Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.
Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.
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DOI：
10.1371/journal.pone.0189600
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Grossi-de-Sa MF
中科院分区：
文献类型：
--
作者：
Almeida Garcia R;Lima Pepino Macedo L;Cabral do Nascimento D;Gillet FX;Moreira-Pinto CE;Faheem M;Moreschi Basso AM;Mattar Silva MC;Grossi-de-Sa MF
RNA干扰（RNAi）技术是一种通过选择性基因下调来控制植物害虫的生物技术。然而，昆虫中RNAi机制的低效与几个障碍有关，包括dsRNA被细胞递送和摄取、dsRNA与细胞膜受体的相互作用以及dsRNA在进食期间暴露于昆虫肠道核酸酶。棉铃象甲（Anthonomus grandis）是一种鞘翅目昆虫，其中RNAi介导的基因沉默不能通过dsRNA进食有效地发挥作用，并且参与机制的因素仍然未知。在此，我们鉴定了棉铃象甲转录组中的三种核酸酶，分别命名为AgraNuc 1、AgraNuc 2和AgraNuc 3，并研究了这些核酸酶对棉铃象甲基因沉默的影响。grandis几丁质合成酶II（AgraChSII）的研究。系统发育分析表明，这三种核酸酶与其他昆虫的DNA/RNA非特异性核酸内切酶家族具有很高的相似性。发现这些核酸酶主要在昆虫的后中肠区域中表达。在核酸酶RNAi介导的基因沉默后两天，肠液对dsRNA的降解显著减少。值得注意的是，在核酸酶基因沉默后，当与对照（非沉默的核酸酶）相比时，口服递送的针对AgraChSII基因的dsRNA导致改善的基因沉默效率。本文所提供的数据表明，A. grandis中肠核酸酶是dsRNA递送的主要障碍之一，并强调需要开发新的RNAi递送策略，重点是保护dsRNA免受肠核酸酶的侵害并增强其口服递送和对作物昆虫害虫的摄取。
RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.