锈赤扁谷盗谷胱甘肽S-转移酶基因CfGSTe1和CfGSTd1与甲酸乙酯耐受性关系

doi:10.3864/j.issn.0578-1752.2026.05.007

摘要/Abstract

摘要：

【背景】 谷胱甘肽S-转移酶（GST）作为昆虫体内的重要代谢酶类，在害虫对化学药剂耐受性的形成中发挥重要作用。甲酸乙酯因具有高效、低毒和低残留等特点，被视为具有应用潜力的绿色储粮熏蒸剂。【目的】 分析锈赤扁谷盗（Cryptolestes ferrugineus）谷胱甘肽S-转移酶基因CfGSTe1和CfGSTd1与甲酸乙酯耐受性之间的关系，阐明锈赤扁谷盗对甲酸乙酯耐受性的分子机制。【方法】 以不同磷化氢抗性水平的锈赤扁谷盗为材料，测定其对甲酸乙酯的药剂敏感性。通过顺丁烯二酸二乙酯协同试验，评估其对甲酸乙酯熏蒸杀虫作用的增效效应，并分析甲酸乙酯处理对GST活性的影响。基于锈赤扁谷盗转录组数据鉴定获得两个关键谷胱甘肽S-转移酶基因（CfGSTe1和CfGSTd1），并进行氨基酸序列和系统发育分析。进一步利用实时荧光定量PCR解析两基因的时空表达特征及其在甲酸乙酯熏蒸胁迫下的转录响应变化。最后，通过RNA干扰（RNAi）技术分别沉默CfGSTe1和CfGSTd1，分析其对锈赤扁谷盗甲酸乙酯耐受性的影响。【结果】 生物测定结果表明，不同磷化氢抗性水平的锈赤扁谷盗对甲酸乙酯敏感性无显著差异，证实两种熏蒸剂间不存在交互抗性。增效剂顺丁烯二酸二乙酯可显著提高甲酸乙酯的熏蒸杀虫活性，且在药剂胁迫下锈赤扁谷盗GST活性显著升高。序列及进化树分析表明CfGSTe1和CfGSTd1分别编码216和215个氨基酸，均含有GST催化活性保守位点，属于Epsilon和Delta家族。实时荧光定量PCR结果显示，两基因均在成虫阶段显著高表达，主要在中肠、脂肪体和马氏管中表达，并可被甲酸乙酯熏蒸显著诱导。通过RNAi技术分别有效沉默CfGSTe1和CfGSTd1后，锈赤扁谷盗对甲酸乙酯耐受性显著降低，表现为熏蒸处理后成虫死亡率明显升高。【结论】 谷胱甘肽S-转移酶基因CfGSTe1和CfGSTd1可能在锈赤扁谷盗对甲酸乙酯解毒代谢过程中发挥重要作用，暗示其与昆虫甲酸乙酯耐受性间存在密切关系。

关键词: 锈赤扁谷盗, 甲酸乙酯, 谷胱甘肽-S-转移酶, RNA干扰, 实时荧光定量PCR

Abstract:

【Background】 Glutathione S-transferases (GSTs) are key detoxification enzymes in insects and play important roles in the development of insect tolerance to chemical insecticides. Ethyl formate (EF), characterized by high efficacy, low toxicity, and low residue, is regarded as a promising green fumigant for stored-grain pest control.【Objective】 To elucidate the molecular mechanisms underlying pest tolerance to EF, this study focuses on the important stored-grain pest Cryolestes ferrugineus, aiming to analyze the relationship between GST genes (CfGSTe1 and CfGSTd) and EF tolerance.【Method】 The present study conducted bioassays to determine the susceptibility of C. ferrugineus with three different levels of phosphine resistance to EF. Through synergistic assays with diethyl maleate (DEM), the potential enhancement of EF fumigation efficacy was evaluated, and the effects of EF treatment on GST activity were analyzed. According to the previous transcriptome data of C. ferrugineus, two key GST genes (CfGSTe1 and CfGSTd1) were identified and subjected to amino acid sequence and phylogenetic analyses. The temporal and spatial expression patterns of these two genes, as well as their transcriptional responses to EF fumigation stress, were further analyzed by using real-time quantitative PCR (RT-qPCR). Finally, the effects of CfGSTe1 and CfGSTd1 on EF tolerance were analyzed by individually silencing these genes using RNA interference (RNAi) technology.【Result】 Bioassay results showed that C. ferrugineus with varying levels of phosphine resistance exhibited no significant differences in sensitivity to EF, confirming the absence of cross-resistance between the two fumigants. The synergist DEM significantly enhanced the fumigant toxicity of EF, and the GST activity in insects was markedly increased under EF stress, suggesting that GSTs play an important role in the detoxification metabolism of EF. Sequence and phylogenetic analyses indicated that CfGSTe1 and CfGSTd1 encode 216 and 215 amino acids, respectively, both containing conserved GST catalytic sites and belonging to the Epsilon and Delta families. The RT-qPCR results indicated that both genes were highly expressed at the adult stage, primarily in the midgut, fat body, and Malpighian tubules, and could be significantly induced by EF exposure. After effectively silencing CfGSTe1 and CfGSTd1 via RNAi, the tolerance of C. ferrugineus to EF was significantly reduced, as evidenced by markedly increased adult mortality following fumigation.【Conclusion】 The CfGSTe1 and CfGSTd1 may play important roles in the detoxification metabolism of EF in C. ferrugineus, suggesting a close association with insect tolerance to this fumigant.

Key words: Cryptolestes ferrugineus, ethyl formate (EF), glutathione S-transferase (GST), RNA interference (RNAi), RT-qPCR

张琪, 陈二虎, 孙德宏, 唐培安. 锈赤扁谷盗谷胱甘肽S-转移酶基因CfGSTe1和CfGSTd1与甲酸乙酯耐受性关系[J]. 中国农业科学, 2026, 59(5): 1008-1019.

ZHANG Qi, CHEN ErHu, SUN DeHong, TANG PeiAn. Relationship Between Glutathione S-Transferase Genes CfGSTe1 and CfGSTd1 and Ethyl Formate Tolerance in Cryptolestes ferrugineus[J]. Scientia Agricultura Sinica, 2026, 59(5): 1008-1019.

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种群Population	经纬度<BOLD>L</BOLD>ongitude and latitude	采集时间Collection time	抗性倍数Resistance ratio	抗性水平Resistance level
SH	31.25° N, 121.46° E	2021-06	1.4	低抗Low resistance
ST	28.72° N, 112.81° E	2021-08	14.0	中抗Moderate resistance
XY	28.62° N, 112.94° E	2021-08	45.8	高抗High resistance

引物名称Primer name	引物序列Primer sequence (5′ to 3′)	决定系数Determination coefficient (R²)	扩增效率Amplification efficiency (%)
CfGSTe1-F	TACTGAACGACTACCAACT	1.000	92.38
CfGSTe1-R	GCATTTCCTTAATCCATTCG	0.998	101.87
CfGSTd1-F	CTTGCTGATTTGGCTTTGG	0.996	92.76
CfGSTd1-R	CATTGGCTTCTTCGTAACC	0.999	93.29
RPS13-F	ATCCGTAAGCATTTGGAACG	0.996	99.19
RPS13-R	AGCCACTAAGGCTGAAGCTG	0.998	102.74
EF1α-F	CCAGGCATGGTAGTGACCTT	0.991	94.26
EF1α-R	TTGGAGGGTTGTTTTTGGAG	0.993	97.76

引物名称 Primer name	引物序列 Primer sequence (5′ to 3′)
dsCfGSTe1-F	ggatcctaatacgactcactataggCATACTGTTCCCACTTT
dsCfGSTe1-R	ggatcctaatacgactcactataggCACCAAACTGAAATCTG
dsCfGSTd1-F	ggatcctaatacgactcactataggGGAGAACACCTTACACCAGAA
dsCfGSTd1-R	ggatcctaatacgactcactataggTTGACCTTAGCGTACCACTT
dsGFP-F	ggatcctaatacgactcactataggATGGTGAGCAAGGGCGAGA
dsGFP-R	ggatcctaatacgactcactataggTTACTTGTACAGCTCGTCCA

种群<BOLD>P</BOLD>opulation	LC₅₀（95%置信区间95% CI）(μL·L^-1）	回归方程Regression equation (y=)	决定系数Determination coefficient (R²)
XY	21.68 (21.05-22.32)	15.47x-15.67	0.990
ST	21.58 (21.01-22.16)	18.55x-19.74	0.992
SH	23.70 (22.68-24.79)	11.24x-10.45	0.998