Molecular genetic studies of DNA: DNA relatedness and symbiotic genes of Rhizobium japonicum

摘要

Rhizobium japonicum is the bacterial symbiont of the soybean, an agronomically important plant, and fixes atmospheric nitrogen in exchange for nutrients from the host plant. A complex series of molecular events occur in the recognition process involving the bacterium and the plant root system which results in the eventual formation of a specialized, bacteria inhabited, nodule structure. The goal of this study was directed toward understanding the location and organization of the genes involved in the symbiosis event. The conservation of these genes, and the DNA sequences surrounding them, was examined by molecular hybridization analysis. In addition, large regions of a plasmid from a fast-growing R. japonicum strain were physically and genetically mapped;Structural nitrogen fixation (nif) and nodulation (nod) genes were hybridized to purified plasmid and total DNA from slow- and fast-growing R. japonicum. The slow-growing R. japonicum strains harbor nif genes on the chromosome, or large megaplasmid, and the fast-growing R. japonicum strains have nif genes located on large plasmids. Hybridization of the symbiotic gene probes to EcoRI restriction enzyme digestions of plasmid and total DNA from both types of strains shows distinct conservation of the symbiotic genes. The slow-growing strains, however, did not hybridize strongly to the nod probe, indicating divergence at this locus. The DNA sequences surrounding the symbiotic genes are conserved with respect to hybridization and restriction digestion patterns. Plasmid DNA sequences in slow-growing strains, which do not contain genes known to be involved in symbiosis, are highly conserved in most of the strains examined. The plasmids isolated from fast-growing R. japonicum are also highly conserved;A large, 350 kb plasmid from fast-growing strain PRC193 was cosmid cloned and large regions were mapped. Restriction enzymes HindIII, HpaI and KpnI were used to determine overlapping sequences of the insert DNA in the clones. A novel, hybridization procedure was used which specifically determined the presence and size of repeated sequences in the plasmid. Nif and nod probes were used to assign the symbiotic gene sequences to locations on the restriction enzyme map.