Bilgi Vineeta, Fosu-Nyarko John, Jones Michael G K
Plant Biotechnology Research Group, Western Australia State Agricultural Biotechnology Centre, School of Veterinary and Life Sciences, Murdoch University, Perth, WA 6150, Australia.
Int J Mol Sci. 2017 Jan 3;18(1):80. doi: 10.3390/ijms18010080.
RNA interference (RNAi) is an effective tool to study gene function. For in vitro studies of RNAi in insects, microinjection of double-stranded (ds)RNA may cause stress. Non-persuasive oral delivery of dsRNA to trigger RNAi is a better mode of delivery for delicate insects such as aphids because it mimics natural feeding. However, when insects feed ad libitum, some individuals may not feed. For accurate measurement of gene knockdown, analysis should only include insects that have ingested dsRNA. The suitability of eleven dyes was assessed to trace ingestion of dsRNA in an artificial feeding system for green peach aphids (GPA, ). Non-toxic levels of neutral red and acridine orange were suitable tracers: they were visible in the stylet and gut after feeding for 24 h, and may also attract aphids to feed. Nymphs stained with neutral red (0.02%) were analysed for target gene expression after feeding on sucrose with dsRNA (V-ATPase, ). There was a greater reduction in expression and reproduction compared to nymphs fed the diet without dye. The results confirm the importance of identifying aphids that have ingested dsRNA, and also provide evidence that the gene is a potential target for control of GPAs.
RNA干扰（RNAi）是研究基因功能的有效工具。对于昆虫RNAi的体外研究，显微注射双链（ds）RNA可能会造成应激。对于蚜虫等脆弱昆虫而言，以非强制方式口服dsRNA来触发RNAi是一种更好的给药方式，因为它模拟了自然取食过程。然而，当昆虫随意取食时，一些个体可能不会取食。为了准确测量基因敲低情况，分析应仅包括摄入dsRNA的昆虫。评估了11种染料在桃蚜人工饲养系统中追踪dsRNA摄入情况的适用性。中性红和吖啶橙的无毒水平是合适的示踪剂：喂食24小时后，它们在口针和肠道中可见，并且可能还会吸引蚜虫取食。用中性红（0.02%）染色的若蚜在以含dsRNA（V-ATPase）的蔗糖为食后，分析其靶基因表达情况。与喂食不含染料食物的若蚜相比，其基因表达和繁殖能力的降低更为明显。结果证实了识别摄入dsRNA的蚜虫的重要性，同时也提供了证据表明该基因是控制桃蚜的潜在靶点。
