupted in nifR3,and Non-polar mutant strain HNR33 was constructed.A recombinant plasmid pHNBR3 was firstly constructed by cloning the nifR3 gene into the expression vector pLAFR3.The pHNBR3 was then transformed into the mutant strain HNR33 to construct the complemental strain HNBR3.Effects of nitrogen sources on the growth of the mutants were tested.There was little difference between the wild strain HN01 and the mutant strains HNR33 and HNBR3 when ammonium chlorite or nitrate was used as sole nitrogen source.Plant assays were carried out with HN01,HNR33 and HNBR3 to investigate their symbiotic nitrogen fixation.There were significant difference on the total number of the nodules and nitrogenase activity tested between the mutants and wild strain.On the other hand,little difference on the nodulation efficiency and the nodule weight per plant was found between them.These results suggest that the nifR3 genes are required for efficient nitrogen fixation and nodulation in HN01.