ace2基因敲除对棉铃虫乙酰胆碱酯酶活力及其杀虫剂敏感性的影响
Effect of ace2 gene knockout on AChE activity and insecticide sensitivity in Helicoverpa armigera
董荣荣** 管 放 萧 欣 杨亦桦***
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DOI：10.7679/j.issn.2095-1353.2026.002
作者单位：南京农业大学植物保护学院，南京210095
中文关键词：棉铃虫；ace2；CRISPR/Cas9；AChE；有机磷；氨基甲酸酯
英文关键词： Helicoverpa armigera; ace2; CRISPR/Cas9; AChE; organophosphate; carbamate
中文摘要：
【目的】 为明确AChE2是否是棉铃虫Helicoverpa
armigera体内杀虫剂的主要作用靶标。【方法】 利用qRT-PCR技术分析棉铃虫体内乙酰胆碱酯酶基因ace1和ace2的时空表达模式；采用CRISPR/Cas9基因编辑技术敲除棉铃虫ace2，并测定敲除品系ace1基因的表达量、乙酰胆碱酯酶活力及对6种杀虫剂（辛硫磷、毒死蜱、灭多威、茚虫威、功夫菊酯和氯虫苯甲酰胺）的敏感性变化。【结果】 棉铃虫ace1和ace2基因在幼虫不同龄期和不同组织中均有表达，且ace1的表达量普遍高于ace2；利用基因编辑和分子标记辅助筛选，成功构建了ace2基因1-5号外显子~34 000 bp大片段缺失的敲除纯合品系Haace2-KO。相对于野生型对照品系SCD，Haace2-KO品系的ace1基因表达量没有显著变化（P >
0.05），乙酰胆碱酯酶活力是对照品系SCD的0.88倍，且二者无显著差异（P > 0.05）；敲除ace2基因，棉铃虫对辛硫磷、毒死蜱、灭多威、茚虫威、功夫菊酯和氯虫苯甲酰胺的敏感性没有显著差异（0.5-1.9倍）。【结论】 ace2基因的敲除对棉铃虫乙酰胆碱酯酶活力和抗胆碱酯酶杀虫剂的敏感性均没有影响，说明AChE2不是参与棉铃虫体内神经传导的关键酶。
英文摘要：
[Aim] To clarify the role of the HaAChE2 gene in
the cotton bollworm (Helicoverpa armigera),
and more specifically, to determine if it is the main acetylcholinesterase gene
in this pest. [Methods] We first
used qRT-PCR to investigate the expression pattern of the ace1 and ace2 genes. We
then constructed an ace2 gene
knockout (KO) strain using the CRISPR/Cas9 system and compared the expression
of the ace1 gene, AChE activity and
the toxicity of six insecticides including phoxim, chlorpyrifos and methomyl, between
the knockout strain and the wild-type SCD strain. [Results] Both ace genes are expressed in different larval stages and tissues. Expression of ace1 was generally higher than that of ace2. A homozygous knockout strain
(Haace2-KO) with a ~34 000 bp deletion from exon 1 to exon 5 on the ace2 gene was successfully obtained
using CRISPR/Cas9-mediated gene editing and molecular marker-assisted
selection. There was no significant difference in the expression of the ace1 gene or AChE activity between the Haace2-KO
strain and the wild-type SCD strain (P > 0.05). Bioassay results show
that there was no significant difference in the toxicity ratios of phoxim,
chlorpyrifos, methomyl, indoxacarb, beta-cyhalothrin and chlorantraniliprole
(0.5 to 1.9 fold) between the Haace2-KO strain and the SCD strain. [Conclusion] The results indicate that knockout of
the ace2 gene has no effect on either
AChE activity, or anti-cholinesterase insecticide sensitivity, in H. armigera. These findings
provide direct evidence that AChE2 is not involved in enzymatic reactions in
the nervous system of H. armigera.