Zuo Y-Y, Huang J-L, Wang J, Feng Y, Han T-T, Wu Y-D, Yang Y-H
College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
Insect Mol Biol. 2018 Feb;27(1):36-45. doi: 10.1111/imb.12338. Epub 2017 Jul 28.
P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta-cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua.
P-糖蛋白[P-gp或ATP结合盒转运体B1（ABCB1）]是癌细胞多药耐药性的重要参与者，但这种节肢动物转运体的确切功能尚不清楚。本研究的目的是利用成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）基因编辑技术，确定P-gp对甜菜夜蛾（Spodoptera exigua）对杀虫剂敏感性的重要性。我们克隆了一个开放阅读框（ORF），其编码的甜菜夜蛾P-糖蛋白（SeP-gp）预计具有P-gp和其他昆虫ABCB1转运体共有的结构特征。利用CRISPR/Cas9基因编辑系统建立了一个在SeP-gp ORF中发生四个核苷酸移码缺失的敲除品系，以测试其在确定对化学杀虫剂或苏云金芽孢杆菌（Bt）杀虫蛋白敏感性方面的潜在作用。比较生物测定结果表明，敲除SeP-gp可使甜菜夜蛾对阿维菌素和甲氨基阿维菌素苯甲酸盐（EB）的敏感性显著提高约三倍，但对多杀菌素、氯虫苯甲酰胺、高效氯氰菊酯、丁硫克百威、茚虫威、毒死蜱、辛硫磷、丁醚脲、氟铃脲、氯虫酰胺或两种Bt毒素（Cry1Ca和Cry1Fa）的敏感性没有影响。我们的数据支持SeP-gp在甜菜夜蛾对阿维菌素和EB敏感性方面的重要作用，并表明SeP-gp的过表达可能导致甜菜夜蛾对阿维菌素和EB产生抗性。
