MOLECULAR GENETICS STUDIES ON THE NODULE FORMATION EFFICIENCY GENE OF Bradyrhizobium Japonicum STRAIN GX201

In this study, a recombinant plasmid pGXN201 was first mutated by using of transposon Tn5gusA5 mutagenesis, and three mutant plasmids , pGXN215, pGXN216 and pGXN217 were achieved. The 3.4kb EcoRI DNA fragment of the pGXN201 and the mutant EcoRI DNA fragments of the three mutant plasmids were subcloned into the EcoRI sites of the clone vectors Ml3 and the pGEM3Z-f(+) respectively.Through DNA sequencing and DNA sequence analysis by comparing with the Bradyrhizobium Japonicum DNA sequences reported, we found that there are four intact Open Reading Frames in the 3.4kb fragment and it was highly homologous to the Bradyrhizobium Japonicum DNA sequence reported, and the Tn5gusA5 containing in the pGXN217 was inserted in one of the four Open Reading Frames .This Open Reading Frame located on about 5.5kb upstream of the nfeC gene, which includes 207bp nucleotides, coding 68 putative amino acids and this is completely identical to that of the Bradyrhizobium Japonicum DNA sequence reported.pGXN217 was transferred into Bradyrhizobium Japonicum strain GX201 by triparental mating using the helper plasmid pRK2073. Marker exchange was achieved by selection on YMA containing Sm, Km ,Gm, Spc et al four kinds of antibiotics. Mutants were confirmed by southern blotting and hybridization with the wild-type region and the Tn5 fragment respectively. The mutant strain GX217 was achieved. The 3.4kb EcoRI fragment of the plasmid pGXN201 was firstly subcloned into the EcoRI site of the plARF3, then the recombinant plasmid was introduced into the mutant strain GX217 by triparental mating using the helper pRK2073 and the mutant strain rGX217 was achieved.Plant assay was performed with the wild-type strain GX201,the mutant strain GX217 and the function recovered strain rGX217.The results indicate that the mutant strain GX217 showed a day delay in nodulation time and its abilities of nodule formation efficiency and competitive nodulation were apparently decreased compared with the parental strain GX201 respectively. We conclude that the gene we studied is a new loci required for efficient nitrogen fixation and for competitive nodulation of soybeans by Bradyrhizobium Japonicum strain GX201.