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脂质帮助双链 RNA 从内体中逃逸,并提高秋粘虫 Spodoptera frugiperda 中的 RNA 干扰。

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

机构信息

Department of Entomology, University of Kentucky, Lexington, Kentucky, 40546, USA.

出版信息

Arch Insect Biochem Physiol. 2020 Aug;104(4):e21678. doi: 10.1002/arch.21678. Epub 2020 Apr 15.

Abstract

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.

摘要

RNA 干扰 (RNAi) 是一种了解基因功能的有效方法,在害虫管理方面具有巨大的潜力。虽然 RNAi 在鞘翅目昆虫中效率高且系统性强,但在鳞翅目昆虫中效率较低。在这项研究中,我们通过使用 Cellfectin II (CFII) 转染试剂对秋粘虫(FAW)Spodoptera frugiperda 细胞中的 RNAi 进行了改进的可能性进行了探索。CFII 配方的 dsRNA 受到来自 Sf9 细胞条件培养基、FAW 幼虫血液和中肠腔内容物中存在的内切酶的保护,免受降解。脂质配方的 dsRNA 也显示出在 Sf9 细胞和 FAW 组织中的内体中积累减少。使 Sf9 细胞和组织暴露于 CFII 配方的 dsRNA 导致内源性基因的显著敲低。将 dsIAP 配方喂食给 FAW 幼虫会导致 iap 基因的敲低、生长迟缓和死亡。在用 CFII 连接的 P-UTP 标记的 dsGFP 处理的 Sf9 细胞和 Spodoptera frugiperda 幼虫中检测到 dsRNA 加工成 siRNA。总体而言,本研究得出的结论是,使用 CFII 转染试剂配制 dsRNA 有助于 dsRNA 逃避内体积累,并提高 FAW 细胞和组织中的 RNAi 效率。

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