The conserved region of β-Gal gene of papaya (Carica papaya L.) pulp was cloned, which encoded a key enzyme of β-galactosidase involved in the cell wall degradation. The RNAi intermediate expression vector of pKAN/RG was constructed containing β-Gal gene in an inverted repeat orientation with the help of pKANNIBAL vector. hptⅡ gene of the modified pCAMBIA 1300 vector was replaced by the hairpin structure of pKAN/RG, which resulted in the formation of intermediate expression vector of p1300-/MFRG. Single T-DNA region of p1300-/MFRG was isolated and incorporated into the pCAMBIA 2301 vector to produce the RNAi Two-T-DNA plant expression vector of p2301/TTRG. The transformation of p2301/TTRG into Agrobacterium tumefaciens EHA 105 was confirmed by restriction enzyme analysis and PCR assay. The embryogenic calli of papaya which had kanamycin resistance and GUS expression were obtained by genetic transformation.