Rop gene plays an important role in the process of symbiotic interaction between legume and rhizobium. A Rop gene Rac1 was amplified from the root cDNA of model legume Lotus japonicus and ligated to the prokaryotic expression vector pET28a. The engineering bacterium carrying Rac1 gene in E. coli BL21 (DE3) was obtained. The expression conditions of Rac1 protein was optimized, and the protein was purified by affinity chromatography. Rac1 protein expression levels of overexpression plant were detected by using the prepared antiRac1 polyclonal antibody. The results showed: (1) the prokaryotic expression vector of pET28aRac1 was constructed successfully by double enzyme digestion and DNA sequencing. (2) The optimal expression condition was induction temperature at 20 ℃, time at 6 h, and IPTG concentration at 0.1 mmol/L. The recombinant protein was highefficiency expressed in the form of soluble protein. The purified Rac1 protein was obtained by affinity chromatography and detected by SDSPAGE. The band size was about 25 kD, and the bands were clear and single. (3) Western blotting analysis showed that the polyclonal antibody could specifically react with the corresponding antigen, and the titer was high. (4) Agrobacterium mediated transformation of hairy roots method was used to obtain the positive hairy roots of Rac1 overexpression plant. The total protein of positive hairy roots was extracted and detected by Western blotting. The results showed that the expression of Rac1 protein in the Rac1 overexpression plant was significantly higher than that in the empty vector control, which proved the construction of overexpression vector was effective from translation level. The results showed that the preparation of Rac1 polyclonal antibody could specificity detect the native Rac1 protein in Lotus japonicus, which will provide a powerful tool for the further study of the biological function of Rac1 in the symbiosis signal transduction pathway.