RNA干扰(RNAi)是一种自然的基因调控机制,在真核生物中高度保守。由于基因沉默机制的阐明,RNAi成为昆虫反向遗传学的重要工具。通过摄入转基因植物产生的双链RNA(dsRNA)来证明有效的靶基因沉默表明,RNAi可能用于害虫管理。尤其是在农业方面。然而,在昆虫中通过RNAi进行基因沉默的效率可能会根据目标分类群的不同而有所不同,和鳞翅目物种已被证明对RNAi相当顽固。开发转基因植物是一个耗时耗力的过程,因此,需要替代的口服递送系统来开发和优化RNAi设置,例如选择有效的靶基因,和dsRNA设计,长度,和稳定性,在其他特征中。我们已经开发了传递系统来评估dsRNA,以沉默来自番茄(Solanumlycopersicum)和甘蔗(Saccharum×officinarum)的两种重要鳞翅目作物害虫的基因:Tutaabsoruta(Meyrick),南美番茄pin虫,和迪亚糖(Fabricius),甘蔗剥夺者,分别。本文描述的方案可以用于相似的物种,并且包括(a)通过含有dsRNA的液滴直接口服递送;(b)通过吸收dsRNA溶液的番茄小叶口服递送;(c)通过表达dsRNA的大肠杆菌递送;和(d)通过表达dsRNA的转基因植物递送。
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.