Zuo Ya-Yun, Xue Yu-Xin, Wang Ze-Yu, Ren Xuan, Aioub Ahmed A A, Wu Yi-Dong, Yang Yi-Hua, Hu Zhao-Nong
Institute of Pesticide Science, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, 712100, China.
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Insect Sci. 2022 Apr;29(2):478-486. doi: 10.1111/1744-7917.12922. Epub 2021 May 17.
Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) gene-editing technology. Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230-fold) and spinetoram (792-fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.
多杀菌素，包括多杀霉素和乙基多杀菌素，作用于昆虫中枢神经系统，逐渐麻痹或杀死目标昆虫。在许多农业害虫中，多杀霉素抗性与烟碱型乙酰胆碱受体（nAChR）α6亚基的功能丧失突变有关。通过基因编辑，已证实nAChR α6是五种昆虫中多杀菌素的作用靶标。最近，在棕榈蓟马和番茄潜叶蛾的多杀霉素抗性品系中，发现了nAChR α6第9外显子的一个点突变（G275E）。迄今为止，尚未获得体内功能证据来支持该突变与鳞翅目害虫对多杀菌素的抗性有关。在本研究中，利用成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）基因编辑技术，将G275E突变引入到甜菜夜蛾的nAChR中。逆转录聚合酶链反应和测序证实，在编辑后的275E品系中，该突变存在于nAChR转录本的第9外显子中。生物测定结果表明，与未编辑的背景品系相比，275E品系对多杀霉素（230倍）和乙基多杀菌素（792倍）具有高度抗性，直接证实了nAChR α6亚基的G275E突变赋予甜菜夜蛾高水平的多杀菌素抗性。遗传分析表明，抗性性状为常染色体遗传且不完全隐性。本研究采用反向遗传学方法，验证了甜菜夜蛾nAChR α6中G275E突变在多杀菌素抗性中的功能作用，并提供了另一个利用CRISPR/Cas9基因编辑技术来确认候选靶位点突变在杀虫剂抗性中作用的实例。
