Construction Of RNA Interference Vector Against Tobacco Mosaic Virus、potato Virus M And Cucumber Mosaic Virus And Transformation In Pepino (Solanum Muricatum Ait.)

Pepino(Solanum muricatum Ait.) is a perennial herb with vegetative propagation. It iseasy to be infected by many viruses due to continuous planting. In order to find a newmethod for defending viral diseases, the purpose of this experiment was to study three kindsof harmful viruses which were common in pepino. The RNAi target sequences that containsthe more effective small interfering RNA[siRNA, long19nt] fragments were filtered basedon the genes sequence of Tobacco mosaic virus (TMV) P126, potato virus M (PVM) CP andCucumber mosaic virus (CMV)2b in GenBank. The splicing-by-overlap extension PCR wasused to fuse three target sequences in together, and at the same time to construct a invertedrepetitive structure with pdk (pyruvate orthophosphate dikinase) intro of plant RNAiexpression vector pCEIHFR which was supposed to form intron splicing hpRNA (ihpRNA)after transcription.1. Selecting and cloning the target fragments from the gene sequence of TMV、PVMand CMV. Importing respectively the GenInfo Identifier is371786069、361071296and83282715into E-RNAi website to filter the target segments. These fragments contain moreeffective small interfering RNAs which can be used of building interference vector fragment.2. Fusion of target fragments and construction of expression vector. Using thetechnology of splicing-by-overlap extension PCR to building the fusion gene PC and TPCone after another, then amplification the reverse insertion fragment R. Then both the sensefragment TPC(F) and the antisense fragment CPT(R) were inserted into the two sides ofintron of vector pHANNIBAL in priority order, and the recombinant plasmid pHAIFR wasconstructed. The sense and antisense fragments with intron were further inserted into thevector pCEPSPS, and the recombinant vector pCEIHFR was acquired ultimately.3. Transformation of Pepino (Solanum muricatum). The recombinant plant-expressionvector pCEIHFR was introduced into Agrobacterium tumefaciens LBA4404by directlytransferring. Finally, the regenerated pepino plants were obtained and laying the foundationfor subsequent experiments.