Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua
Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua
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DOI：
10.1111/1744-7917.12922
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发表时间：
2021-05
期刊：
影响因子：
4
通讯作者：
Yayun Zuo;Yuxin Xue;Ze-yu Wang;Xuan Ren;A. Aioub;Yidong Wu;Yihua Yang;Zhaonong Hu
中科院分区：
文献类型：
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作者：
Yayun Zuo;Yuxin Xue;Ze-yu Wang;Xuan Ren;A. Aioub;Yidong Wu;Yihua Yang;Zhaonong Hu
多杀菌素，包括多杀菌素和乙基多杀菌素，作用于昆虫中枢神经系统，逐渐麻痹或杀死目标昆虫。多杀菌素耐药性与许多农业害虫中尼古丁乙酰胆碱受体（nAChR）α6亚基的功能丧失突变有关。使用基因编辑，nAChR α6已被验证为五种昆虫中多杀菌素的靶标。最近，在多杀菌素抗性的蓟马帕尔米和Tuta absoluta菌株中鉴定出nAChR α6外显子9的点突变（G275 E）。到目前为止，没有在体内功能的证据已经获得支持，这种突变是在鳞翅目害虫多杀菌素抗性。在这项研究中，G275 E突变被引入到甜菜夜蛾的nAChR使用成簇规则间隔短回文重复序列（CRISPR）/ CRISPR相关蛋白9（Cas9）基因编辑技术。逆转录-聚合酶链反应和测序证实，该突变存在于编辑的275 E菌株中nAChR转录本的外显子9中。生物测定结果表明，与未编辑的背景菌株相比，275 E菌株对多杀菌素（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G275 E突变赋予S. exigua.抗性遗传分析表明，抗性性状为常染色体不完全隐性遗传。本研究采用反向遗传学方法验证了S.该研究提供了另一个使用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所起作用的例子。
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.