College of Plant Protection/Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
Pest Manag Sci. 2013 Sep;69(9):1034-9. doi: 10.1002/ps.3553. Epub 2013 Jul 4.
One of the key limiting factors for using RNA interference technology to develop new pest control practices is the toxicological effectiveness. In order to explore potential methods to improve the efficiency of RNAi in insects, we cloned two dsRNA binding proteins from Laodelphax striatellus and investigated their functions.
In this study, the genes homologous to the dsRNA binding proteins TRBP and STAU in L. striatellus were first cloned and designated as LsTRBP and LsSTAU. Using the chitinase gene Chi, which has been confirmed to have lethal effects when knocked down by RNAi, as a reporter, the functions of these two dsRNA binding proteins were then investigated by using dsRNA feeding. The results showed that feeding with dsChi alone caused significant lethal effects, with a mortality four-fold higher than that of the control, whereas feeding with dsTRBP or dsSTAU alone did not yield any significant lethal effects. When dsChi was fed in the mixture 1:1 with dsSTAU, it also resulted in high mortality, which was a little lower than feeding with dsChi alone, but not statistically significant. However, when fed in the 1:1 mixture with dsTRBP, the lethal effect of dsChi was completely eliminated. Further analysis of gene expression found that single dsRNA depressed only its own target gene. When treated in 1:1 mixture, dsSTAU or dsChi did not change significantly the depression of the other dsRNA, but dsTRAB counteracted almost all the effect of dsChi.
These results indicate that the loss of LsTRBP can significantly inhibit dsChi-mediated genetic interference. These findings not only suggest that the LsTRBP gene in L. striatellus plays an important role in regulating RNAi but also imply that the metabolic regulation of dsRNAs with respect to genetic interference in L. striatellus is similar to that in higher animal species.
利用 RNA 干扰技术开发新的害虫防治方法的关键限制因素之一是毒理学效果。为了探索提高昆虫中 RNAi 效率的潜在方法，我们从褐飞虱中克隆了两个 dsRNA 结合蛋白，并研究了它们的功能。
本研究首次克隆了褐飞虱 dsRNA 结合蛋白 TRBP 和 STAU 的同源基因，并将其命名为 LsTRBP 和 LsSTAU。利用已证实通过 RNAi 敲低具有致死效应的几丁质酶基因 Chi 作为报告基因，通过 dsRNA 喂养研究了这两种 dsRNA 结合蛋白的功能。结果表明，单独喂食 dsChi 会导致显著的致死效应，死亡率比对照高四倍，而单独喂食 dsTRBP 或 dsSTAU 则没有产生任何显著的致死效应。当 dsChi 与 dsSTAU 以 1:1 的比例混合喂养时，也会导致高死亡率，略低于单独喂食 dsChi，但无统计学意义。然而，当与 dsTRBP 以 1:1 的比例混合喂食时，dsChi 的致死效应完全消除。进一步的基因表达分析发现，单独的 dsRNA 仅抑制其自身的靶基因。当以 1:1 的比例混合处理时，dsSTAU 或 dsChi 对另一种 dsRNA 的抑制作用没有明显变化，但 dsTRAB 几乎抵消了 dsChi 的所有作用。
这些结果表明，LsTRBP 的缺失可显著抑制 dsChi 介导的遗传干扰。这些发现不仅表明褐飞虱中的 LsTRBP 基因在调节 RNAi 中起着重要作用，还暗示了褐飞虱中 dsRNA 对遗传干扰的代谢调节与高等动物物种相似。
