Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Piracicaba, SP, Brazil.
Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba, SP, Brazil.
Methods Mol Biol. 2022;2360:317-345. doi: 10.1007/978-1-0716-1633-8_23.
RNA interference (RNAi) is a natural mechanism of gene regulation, highly conserved in eukaryotes. Since the elucidation of the gene silencing mechanism, RNAi became an important tool used in insect reverse genetics. The demonstration of effective target-gene silencing by ingestion of double-stranded RNA (dsRNA) produced by transgenic plants indicated the RNAi potential to be used in insect pest management, particularly in agriculture. However, the efficiency of gene silencing by RNAi in insects may vary according to the target taxa, and lepidopteran species have been shown to be quite recalcitrant to RNAi. Developing transgenic plants is a time-consuming and labor-intensive process, so alternative oral delivery systems are required to develop and optimize RNAi settings, such as selecting an efficient target gene, and dsRNA design, length, and stability, among other features. We have developed delivery systems to evaluate dsRNAs to silence genes from two important lepidopteran crop pests of tomato (Solanum lycopersicum) and sugarcane (Saccharum × officinarum): Tuta absoluta (Meyrick), the South American Tomato Pinworm, and Diatraea saccharalis (Fabricius), the Sugarcane Borer, respectively. The protocol described here can be used in similar species and includes (a) direct oral delivery by droplets containing dsRNA; (b) oral delivery by tomato leaflets that absorbed dsRNA solution; (c) delivery by Escherichia coli expressing dsRNA; and (d) delivery by transgenic plants expressing dsRNA.
RNA 干扰 (RNAi) 是一种基因调控的自然机制，在真核生物中高度保守。自从阐明基因沉默机制以来，RNAi 已成为昆虫反向遗传学中使用的重要工具。通过摄入转基因植物产生的双链 RNA (dsRNA) 有效沉默靶基因的证明表明，RNAi 有可能用于昆虫害虫管理，特别是在农业中。然而，RNAi 在昆虫中的基因沉默效率可能因靶标分类群而异，并且鳞翅目物种对 RNAi 的抗性相当强。开发转基因植物是一个耗时耗力的过程，因此需要替代口服递送系统来开发和优化 RNAi 方案，例如选择有效的靶基因和 dsRNA 的设计、长度和稳定性等特征。我们已经开发了递送系统来评估 dsRNA 以沉默番茄 (Solanum lycopersicum) 和甘蔗 (Saccharum × officinarum) 两种重要鳞翅目作物害虫的基因：Tuta absoluta (Meyrick)，即南美番茄潜叶蛾，以及 Diatraea saccharalis (Fabricius)，即甘蔗螟。这里描述的方案可用于类似的物种，包括 (a) 通过含有 dsRNA 的液滴直接口服递送；(b) 通过吸收 dsRNA 溶液的番茄小叶进行口服递送；(c) 通过表达 dsRNA 的大肠杆菌进行递送；和 (d) 通过表达 dsRNA 的转基因植物进行递送。
