ABSTRACT

RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 (DvSSJ1) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

摘要

RNA 干扰 (RNAi) 是一种保护免受有害遗传元素和调节基因表达的自然机制，可以通过同源双链 RNA (dsRNA) 的传递人为触发。这种机制可以作为一种高度特异性和环境友好的害虫防治策略加以利用。为此，需要用于生产大量重组 dsRNA 的系统。我们描述了一种在大肠杆菌中有效产生大量环状 dsRNA 的系统，并证明了这些分子对西方玉米根虫（WCR，Diabrotica virgifera virgifera LeConte）的有效杀虫活性，这是一种对玉米作物具有高度破坏性的害虫。在我们的系统中，dsRNA 的两条链在大肠杆菌中表达嵌入茄子潜伏病毒(ELVd) 的非常稳定的支架内，这是一种小的环状非编码 RNA。通过使用编码来自嗜热四膜虫的 I 组自催化内含子的 cDNA作为间隔子，实现了具有长反向重复的相应质粒在大肠杆菌中的稳定性。茄子 tRNA 连接酶的共表达促进了大肠杆菌细胞中的 RNA 环化和大规模积累，茄子 tRNA 连接酶是在宿主植物复制过程中连接 ELVd 的酶。插入的内含子在转录过程中有效地从 RNA 产物中自我剪接。含有与平滑隔膜连接 1 ( DvSSJ1)同源的 dsRNA 部分的环状 RNA) 基因对 WCR 幼虫表现出优异的杀虫活性。最后，我们表明，可以使用第二个嗜热嗜热菌自剪接内含子以置换的形式将类病毒支架与最终的环状 dsRNA 产物分离。