Abstract:The TMV-replicase gene was regarded target-directed sequence for RNA interference in this paper. The target sequence was linked to pMD18-T Simple Vector and replicated in E.coli DH 5α. after obtained by RT-PCR reaction. The pMD18-T Simple vector (which contains target sequence) was digested restrictively by double-endonuclease, after purifying the digested product was inserted into pUCCRNAi vector by the counterrepeat way. Then the counterrepeated structure of target sequence was inserted into pC2300-35s-OCS expression vector after enzyme disgested. The recombination plamid was transferred into Agrobacterium tumefaciens which only contained assistant plasmid in order to construct double-plasmid expression vector. With PCR reaction and restriction endonuclease digestion, the target gene sequence was comfirmed in engineering-bacterium.