Mesorhizobium loti MAFF303099 was used to construct a random mutant library with transposon Tn5-sacB mutagenesis technique.A total of about 10000 independent mutants were obtained.A method for rapidly identifying mutants from the library was established.The mutant of the symbiotic nodule gene nodB was identified through screening the mutant library.The results showed that the transposon Tn5-sacB was inserted into the genome DNA of all the mutants in the mutant library,with the tri-parental mating transformation efficiency of 3.75×10-5.The MAFF303099 mutant of nodB did not form nodules on Lotus japonicus roots comparing with the wildtype.