Construction Of Brassica Oleracea Expressing DsRNA And Its Resistance Against Plutella Xylostella

Brassica oleracea is cultivated all over the world and occupies an important position in vegetable production,which is seriously damaged by Plutella xylostella.Recently,transgenic plants expressing ds RNA targeting genes in pest have received more and more attention,and have great potential in the pest control.It is essential to develop transgenic B.oleracea expressing ds RNA for the control of P.xylostella.In this experiment,the juvenile hormone cyclohydrolase（JHEH）and ecdysone receptor（ECR）genes in P.xylostella were selected as target genes with the fragments of 437 bp and 412 bp as the targets for RNA interference,respectively.The att B1 and att B2 sequences was added to the both ends of the target fragment and then the Gateway homologous recombination reaction was performed to successfully construct the plant expression vector expressing the corresponding ds RNA.In this experiment,the seed sterilization was optimized to find out the best sterilization was using 3%Na Cl O.On the basis of the optimized seed sterilization,the method of agrobacterium transformation was used to construct transgenic B.oleracea expressing the ds RNA of target gene fragment in P.xylostella.The genomic DNA and m RNA of the transgenic B.oleracea were used to verify the presence of ds RNA-expressing unit by the PCR and RT-PCR,respectively.Four strains of transgenic B.oleracea expressing ds RNA of Px JHEH and 2 strains expressing the ds NRA of Px ECR were successfully obtained.After the 4^th instar larvae of P.xylostella fed on the transgenic plant leaves expressing the ds RNAs of Px JHEH and Px ECR for 5 d,the mortalities of P.xylostella reached 15.00%and 6.67%,respectively.The larvae feeding on the transgenic plants had a significantly higher mortality rate comparing to the larvae feeding on the control plant.The expression level of Px JHEH in P.xylostella feeding on transgenic plant expressing ds RNA of Px JHEH was suppressed to 55.3%,and the expression level of Px ECR in P.xylostella feeding on transgenic plant expressing ds RNA of Px ECR was suppressed to 64.33%compared with the control.The above results indicate that transgenic cabbage expressing ds RNA targeting the pest gene is feasible for the control of P.xylostella,which provides new ideas and approaches for the prevention and control of P.xylostella in the future.