Research Institute of Applied Biology, Shanxi University, Taiyuan, China.
College of Life Science, Shanxi University, Taiyuan, China.
Pest Manag Sci. 2019 Jun;75(6):1707-1717. doi: 10.1002/ps.5291. Epub 2019 Jan 8.
The efficiency of RNA interference (RNAi) varies considerably among different insect species, and there is growing evidence to suggest that degradation of double-stranded (dsRNA) prior to uptake is an important factor that limits the efficiency of RNAi in insects. In Locusta migratoria, RNAi is highly efficient when dsRNA is delivered by injection, but not by feeding. However, detailed mechanisms causing such differential RNAi efficiency are still elusive.
We identified and characterized the full-length complementary DNAs (cDNAs) of two new dsRNA nuclease (dsRNase) genes from L. migratoria, which were named LmdsRNase1 and LmdsRNase4. Transcript analyses revealed that LmdsRNase1 and LmdsRNase4 were highly expressed in hemolymph with relatively lower expression in other tested tissues. Our study using heterologously expressed LmdsRNase1 and LmdsRNase4 fusion proteins showed that LmdsRNase1 can degrade dsRNA rapidly at an optimal pH of 5, whereas LmdsRNase4 had no activity at any of the pH values examined. In comparing the substrate specificity of the four LmdsRNases, we found that only LmdsRNase1 and LmdsRNase2 digested dsRNA; however, our experiments suggested that the physiological pH of hemolymph (7.0) suppresses LmdsRNase1 activity permitting significant dsRNA stability in this tissue. Conversely, the physiological pH of midgut juice (6.8) is ideal for LmdsRNase2 activity, resulting in degradation of dsRNA in midgut.
The physiological pH of different insect tissues or compartments can significantly alter the stability of dsRNA by influencing LmdsRNase activity in L. migratoria. Thus, new strategies to overcome such obstacles are expected to help implement RNAi-based technologies for insect pest management. © 2018 Society of Chemical Industry.
RNA 干扰（RNAi）在不同昆虫物种之间的效率差异很大，越来越多的证据表明，双链 RNA（dsRNA）在被摄取之前的降解是限制昆虫中 RNAi 效率的一个重要因素。在飞蝗中，dsRNA 通过注射给药时 RNAi 的效率很高，但通过喂食则不然。然而，导致这种差异 RNAi 效率的详细机制仍然难以捉摸。
我们从飞蝗中鉴定并表征了两个新的 dsRNA 核酸酶（dsRNase）基因的全长 cDNA，分别命名为 LmdsRNase1 和 LmdsRNase4。转录分析显示，LmdsRNase1 和 LmdsRNase4 在血液中高度表达，而在其他测试组织中表达相对较低。我们使用异源表达的 LmdsRNase1 和 LmdsRNase4 融合蛋白进行的研究表明，LmdsRNase1 可以在最佳 pH 值 5 下快速降解 dsRNA，而 LmdsRNase4 在检测到的任何 pH 值下均无活性。在比较四种 LmdsRNases 的底物特异性时，我们发现只有 LmdsRNase1 和 LmdsRNase2 可以消化 dsRNA；然而，我们的实验表明，血液的生理 pH 值（7.0）抑制了 LmdsRNase1 的活性，从而使该组织中的 dsRNA 具有显著的稳定性。相反，中肠汁液的生理 pH 值（6.8）非常适合 LmdsRNase2 的活性，导致中肠 dsRNA 的降解。
在飞蝗中，不同昆虫组织或隔室的生理 pH 值可以通过影响 LmdsRNase 的活性来显著改变 dsRNA 的稳定性。因此，预计克服这些障碍的新策略将有助于实施基于 RNAi 的技术来管理昆虫害虫。 © 2018 化学工业协会。
