Institute of Applied Biology, Shanxi University, Taiyuan, China.
College of Life Science, Shanxi University, Taiyuan, China.
Insect Sci. 2021 Dec;28(6):1677-1689. doi: 10.1111/1744-7917.12882. Epub 2020 Nov 19.
The efficiency of RNA interference (RNAi) varies substantially among different insect species. Rapid degradation of double-stranded RNA (dsRNA) by dsRNA-degrading nucleases (dsRNases) has been implicated to cause low RNAi efficiency in several insect species. In this study, we identified four dsRNase genes (OfdsRNase1, OfdsRNase2, OfdsRNase3 and OfdsRNase4) from the Asian corn borer (Ostrinia furnacalis) transcriptome database. Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides. Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae. RNAi efficiency was investigated in 2-d-old fifth-instar larvae (high expression of dsRNase2) and 2-d-old pupae (low expression of dsRNase2) by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein (OfLgl). Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae, but not in larvae, suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages. This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene, OfHex1, was significantly improved after knockdown of OfdsRNase2. When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro, only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA. Taken together, our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O. furnacalis larvae.
RNA 干扰（RNAi）的效率在不同的昆虫物种中差异很大。双链 RNA（dsRNA）降解酶（dsRNases）对双链 RNA 的快速降解已被认为是导致几种昆虫物种中 RNAi 效率低下的原因。在本研究中，我们从亚洲玉米螟（Ostrinia furnacalis）转录组数据库中鉴定出了四个 dsRNase 基因（OfdsRNase1、OfdsRNase2、OfdsRNase3 和 OfdsRNase4）。生物信息学分析表明，每个推断的蛋白质序列都含有内切核酸酶 NS 结构域和信号肽。基因表达分析显示，OfdsRNase2 仅在幼虫的中肠中表达。通过喂食或注射靶向编码致死性巨型幼虫蛋白（OfLgl）的标记基因的 dsRNA，在 2 日龄的五龄幼虫（dsRNase2 高表达）和 2 日龄的蛹（dsRNase2 低表达）中研究了 RNAi 效率。我们的结果表明，OfLgl 仅部分沉默了蛹中 OfLgl 的表达，但在幼虫中则不然，这表明 OfdsRNase2 可能导致幼虫阶段的 RNAi 效率降低。这一假设得到了我们使用组织培养技术进行的 RNAi-of-RNAi 实验的支持，在该实验中，敲低 OfdsRNase2 后，对报告基因 OfHex1 的沉默效率显著提高。当进行双荧光素酶测定以评估这四种 dsRNases 在体外的作用时，只有在 S2 细胞中表达的 OfdsRNase2 才能通过降解 dsRNA 显著影响 RNAi 效率。综上所述，我们的研究结果表明，OfdsRNase2 在中肠对 dsRNA 的降解导致了亚洲玉米螟幼虫中 RNAi 效率低下。
