| It is a promising approach to use RNAi technique to control insect pest by expressing ds RNA in microorgism or entomopathogen.Using Bacillus thuringiensis（Bt）-mediated RNA interference can make double-effect of toxin and RNAi in poisoning of pests.In this experiment,the ds RNA targeting Plutella xylostella arginine kinase gene（Px AK）,integrinβ1 subunit gene（Px Intβ1）,and both genes were expressed in Bt HD73,separately,to inhibit the expression of Px AK/Px Intβ1 in P.xylostella.The toxicity of these Bt strains against P.xylostella was compared to clarify the control effect of Bt-mediated RNAi technology by targeting multiple genes simultaneously.In this experiment,three gene fragments of Px AK and Px Intβ1,and the fusion fragment of Px AK and Px Intβ1（Px AK-Intβ1）were amplified from the pHells-ds AK-β1 plasmid stored in the laboratory.The optimized pro3a7 promoter was selected to express ds RNA.The forward promoter,gene fragment and reverse promoter were ligated in order.The ligated fragment was inserted into the vector pHT315 to form a dual-promoter RNAi vector.The RNAi vector pHT315-ds AK,pHT315-ds Intβ1 and pHT315-ds AK-Intβ1were obtained individually.The three RNAi vectors pHT315-ds AK,pHT315-ds Intβ1,pHT315-ds AK-Intβ1 and pHT315 vector were separately hypomethylated and transferred to the strain HD73 of Bt to obtain four engineering strains HD73-pHT315-ds AK,HD73-pHT315-ds Intβ1,HD73-pHT315-ds AK-Intβ1 and HD73-pHT315.The engineered bacteria normally e xpressed the toxic protein,and most of the toxin protein crystals ha d the same shape with HD73.HD73-pHT315-ds AK,HD73-pHT315-ds Intβ1,and HD73-pHT315-ds AK-Intβ1 strains all expressed ds RNA of the target gene,and HD73-pHT315 and HD73 did not express ds RNA as the control.The four engineered bacteria and HD73 were cultured and fed to the larvae.The LC50of the HD73-pHT315-ds AK-Intβ1 was found to be the smallest,while the LC50of HD73-pHT315 was the maximum.After treating the larvae with a 3 OD bacterial solution,the relative expression of the target gene in the larvae was measured.It was found that after 24hours of feeding,the expression of AK and Intβ1 genes were inhibited by the treatment of HD73-pHT315-ds AK-Intβ1 compared with the control of HD73;treatments of HD73-pHT315-ds AK and HD73-pHT315-ds Intβ1 inhibited the expression of their respective target genes in the larvae;HD73-pHT315 and HD73 treatments did not affect the gene expression in the larvae.The two bacterial liquids HD73-pHT315-ds AK and HD73-pHT315-ds Intβ1 were mixed in a ratio of 1:1 to treat the P.xylostella,and compared with the HD73-pHT315-ds AK-Intβ1 treatment group on the lethality of the P.xylostella,it was found that after 12h,the death rate of the latter to P.xylostella was significantly higher than the former.In summary,HD73-pHT315-ds AK-Intβ1 had a stronger insecticidal activity against P.xylostella than the engineered bacteria only targeting single gene,indicating a better application for pest control. |
