甜菜夜蛾SeGluCl剪接变异体的敲除及其对杀虫剂敏感性的影响  

Knockout of SeGluCl splicing variants and its effects on the insecticide susceptibility in the beet armyworm,Spodoptera exigua(Lepidoptera:Noctuidae)

作　　者：安俊龙 殷旭 高新培 叶尔那·阿扎题 陈礼钰 裴雅琨 胡兆农[1,2] 左亚运 AN Jun-Long;YIN Xu;GAO Xin-Pei;ERNA·Azati;CHEN Li-Yu;PEI Ya-Kun;HU Zhao-Nong;ZUO Ya-Yun(College of Plant Protection,Northwest A&F University,Yangling 712100,China;Key Laboratory for Botanical Pesticide R&D of Shaanxi Province,Yangling 712100,China)

机构地区：[1]西北农林科技大学植物保护学院,杨凌712100 [2]陕西省植物源农药研究与开发重点实验室,杨凌712100

出　　处：《昆虫学报》2023年第1期55-62,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(32001941);中国博士后科学基金项目(2020M683586);大学生创新创业训练计划创新训练项目(202110712011)。

摘　　要：【目的】本研究旨在利用CRISPR/Cas9基因编辑技术,探索甜菜夜蛾Spodoptera exigua谷氨酸门控氯离子通道(glutamate-gated chloride channels,GluCls)基因不同转录变异体编码的SeGluCls对杀虫剂敏感性是否存在差异。【方法】采用RT-PCR和RACE技术获得甜菜夜蛾SeGluCl的全长cDNA序列,并对其进行生物信息学分析;利用CRISPR/Cas9基因编辑技术敲除SeGluCl两个剪接变异体SeGluCl 3a和SeGluCl 3b,并构建甜菜夜蛾敲除品系(3a-KO和3b-KO),生物测定甜菜夜蛾敏感品系WH-S(对照品系)及3a-KO和3b-KO品系的3龄幼虫对阿维菌素、甲维盐和氟虫腈3种杀虫剂敏感性的差异。【结果】获得了甜菜夜蛾SeGluCl全长cDNA序列,并发现其2种剪接变异体(SeGluCl 3a和SeGluCl 3b),SeGluCl 3a(GenBank登录号:OM304353)的开放阅读框(open reading frame,ORF)长1362 bp,编码454个氨基酸,SeGluCl 3b(GenBank登录号:OM304354)的ORF长1365 bp,编码455个氨基酸。两种剪接变异体SeGluCl 3a和SeGluCl 3b编码的SeGluCls都包含4个跨膜区和1个半胱氨酸环。系统发育分析表明,SeGluCl与斜纹夜蛾S.litura和草地贪夜蛾S.frugiperda的GluCl亲缘关系最近。与对照品系WH-S相比,3a-KO和3b-KO敲除品系对阿维菌素、甲维盐和氟虫腈的敏感性无显著差异。【结论】甜菜夜蛾SeGluCl剪接变异体SeGluCl 3a和SeGluCl 3b编码的SeGluCls对阿维菌素、甲维盐和氟虫腈这3种杀虫剂的敏感性无差异。【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 1362 bp in length encoding 454 amino acids.The ORF of SeGluCl 3b(GenBank accession no.:OM304354)is 1365 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.

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 

分 类 号：Q965.9[生物学—昆虫学]