Construction Of RNAi Vectors Of Bactrocer A Dorsalis Channel Gene And Cytochrome Gene Then Genetic Transformation In Citrus

Bactrocera dorsalis, the Oriental fruit fly, is widely distributed in the citrus production areas in China. It is a serious threat to Chinese citrus industry and which lead to a huge economic losses every year. In addition, it can also endanger more than250kinds of fruits and crop, such as oranges, mangoes, carambola and so on. Now, chemical control is the important measure to control the B. dorsalis. But the chemical pesticides applied improperly, not only affect fruit safety, at the same time can also lead to serious drug resistance. Therefore, chemical control is hardly to achieve the sustainable control of B. dorsalis. Sodium channel genes main function of target, when use DDT and synthetic pyrethroid insecticides to control insect. Cytochrome p450monoxygenases are similar gens protein in structure properties, reports indicate that those are concerned with resistance for organochlorine and strain insecticide resistance. Application of RNAi technology can affect the insects sodium channel gene and Cytochrome P450genes expression normally, then, it can enhance the sensibility of insects and affect its growth and reproduction. Therefore, we can use genetic transformation mediated by agrobacterium tumefaciens plants to get some B. dorsalis resistant plants. This test will take sodium channels JN416983and P450CYP6EK1gene specific sequence as the target sequence, to construct the two genes RNA interference vectors. Then, we use genetic transformation mediated by agrobacterium tumefaciens plants to get some new resistance materials for B. dorsalis.1. Construction of RNAi vectorsSearch the sodium channel genes and cytochrome P450genes sequence of B. dorsalis on NCBI, compare the conservative sequence, design specific primers, on the restriction site was added the upstream sense primer and down stream sense primer to construction of RNAi vectors.A. Construction of PFGC5941-C1R1/C2R2vectorWe added restriction enzymes on upstream and downstream of forward and reverse fragments purpose of sodium channel gene is Xhol, Ncol, Xbal and BamHI. Cloning with forward and reverse duplicate genes. Target genes of C1R1vector was digested by XhoI and Ncol and the PFGC5941vector was digested by XhoI and NcoI. Connect them with T4connection enzymes, then transferred the connection product into E. Coli DH5a, randomly selected monoclonal colony, PCR detection screening of the right single colony. Extraction of plasmid, the "PFGC5941-forward fragment C1R1" vector was digested to prove the vector is correct; the "PFGC5941-forward fragment C1R1" vector was digested by Xbal and BamHI, and in the meanwhile the reverse fragment C2R2was digested by XbaI and BamHI. Connect them with T4connection enzymes, then transferred the connection product into E. Coli DH5a, randomly selected monoclonal colony, obtaining the "PFGC5941-forward fragment C1R1/reverse fragment C2R2" vector through digestion.B. Construction of PFGC5941-P1F1/P2F2vectorUsing the same method as mentioned above. We added restriction enzymes on upstream and downstream of forward fragment purpose of cytochrome P450gene is XhoI and NcoI, added restriction enzymes on upstream and downstream of reverse fragment purpose of sodium channel gene is XbaI and BamHI. After Double enzyme and connect them with T4connection enzymes, transferred the connection product into E. Coli DH5a. Extraction of plasmid, Finally, obtaining the "PFGC5941-forward fragment P1F1/reverse fragment P2F2" vector through digestion and PCR.2. Transformation mediated by agrobacterium tumefaciens plants and verification of transgenic plants.PFGC5941-C1R1/C2R2and PFGC5941-P1F1/P2F2vectors were transferred into agrobacterium EHA105with electric shocks. Infecting JinCheng epicotyl segments with Agrobacterium which containing the plasmid PFGC5941-forward fragment C1R1reverse fragments C2R2after PCR detection. All the explants were cultured with herbicides Basta to Screening positive for adventitious bud. There were15adventitious bud which have resistance herbicide. We designed the specific primers L1/R1according to the BAR gene in PFGC5941vector, after PCR detection we obtained9strains transgenic plants.Infecting Jincheng epicotyl segments with Agrobacterium which containing the plasmid PFGC5941-forward fragment PIF1/reverse fragments P2F2as above method. All the epicotyl segments were cultured with herbicides Basta to Screening positive for adventitious bud. There were12adventitious bud which have resistance herbicide. Finally, we obtained14strains transgenic plants using the specific primers L1/R1for PCR analysis.