Embrapa Genetic Resources and Biotechnology, Brasilia 70770-917, DF, Brazil.
Biotechnology and Molecular Biology Department, Federal University of Brasilia (UnB), Brasilia 70910-900, DF, Brazil.
Int J Mol Sci. 2022 Nov 8;23(22):13713. doi: 10.3390/ijms232213713.
Cotton is the most important crop for fiber production worldwide. However, the cotton boll weevil (CBW) is an insect pest that causes significant economic losses in infested areas. Current control methods are costly, inefficient, and environmentally hazardous. Herein, we generated transgenic cotton lines expressing double-stranded RNA (dsRNA) molecules to trigger RNA interference-mediated gene silencing in CBW. Thus, we targeted three essential genes coding for chitin synthase 2, vitellogenin, and ecdysis-triggering hormone receptor. The stability of expressed dsRNAs was improved by designing a structured RNA based on a viroid genome architecture. We transformed cotton embryos by inserting a promoter-driven expression cassette that overexpressed the dsRNA into flower buds. The transgenic cotton plants were characterized, and positive PCR transformed events were detected with an average heritability of 80%. Expression of dsRNAs was confirmed in floral buds by RT-qPCR, and the T cotton plant generation was challenged with fertilized CBW females. After 30 days, data showed high mortality (around 70%) in oviposited yolks. In adult insects fed on transgenic lines, chitin synthase II and vitellogenin showed reduced expression in larvae and adults, respectively. Developmental delays and abnormalities were also observed in these individuals. Our data remark on the potential of transgenic cotton based on a viroid-structured dsRNA to control CBW.
棉花是全球纤维生产最重要的作物。然而，棉铃象鼻虫（CBW）是一种害虫，在受感染地区会造成重大的经济损失。目前的控制方法成本高、效率低且对环境有害。在此，我们生成了表达双链 RNA（dsRNA）分子的转基因棉花品系，以触发 CBW 中的 RNA 干扰介导的基因沉默。因此，我们针对编码几丁质合酶 2、卵黄蛋白原和蜕皮激素触发激素受体的三个必需基因。通过设计基于类病毒基因组结构的结构化 RNA，提高了表达的 dsRNA 的稳定性。我们通过将驱动表达盒的启动子插入到花蕾中，转化棉花胚胎，该表达盒过量表达 dsRNA。对转基因棉花植物进行了特征分析，并通过平均遗传率为 80%的阳性 PCR 转化事件进行了检测。通过 RT-qPCR 证实了 dsRNA 在花蕾中的表达，并对 T 型棉花植株世代进行了受精 CBW 雌性的挑战。30 天后，数据显示产卵蛋黄中的死亡率很高（约 70%）。在以转基因系为食的成年昆虫中，几丁质合酶 II 和卵黄蛋白原在幼虫和成虫中分别显示出表达降低。这些个体还观察到发育延迟和异常。我们的数据表明，基于类病毒结构 dsRNA 的转基因棉花具有控制 CBW 的潜力。
