BACKGROUND: RNA interference (RNAi)-based pest management requires efficient delivery and large-batch production of double-stranded (ds)RNA. We previously developed a nanocarrier-mediated dsRNA delivery system that could penetrate an insect's body and efficiently silence gene expression. However, there is a great need to improve the plasmid-Escherichia coli system for the mass production of dsRNA. Here, for efficient dsRNA production, we removed the rnc gene encoding endoribonuclease RNase III in E. coli BL21(DE3) and matched with the RNAi expression vector containing a single T7 promoter. RESULTS:The novel pET28-BL21(DE3) RNase III-system was successfully constructed to express vestigial (vg)-dsRNA against Harmonia axyridis. dsRNA was extracted and purified from cell cultures in four E. coil systems, and the yields of dsRNA in pET28-BL21(DE3) RNase III-, pET28-HT115(DE3), L4440-BL21(DE3) RNase III-and L4440-HT115(DE3) were 4.23, 2.75, 0.88 and 1.30 ∼g mL −1 respectively. The dsRNA expression efficiency of our novel E. coil system was three times that of L4440-HT115 (DE3), a widely used dsRNA production system. The RNAi efficiency of dsRNA produced by our system and by biochemical synthesis was comparable when injected into Harmonia axyridis. CONCLUSION: Our system expressed dsRNA more efficiently than the widely used L4440-HT115(DE3) system, and the produced dsRNA showed a high gene-silencing effect. Notably, our pET28-BL21(DE3) RNase III-system provides a novel method for the mass production of dsRNA at low cost and high efficiency, which may promote gene function analysis and RNAi-based pest management.

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The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4–104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3–100%. Additionally, the recombination efficiencies were 2.5–45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome.