According to the pathogen–derived resistance, a framework of a universal RNAi vector with hairpin structure was constructed by inserting a CaMV35s promoter–intron–terminator into a plasmid of pCAMBIA 2301. By adopting the conservation sequence of virus gene p25, p20 and p23 in Citrus tristeza as templates, three RNAi vectors, including ds2301–p25, ds2301–p20 and ds2301–p23, were separately obtained through inserting three pairs of positive–sense strand and antisense strand into the RNAi vector framework, and the ds2301–p23 was infiltrated into Mexican lime leaves at the same time. Northern blot detection approach was adopted to find the specific siRNA in probe of gene p23 labeled with digoxigenin. The results showed that the transient expression of Agrobacterium in Mexican leaves could triggered RNAi and generated specific siRNA, which indicated that the framework of RNAi vector was successfully constructed, and the three vectors with CTV virus gene segments could be applied in the analysis of gene function and the selection of resistance resource.