关键词: 甜菜夜蛾, 剪接变异体, 谷氨酸门控氯离子通道, CRISPR/Cas9系统

Abstract: 【Aim】The purpose of this study is to explore whether glutamate-gated chloride channels (GluCls) of the beet armyworm, Spodoptera exigua (SeGluCls) encoded by different transcription variants of SeGluCl have different susceptibilities to insecticides using CRISPR/Cas9 gene editing technology. 【Methods】 The full-length cDNA sequence of SeGluCl from S. exigua was obtained using RT-PCR and RACE, and analyzed by bioinformatics. Two splicing variants SeGluCl 3a and SeGluCl 3b of SeGluCl were knocked out by CRISPR/Cas9 gene editing technique, and two knockout strains (3a-KO and 3b-KO) of S. exigua were established. The difference in the susceptibilies of the 3rd instar larvae of the susceptible strain WH-S (control strain) and 3a-KO and 3b-KO strains to three insecticides abamectin, emamectin benzoate and fipronil was determined by bioassay. 【Results】 The full-length cDNA sequence of SeGluCl of S. exigua was obtained, and its two splicing variants (SeGluCl 3a and SeGluCl 3b) were found. The open reading frame (ORF) of SeGluCl 3a (GenBank accession no.: OM304353) is 1 362 bp in length encoding 454 amino acids. The ORF of SeGluCl 3b (GenBank accession no.: OM304354) is 1 365 bp in length encoding 455 amino acids. The two SeGluCls encoded by the splicing variants SeGluCl 3a and SeGluCl 3b contain four transmembrane regions and one cysteine loop. Phylogenetic analysis indicated that SeGluCl was most closely related to GluCls of S. litura and S. frugiperda. Compared to the control strain WH-S, the knockout strains (3a-KO and 3b-KO) showed no significant difference in the susceptibilities to abamectin, emamectin benzoate and fipronil. 【Conclusion】 There is no difference in the susceptibility of SeGluCls encoded by the splicing variants SeGluCl 3a and SeGluCl 3b of SeGluCl to the three insecticides abamectin, emamectin benzoate and fipronil in S. exigua.

Key words: Spodoptera exigua, splicing variant, glutamategated chloride channel, CRISPR/Cas9 system