背景:最近,基于RNA干扰(RNAi)的生物农药,一种特定物种的害虫控制替代方案,在美国和加拿大已经放松管制和商业化。山楂蜘蛛螨,两栖动物,是酒类植物的主要害虫,主要由合成农药控制。为了解决Viennensis出现的抗药性问题,我们启动了一个开发基于RNAi的生物农药的项目。
结果:在这项研究中,我们(i)使用叶盘开发了一种用于A.viennensis的饮食RNAi系统,(ii)评估了多个对照基因的适用性,以区分该RNAi系统内的序列特异性沉默与非特异性效应,和(iii)筛选靶基因候选物。因此,β-葡糖醛酸酶(GUS),一种来自大肠杆菌的酶和广泛使用的植物报告基因是对A.viennensisRNAi的适当控制,而绿色荧光蛋白(GFP),不适合,因为它的死亡率明显高于其他对照。对于靶基因筛选,所有候选人都被确认被压制,包括两个管家基因(液泡型H-ATPase亚基A(V-ATPaseA)和甘油醛3-磷酸脱氢酶,(GAPDH)),和三个与发育相关的基因(ATP依赖性RNA解旋酶DDX3Y(Belle),CREB结合蛋白(CBP),和法尼酸O-甲基转移酶(FaMet))。与其他候选物相比,敲除V-ATP酶A导致最高的死亡率(~90%)和降低的繁殖力(超过90%)。至于与发育相关的基因,压制Belle和CBP,导致大约65%的死亡率,以及减少86%和40%的繁殖力,分别。FaMet的沉默,然而,对A.viennensis的生物学影响可忽略不计。
结论:共同努力不仅建立了有效的dsRNA递送方法,但也为基于RNAi的生物农药提供了潜在的靶基因,一种对亚洲和欧洲的果树和木本观赏植物具有破坏性的入侵性害虫。©2023化学工业学会。
BACKGROUND: Recently, RNA interference (RNAi)-based biopesticide, a species-specific pest control alternative, has been deregulated and commercialized in the US and Canada. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is a major pest for rosaceous plants, which has been controlled primarily by synthetic pesticides. To address the emerging resistance issues in A. viennensis, we initiated a project to develop RNAi-based biopesticides.
RESULTS: In this study, we (i) developed a dietary RNAi system for A. viennensis using leaf disc, (ii) assessed the suitability of multiple control genes to distinguish sequence-specific silencing from non-specific effects within this RNAi system, and (iii) screened for the target gene candidates. As a result, β-Glucuronidase (GUS), an enzyme derived from E. coli and a broadly used reporter for plants is the appropriate control for A. viennensis RNAi, while green fluorescent protein (GFP), is not suitable due to its significantly higher mortality than the other controls. For target gene screening, suppression was confirmed for all the candidates, including two housekeeping genes (Vacuolar-type H + -ATPase subunit A (V-ATPase A) and Glyceraldehyde 3-phosphate dehydrogenase, (GAPDH)), and three genes associated with development (ATP-dependent RNA Helicase DDX3Y (Belle), CREB-binding protein (CBP), and Farnesoic acid O-methyltransferase (FaMet)). Knocking down of V-ATPase A resulted in the highest mortality (~ 90%) and reduced fecundity (over 90%) than other candidates. As for the genes associated with development, suppression of Belle and CBP, led to approximately 65% mortality, as well as 86% and 40% reduction in fecundity, respectively. Silencing of FaMet, however, had negligible biological impacts on A. viennensis.
CONCLUSIONS: The combined efforts not only establish an effective dsRNA delivery method, but also provide potential target genes for RNAi-based biopesticides against A. viennensis, a devastating invasive pest for fruit trees and woody ornamental plants throughout Asia and Europe. © 2023 Society of Chemical Industry.