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A novel plasmid– Escherichia coli system produces large batch dsRNAs for insect gene silencing
Author(s) -
Ma ZhongZheng,
Zhou Hang,
Wei YanLong,
Yan Shuo,
Shen Jie
Publication year - 2020
Publication title -
pest management science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.296
H-Index - 125
eISSN - 1526-4998
pISSN - 1526-498X
DOI - 10.1002/ps.5792
Subject(s) - rna silencing , biology , rna interference , rnase p , microbiology and biotechnology , plasmid , gene silencing , escherichia coli , gene expression , rna , gene , genetics
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 μg 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. © 2020 Society of Chemical Industry