Department of Entomology, University of Nebraska - Lincoln, Lincoln, NE 68583-0816, USA.
J Insect Sci. 2010;10:162. doi: 10.1673/031.010.14122.
To test the efficacy of RNA interference (RNAi) as a method for target-site screening in Diabrotica virgifera virgifera LeConte (Coleptera: Chrysomelidae) larvae, genes were identified and tested for which clear RNAi phenotypes had been identified in the Coleopteran model, Tribolium castaneum. Here the cloning of the D. v. vergifera orthologs of laccase 2 (DvvLac2) and chitin synthase 2 (DvvCHS2) is reported. Injection of DvvLac2-specific double-stranded RNA resulted in prevention of post-molt cuticular tanning, while injection of DvvCHS2-specific dsRNA reduced chitin levels in midguts. Silencing of both DvvLac2 and DvvCHS2 was confirmed by RT-PCR and quantitative RT-PCR. As in T. castaneum, RNAi-mediated gene silencing is systemic in Diabrotica. The results indicate that RNAi-induced silencing of D. v. vergifera genes provides a powerful tool for identifying potential insecticide targets.
为了测试 RNA 干扰 (RNAi) 作为一种在 Diabrotica virgifera virgifera LeConte（鞘翅目：金龟子科）幼虫中进行靶位筛选的方法的功效，鉴定并测试了在鞘翅目模式生物 Tribolium castaneum 中已明确具有 RNAi 表型的基因。本文报道了漆酶 2（DvvLac2）和几丁质合成酶 2（DvvCHS2）的 D. v. vergifera 同源基因的克隆。DvvLac2 特异性双链 RNA 的注射导致蜕皮后表皮鞣制的预防，而 DvvCHS2 特异性 dsRNA 的注射降低了中肠的几丁质水平。通过 RT-PCR 和定量 RT-PCR 证实了 DvvLac2 和 DvvCHS2 的沉默。与 T. castaneum 一样，Diabrotica 中的 RNAi 介导的基因沉默是全身性的。结果表明，D. v. vergifera 基因的 RNAi 诱导沉默为鉴定潜在的杀虫剂靶标提供了一种强大的工具。
