Wang Ying, Baker Nicholas, Amdam Gro V
School of Life Sciences, Arizona State University, Arizona, USA.
J Vis Exp. 2013 Jul 25(77):50446. doi: 10.3791/50446.
This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception. RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species. The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.
本视频展示了RNA干扰（RNAi）的新技术，该技术通过注射双链RNA（dsRNA）在蜜蜂中同时下调两个基因。它还介绍了一种用于测量味觉感知的喙伸展反应（PER）测定法的方案。RNAi介导的基因敲低是一种下调靶基因表达的有效技术。该技术通常用于单个基因操作，但在检测基因之间的相互作用和联合效应方面存在局限性。在本视频的第一部分，我们展示了两种同时敲低两个基因的策略（称为双基因敲低）。我们表明这两种策略都能够有效抑制处于调节反馈回路中的两个基因，即卵黄原蛋白（vg）和超气门蛋白（usp）。这种双基因敲低方法可用于剖析基因之间的相互关系，并可轻松应用于不同的昆虫物种。本视频的第二部分展示了在双基因敲低处理后蜜蜂的喙伸展反应（PER）测定。PER测定是测量蜜蜂味觉感知的标准测试，它是蜜蜂行为成熟速度的关键预测指标。巢内蜜蜂更强的味觉感知表明行为发育增加，这通常与更早开始觅食和花粉觅食专业化的年龄相关。此外，PER测定可用于识别蜜蜂的饱腹或饥饿代谢状态。最后，PER测定与配对不同气味刺激来训练蜜蜂相结合，也广泛用于蜜蜂的学习和记忆研究。
