Department of Disease and Stress Biology, The John Innes Centre, Norwich Research Park, Norwich, United Kingdom.
PLoS One. 2011;6(10):e25709. doi: 10.1371/journal.pone.0025709. Epub 2011 Oct 5.
RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.
METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.
CONCLUSIONS/SIGNIFICANCE: Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.
RNA 干扰 (RNAi) 是一种有价值的反向遗传学工具，可用于研究包括半翅目昆虫（如蚜虫）在内的各种生物体中的基因功能。先前的工作表明，通过将双链 RNA (dsRNA) 或小干扰 RNA (siRNA) 直接注入蚜虫的血淋巴，或通过让这些昆虫食用含有小 RNA 的人工饲料，可以实现豌豆蚜 (Acyrthosiphon pisum) 基因的 RNAi 介导的敲低。
方法/主要发现：在这项研究中，我们开发了用于蚜虫的植物介导 RNAi 技术，以允许在蚜虫的自然环境中进行基因沉默，并在实验过程中尽量减少对这些昆虫的处理。选择绿桃蚜 M. persicae 是因为它有广泛的植物宿主范围，包括模式植物拟南芥和烟草，对于这些植物可以相对较快地生成转基因材料。我们针对在肠道中主要表达的 M. persicae Rack1 和在唾液腺中主要表达的 M. persicae C002 (MpC002) 进行了靶向。将蚜虫喂食到瞬时产生与这些基因对应的 dsRNA 的烟草叶片圆盘上，或喂食到稳定产生 dsRNA 的拟南芥植株上。在转基因烟草和拟南芥上，MpC002 和 Rack-1 的表达被敲低了多达 60%。此外，沉默的蚜虫产生的后代较少，这与这些基因具有必需功能相一致。
结论/意义：在我们的植物介导 RNAi 方法和已发表的蚜虫沉默方法中，都实现了类似水平的基因沉默。此外，烟草叶片圆盘测定法可以开发成一种筛选方法，以评估哪些基因对于蚜虫在植物上的生存是必需的。我们的结果还证明了植物介导 RNAi 方法用于蚜虫控制的可行性。
