Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Department of Biotechnology, Faculty of Life Sciences, University of Central Punjab, Lahore, Pakistan.
Sci Rep. 2022 Jun 21;12(1):10405. doi: 10.1038/s41598-022-14667-z.
RNA interference (RNAi) triggered by exogenous double-stranded RNA (dsRNA) is a powerful tool to knockdown genetic targets crucial for the growth and development of agriculturally important insect pests. Helicoverpa armigera is a pest feeding on more than 30 economically important crops worldwide and a major threat. Resistance to insecticides and Bt toxins has been gradually increasing in the field. RNAi-mediated knockdown of H. armigera genes by producing dsRNAs homologous to genetic targets in bacteria and plants has a high potential for insect management to decrease agricultural loss. The acetylcholinesterase (AChE), ecdysone receptor (EcR) and v-ATPase-A (vAA) genes were selected as genetic targets. Fragments comprising a coding sequence of < 500 bp were cloned into the L4440 vector for dsRNA production in bacteria and in a TRV-VIGS vector in antisense orientation for transient expression of dsRNA in Solanum tuberosum leaves. After ingesting bacterial-expressed dsRNA, the mRNA levels of the target genes were significantly reduced, leading to mortality and abnormal development in larva of H. armigera. Furthermore, the S. tuberosum plants transformed with TRV-VIGS expressing AChE exhibited higher mortality > 68% than the control plants 17%, recorded ten days post-feeding and significant resistance in transgenic (transient) plants was observed. Moreover, larval lethality and molting defects were observed in larva fed on potato plants expressing dsRNA specific to EcR. Analysis of transcript levels by quantitative RT-PCR revealed that larval mortality was attributable to the knockdown of genetic targets by RNAi. The results demonstrated that down-regulation of H. armigera genes involved in ATP hydrolysis, transcriptional stimulation of development genes and neural conduction has aptitude as a bioinsecticide to control H. armigera population sizes and therefore decreases crop loss.
外源双链 RNA（dsRNA）引发的 RNA 干扰（RNAi）是一种强大的工具，可以敲低对农业重要害虫生长和发育至关重要的遗传靶标。棉铃虫是一种世界性的害虫，以 30 多种经济上重要的作物为食，是一个主要的威胁。田间对杀虫剂和 Bt 毒素的抗性逐渐增加。通过产生与细菌和植物中遗传靶标同源的 dsRNA 来敲低棉铃虫基因，具有降低农业损失的昆虫管理的巨大潜力。乙酰胆碱酯酶（AChE）、蜕皮激素受体（EcR）和 v-ATPase-A（vAA）基因被选为遗传靶标。包含编码序列<500bp 的片段被克隆到 L4440 载体中，用于细菌中 dsRNA 的产生，并在 TRV-VIGS 载体中以反义方向用于马铃薯叶片中 dsRNA 的瞬时表达。在摄入细菌表达的 dsRNA 后，靶基因的 mRNA 水平显著降低，导致棉铃虫幼虫死亡和发育异常。此外，转化的马铃薯植物（瞬时）中表达 AChE 的 TRV-VIGS 比对照植物（17%）表现出更高的死亡率（>68%），在喂食后 10 天记录到，并且在转基因植物中观察到显著的抗性。此外，在喂食表达 EcR 特异性 dsRNA 的马铃薯植物的幼虫中观察到幼虫致死率和蜕皮缺陷。通过定量 RT-PCR 分析转录水平表明，幼虫死亡率归因于 RNAi 对遗传靶标的敲低。结果表明，下调参与 ATP 水解、发育基因转录刺激和神经传导的棉铃虫基因具有作为生物杀虫剂的潜力，以控制棉铃虫种群数量，从而降低作物损失。
