Gillet François-Xavier, Garcia Rayssa A, Macedo Leonardo L P, Albuquerque Erika V S, Silva Maria C M, Grossi-de-Sa Maria F
Embrapa Genetic Resources and BiotechnologyBrasília, Brazil.
Department of Cellular Biology, Brasilia Federal University (UnB)Brasília, Brazil.
Front Physiol. 2017 Apr 28;8:256. doi: 10.3389/fphys.2017.00256. eCollection 2017.
Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (), we showed that the chimeric protein PTD-DRBD (peptide transduction domain-dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.
产生双链RNA（dsRNA）的转基因（GM）作物主要作为一种基于RNA干扰（RNAi）的抗作物害虫策略正在接受研究。然而，该策略的局限性包括dsRNA对昆虫肠道核酸酶的敏感性及其较差的昆虫细胞膜穿透性。在研究害虫棉铃象鼻虫时，我们发现嵌合蛋白PTD-DRBD（肽转导结构域-dsRNA结合结构域）与dsRNA结合形成核糖核蛋白颗粒（RNP），可提高昆虫体内RNAi机制的有效性。RNP可能通过掩盖dsRNA来减缓核酸酶活性。此外，PTD介导的在昆虫肠道细胞中的内化在质膜接触后的几分钟内即可实现，从而限制了RNP与肠道核酸酶的接触时间。因此，与裸露的dsRNA相比，RNP经口服给药时昆虫基因沉默效率提高了约2倍。综上所述，这些数据证明了工程化RNP在提高dsRNA稳定性和细胞摄取方面的作用，为设计转基因植物中增强的抗作物害虫RNAi策略开辟了一条道路。
