Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors.
Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors.
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通过细菌表达和口服 RNAi 效应子敲低粘虫几丁质酶基因
DOI：
10.1186/s12896-017-0328-7
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发表时间：
2017-02-09
影响因子：
3.5
通讯作者：
Wuriyanghan H
中科院分区：
文献类型：
--
作者：
Ganbaatar O;Cao B;Zhang Y;Bao D;Bao W;Wuriyanghan H
背景：RNAi（RNA干扰）是一种通过序列特异性方式沉默靶基因的技术。 RNAi技术已被用于开发抗病原作物。 2007年，首次报道了利用RNAi技术开发抗食草昆虫的转基因植物，引发了一系列基于该技术的RNAi机制和针对多种昆虫物种的控制策略的研究。粘虫属于鳞翅目夜蛾科，对具有重要经济意义的农作物构成威胁。最近，中国北方地区玉米生产受到严重威胁，需要新的控制方法。结果：几丁质酶基因主要在肠道组织中表达，据报道是几种昆虫的理想沉默靶标，因此被选为靶基因。针对靶基因的干扰序列被克隆到 L4440 载体中，以产生序列特异性 dsRNA（双链 RNA）。将重组L4440载体转化到大肠杆菌菌株HT115 (DE3)中，该菌株在dsRNA降解活性方面存在缺陷，因此可以防止dsRNA被细胞机器降解。将细菌与人工饲料混合并喂给分离分枝杆菌。我们发现，口服细菌表达的 dsRNA 会在受体昆虫中产生 RNAi 效应。实时定量PCR结果显示，口服表达相应dsRNA的工程菌后，分枝杆菌肠道组织中目标MseChi1和MseChi2基因的表达水平下调。在受体昆虫中检测到序列特异性 siRNA（小干扰 RNA），这支持了 M. separata 中存在 siRNA 介导的沉默效应。此外，MseChi1和MseChi2的敲除导致摄食幼虫死亡率增加和体重减轻。结论：我们报道了一种简单且低成本的实验方法来沉默M. separata内源基因表达。我们的研究既为利用RNAi技术控制M. separata提供了实验基础，也为研究该昆虫重要发育和调控基因的功能丧失提供了有用的研究工具。
Background:RNAi (RNA interference) is a technology for silencing of target genes via sequence-specific manner. RNAi technology has been used for development of anti-pathogenic crops. In 2007, development of transgenic plants resistant to insect herbivore using RNAi technology was first reported, leading to a burst of efforts aimed at exploitation of RNAi mechanism and control strategy against variety of insect species based on this technique. Mythimna separata belongs to noctuidae family of lepidoptera and is posing threat to crops of economic importance. Recently, outbreaks of M. separata severely threatens corn production in Northern China, calling for new control approaches.Results:Chitinase genes were chosen as the target genes as they were expressed predominantly in the gut tissue and were reported to be ideal silencing targets in several insect species. Interfering sequences against the target genes were cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3) which was defective in dsRNA degradation activity, so preserving the dsRNA from degradation by cellular machinery. The bacteria were mixed with artificial diet and were fed to M. separata. We showed that oral delivery of bacterially expressed dsRNA would lead to RNAi effects in the recipient insect. Quantitative real-time PCR results showed that expression level of target MseChi1 and MseChi2 genes in gut tissue of M. separata were down-regulated after oral delivery of engineered bacteria expressing the corresponding dsRNA. Sequence-specific siRNA (small interfering RNA) was detected in recipient insects, supporting the existence of siRNA-mediated silencing effects in M. separata. Furthermore, knockdown of MseChi1 and MseChi2 resulted in increased mortality and reduced body weight of the feeding larvae.Conclusion:We reported a simple and low cost experimental procedure to silence M. separata endogenous gene expression. Our research provides both an experimental foundation for using RNAi technology to control M. separata and also a useful research tool for loss-of-function study of important developmental and regulatory genes in this insect species.