Abstract:genes including RbohA(Lj5g3v1497840),RbohB(Lj6g3v1549190) and RbohC (Lj5g3v1497820) were found to be primarily expressed in nodules based on the transcriptions of Rboh genes in symbiosis in Lotus japonicus. Their function involved in symbiosis were further studied in Lotus japonicus.The subcellular location of proteins,the expression pattern of Rboh genes,the proteins function and the promoter activity of Rboh genes in Lotus japonicas were studied with methods of molecular biology,cell biology and genetics. The results showed that LjRbohA,LjRbohB and LjRbohC proteins were localized at the plasma membrane.The transcript of LjRbohA,LjRbohB and LjRbohC accumulated abundantly in roots and nodules. The activity of LjRbohA and LjRbohB promoters were detected at vascular tissue,nodule,and the lateral root primordia.The numbers of nodules were reduced in LjRbohB,LjRbohALjRbohC and LjRbohALjRbohBLjRbohC knockout transgenic roots compared with control transgenic roots. It is indicated that these three Rbohs might have an overlapping function in regulating nitrogen fixation symbiosis in L. japonicus.