Cloning And Related Funtion Of CysDN In Sinorhizobium Fredii WGF03

Biological nitrogen fixation is the most important form of nitrogen into the biosphere on earth, and it has a special meaning for agricultural production. Sulfur is playing a important role between nitrogen-fixation and nodulation, modern research has already proved that Rhizobium Nod factor reducing end of the sulfur modification determine the host specificity of Rhizobium. Furthermore, the sulfur modification of exopolysaccharide with nodulation and nitrogen fixation have a certain relationship. The purpose of this work is the way to get through mutation to obtain related genes of the sulfate assimilation pathway, we constructed the homologous single exchange mutantion, then complemented the function of these strains, which reveals the sulfur metabolism genes playing a important role in the process in symbiotic rhizobia and legumes. A random insertion mutant library of Sinorhizobium fredii WGF03 was constructed by three parental hybridization with the plasmid pTnMod-RKm' as transposon vector. A mutant strain, could not metabolize sulfate while could use cysteine as sulfur source, was selected by MM medium with different sulfur sources from the library. Extremely obtained an operon related to sulfate assimilation pathway-cysDN, through Molecular cloning and sequence analysis showed that the operon has 92% and 96% similarity with the cysDN gene sequence of Sinorhizobium sp. BR816 at nucleotide and amino acid level respectively. with Rhizobium tropici the cysH, cysD, cysN has 83% of gene homology. Suicide plasmids pK18mob were constructed cysD and cysN homologous single exchange mutant and obtained mutantions cysDF/WGF03, cysDR/WGF03, cysNF/WGF03, cysNR/WGF03. A recombinant plasmid pLAFRJ+cysDN was constructed by using plasmid pLAFRJ ligated with cysDN, and transferred into above mutants respectively to obtained complement strains.The results of growth tests found that the mutants had no growth on the MM medium with sulfate as sulfur source, but the complement strains could grow normally, indicating that cysDN operon related to sulfate assimilation. The plant tests indicated that the mutant strains delayed 1-2 d to nodulate than the wild strain, and nitrogenase activity and nodulation efficiency in mutants were slightly lower than the original strain, but there was no differences on the number and weight of nodules and the dry weight of plant. Sinorhizobium fredii WGF03 would be accounted for 70% in competitive nodulation, while the mutant strain only 30%. This indicate that the mutant operon cysDN has a impact on competitive nodulation ability of rhizobium, but the mutant cysDF/WGF03, cysDR/WGF03, cysNF/WGF03, cysNR/WGF03 nitrogenase activity lower than the original strain WGF03, indicating this operon of WGF03 with nitrogenase activity have a certain relationship.