Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella

作者：Xingliang Wang, Yiming Ma, Falong Wang, Yihua Yang, Shuwen Wu, Yidong Wu · 发表于：Pest Management Science · 年份：2019 · DOI：10.1002/ps.5689 · 被引用次数：53 · 研究领域：CRISPR and Genetic Engineering、Insect Resistance and Genetics、Neurobiology and Insect Physiology Research

BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach. RESULTS: Here, we successfully constructed a homozygous strain (Pxα6-KO) with a 2-nt deletion mutation of nAChRα6 by CRISPR/Cas9 coupled with non-homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3-TM4 loop region. When compared to the background strain IPP-S, the knockout strain Pxα6-KO exhibited 229- and 1462-fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3-fold) effects on the toxicities of imidacloprid, abamectin, β-cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2-nt deletion mutation of nAChRα6 in the Pxα6-KO strain. CONCLUSION: In vivo functional investigation demonstrates the ...