School of Life Sciences, Guangzhou University;
School of Life Sciences, Guangzhou University.
J Vis Exp. 2024 Oct 4(212). doi: 10.3791/67360.
The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of Lepidoptera with a good accumulation of basic research. It is also the first insect in Lepidoptera with its complete genome sequenced and assembled, which provides a solid foundation for gene functional study. Although RNA interference (RNAi) is widely used in reverse gene functional study, it is refractory in silkworms and other Lepidopteran species. Previous successful RNAi-related research to deliver double-stranded RNA (dsRNA) was performed through injection only. Delivery of dsRNA through feeding is never reported. In this article, we describe step-by-step procedures to prepare the chitosan/dsRNA nanoparticles, which are fed to the silkworm larvae by ingestion. The protocol includes (i) selection of the proper stage of silkworm larvae, (ii) synthesis of dsRNA, (iii) preparation of the chitosan/dsRNA nanoparticles, and (iv) feeding the silkworm larvae with chitosan/dsRNA nanoparticles. Representative results, including gene transcript confirmation and phenotype observation, are presented. dsRNA feeding is a simple technique for RNAi in silkworm larvae. Since silkworm larvae are easy to rear and large enough to operate, it provides a good model to demonstrate larval RNAi in insects. In addition, the simplicity of this technique stimulates more student involvement in research, making silkworm larvae an ideal genetic system for use in a classroom setting.
家蚕是一种重要的经济昆虫，在中国已有数千年的历史。同时，家蚕是鳞翅目模式昆虫，基础研究积累良好。它也是第一个完成全基因组测序和组装的鳞翅目昆虫，为基因功能研究提供了坚实的基础。尽管 RNA 干扰（RNAi）在反向基因功能研究中得到了广泛应用，但在蚕和其他鳞翅目物种中却难以实施。以前成功的 RNAi 相关研究是通过注射来递送双链 RNA（dsRNA），而通过摄食来递送 dsRNA 的方法从未有过报道。在本文中，我们描述了通过摄食将壳聚糖/dsRNA 纳米颗粒递送到家蚕幼虫体内的逐步操作程序。该方案包括：（i）选择家蚕幼虫的适当阶段；（ii）dsRNA 的合成；（iii）壳聚糖/dsRNA 纳米颗粒的制备；以及（iv）用壳聚糖/dsRNA 纳米颗粒喂养家蚕幼虫。本文还提供了代表性的结果，包括基因转录的确认和表型观察。dsRNA 喂养是一种在家蚕幼虫中进行 RNAi 的简单技术。由于家蚕幼虫易于饲养且体型足够大，易于操作，因此它为昆虫幼虫 RNAi 提供了一个很好的模型。此外，该技术的简单性激发了更多学生参与研究，使家蚕幼虫成为课堂教学中使用的理想遗传系统。
