一种新型质粒-大肠杆菌系统可批量生产双链 RNA 用于昆虫基因沉默。

A novel plasmid-Escherichia coli system produces large batch dsRNAs for insect gene silencing.

机构信息

Department of Entomology, MOA Key Lab of pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.

出版信息

Pest Manag Sci. 2020 Jul;76(7):2505-2512. doi: 10.1002/ps.5792. Epub 2020 Mar 2.

DOI:10.1002/ps.5792

PMID:32077251

Abstract

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 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.

摘要

背景

基于 RNA 干扰（RNAi）的害虫管理需要高效的双链 RNA（dsRNA）传递和大规模生产。我们之前开发了一种纳米载体介导的 dsRNA 传递系统，该系统可以穿透昆虫的身体并有效沉默基因表达。然而，需要极大地改进质粒-大肠杆菌系统以大规模生产 dsRNA。在这里，为了高效生产 dsRNA，我们在大肠杆菌 BL21(DE3)中去除了编码内切核糖核酸酶 RNase III 的 rnc 基因，并与含有单个 T7 启动子的 RNAi 表达载体匹配。

结果

成功构建了新型 pET28-BL21(DE3)RNase III 系统以表达针对 Harmonia axyridis 的 vestigial(vg)-dsRNA。从四种大肠杆菌系统的细胞培养物中提取和纯化 dsRNA，pET28-BL21(DE3)RNase III-、pET28-HT115(DE3)、L4440-BL21(DE3)RNase III-和 L4440-HT115(DE3)中 dsRNA 的产量分别为 4.23、2.75、0.88 和 1.30μg/mL。我们新型大肠杆菌系统的 dsRNA 表达效率是广泛使用的 dsRNA 生产系统 L4440-HT115(DE3)的三倍。当将 dsRNA 注射到 Harmonia axyridis 中时，我们系统产生的 dsRNA 的 RNAi 效率与生化合成的 dsRNA 相当。