GSTs在花生蚜对双丙环虫酯解毒代谢中的作用

doi:10.13560/j.cnki.biotech.bull.1985.2025-0580

摘要/Abstract

摘要：

目的花生蚜（Aphis craccivora）是全球性重要农业害虫，严重威胁花生等作物的产量，双丙环虫酯为新型高效杀虫剂，对花生蚜等刺吸式口器害虫具有较好的防治效果，明确花生蚜对新型杀虫剂双丙环虫酯的代谢抗性机制，为科学制定田间防控策略提供理论依据。方法采用浸叶法测定花生蚜对双丙环虫酯的敏感性；通过酶活性测定与增效剂毒力实验评估谷胱甘肽S-转移酶（glutathione S-transferase, GSTs）在解毒代谢中的作用；利用实时荧光定量PCR分析GSTs的组织表达谱、龄期表达谱及药剂诱导表达情况；结合RNA干扰技术鉴定关键抗性相关GSTs。结果双丙环虫酯处理后花生蚜GST活性显著升高，增效剂顺丁烯二酸二乙酯（DEM）预处理增加了花生蚜对双丙环虫酯的敏感性，表明GSTs可能参与解毒代谢作用。组织与龄期表达谱分析表明，花生蚜大部分的GSTs在中肠和脂肪体中高表达，且表达水平随发育进程呈显著上升趋势。双丙环虫酯可诱导GSTT2和GSTS2表达上调，RNA干扰GSTT2后，花生蚜对双丙环虫酯的敏感性显著提高。结论 GSTT2在花生蚜响应双丙环虫酯胁迫、降低其敏感性中发挥重要作用，可为进一步探究花生蚜对双丙环虫酯的解毒机制奠定基础。

关键词: 花生蚜, GSTs, 双丙环虫酯, 解毒代谢

Abstract:

Objective Aphis craccivora is a globally prominent agricultural pest that severely threatens the yield of peanuts and other crops. Afidopyropen, as a novel and highly efficient insecticide, demonstrates potent control efficacy against sap-sucking pests. Clarifying the metabolic resistance mechanisms of A. craccivora to afidopyropen is of critical importance for the development of scientifically robust field management strategies. Method The sensitivity of A. craccivora to afidopyropen was evaluated through leaf-dip bioassay. The role of glutathione S-transferases (GSTs) in detoxification metabolism was assessed through enzyme activity assays and synergist toxicity bioassays. Tissue expression pattern, developmental expression pattern, and insecticide-induced expression profiles of GSTs were analyzed via quantitative real-time PCR (RT-qPCR). RNA interference (RNAi) was employed to identify key GSTs associated with tolerance to afidopyropen. Result Afidopyropen treatment significantly increased GST activity in A. craccivora. Pretreatment with the synergist diethyl maleate (DEM) enhanced the susceptibility of A. craccivora to afidopyropen, indicating the involvement of GSTs in detoxification metabolism in A. craccivora. Tissue and temporal expression pattern analyses revealed that most GSTs were highly expressed in the midgut and fat body, with expressions rising markedly during developmental progression. Afidopyropen induced the upregulation of GSTT2 and GSTS2. RNAi-mediated silencing of GSTT2 significantly increased the sensitivity to the afidopyropen in A. craccivora. Conclusion GSTT2 plays an important role in response to afidopyropen stress by reducing its sensitivity to the insecticide. This finding lays the foundation for further investigation into the detoxification mechanisms of A. craccivora against afidopyropen.

Key words: Aphis craccivora, GSTs, afidopyropen, detoxification metabolism

薛超, 段爱玲, 王爱玉, 杨媛雪. GSTs在花生蚜对双丙环虫酯解毒代谢中的作用[J]. 生物技术通报, 2025, 41(12): 304-312.

XUE Chao, DUAN Ai-ling, WANG Ai-yu, YANG Yuan-xue. The Role of GSTs in the Detoxification Metabolism of Aphis craccivora to Afidopyropen[J]. Biotechnology Bulletin, 2025, 41(12): 304-312.

图/表 6

表1 本研究所用引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5′-3′）
RT-qPCR
GSTD1-F	CAGAAGAGCATCCGATTATTTCGGC
GSTD1-R	CGGCGAGAGTTACTTCAGGTCCAG
GSTD2-F	GGTTGCATTGGAAATCGACGAAG
GSTD2-R	TTCATTTTTGCATCTTCGGGGTTAG
GSTT1-F	ACTGGCCAGGCAGCAAATGTTG
GSTT1-R	CCACTTCACATGCAGCAACAAGATC
GSTT2-F	GGCGAGGTGGTTCTGTTGGATTT
GSTT2-R	CTTCCCGTTGTGGATGAGAACTGG
GSTS1-F	CCTGCTATGAATGGAAAACCAGCAA
GSTS1-R	GGACGATTGGCATATGGATCGAAA
GSTS2-F	CTCGAGCCGAACAGATCCGATTC
GSTS2-R	CCGACAAATGGCTACGGACTGATTA
GSTS3-F	CTTCAATTTTCCTGCACTGGCAGAA
GSTS3-R	GGAACTTTTCCAAATGGCATTGTGG
GSTO1-F	CGACATCACTGCTCTGGCTGAACC
GSTO1-R	CAATTTCCAATGTTGGAACTTTGCC
GSTM1-F	TCCACCACGGTGAACACATGAAAC
GSTM1-R	AGCGCTTGCTTCTTCGGGTCTT
GSTM2-F	GGTTGACCTCACCCAGGTAATGGA
GSTM2-R	TGCCAAATGCTACACAGTCTTCTGG
RPS8-F	ACTAAGCTTGGAGCTCGCCGTGTAC
RPS8-R	TCTGACCAACTCATTGTTTGATGCG
RNAi
dsGSTT2-F	TAATACGACTCACTATAGGGCAGATACCAGTTCTCATCCACAA
dsGSTT2-R	TAATACGACTCACTATAGGGCAGATACCAGTTCTCATCCACAA
dsGSTT2-F	TAATACGACTCACTATAGGGAAGTATGAGTCCAAGGAAGAG
dsGSTT2-R	TAATACGACTCACTATAGGGAAGTATGAGTCCAAGGAAGAG
dsEGFP-F	TAATGTCTATAGGGAAGTTCAGCGTGTCCG
dsEGFP-R	TAATGTCTATAGGGCCTTGATGCCGTTC

图1 GST在花生蚜对双丙环虫酯解毒代谢中的作用A：双丙环虫酯处理花生蚜后GST活性变化；B：DEM对双丙环虫酯的增效作用。**P<0.01，*P<0.05，ns表示差异不显著。下同

Fig. 1 Role of GST in the detoxification metabolism of A. craccivora to afidopyropenA: GST activity in A. craccivora after treatment with afidopyropen. B: Enhanced effect of DEM on afidopyropen. **P<0.01. *P<0.05. ns refers to no significant difference. The same below

图2 花生蚜GSTs在不同组织中的表达谱不同字母代表显著水平差异（P<0.05），下同

Fig. 2 Expression patterns of GSTs at different tissues of A. craccivoraThe different letters indicate significant differences (P<0.05), the same below

图3 花生蚜GSTs在不同龄期中的表达谱

Fig. 3 Expression patterns of GSTs at different developmental stages in A. craccivora

图4 LC50浓度丙环虫酯处理花生蚜后GSTs表达量的变化

Fig. 4 Changes in the expressions of GSTs in A. craccivora after treated with imidacloprid of LC50 concentration

图5 GSTT2和GSTS1在花生蚜对双丙环虫酯解毒代谢中的作用A-B：饲喂dsGSTT2后48和72 h GSTT2的表达量；C：饲喂dsGSTT2后花生蚜对双丙环虫酯敏感性的变化；D-E：饲喂dsGSTS1后48和72 h GSTT2的表达量；F：饲喂dsGSTS1后花生蚜对双丙环虫酯敏感性的变化

Fig. 5 Role of GSTT2 and GSTS1 in the detoxification to afidopyropen in A. craccivoraA-B: Expressions of GSTT2 at 48 and 72 h after feeding dsGSTT2. C: Changes in the sensitivity of A. craccivora after feeding dsGSTT2 to afidopyropen. D-E: Expressions of GSTS2 at 48 and 72 h after feeding dsGSTS2. F: Changes in the sensitivity of A. craccivora after feeding dsGSTT1 to afidopyropen

参考文献 40

[1]	Annan IB, Tingey WM, Schaefers GA, et al. Stylet penetration activities by Aphis craccivora (Homoptera: Aphididae) on plants and excised plant parts of resistant and susceptible cultivars of cowpea (Leguminosae) [J]. Ann Entomol Soc Am, 2000, 93(1): 133-140.
[2]	Lu ZZ, Feng LK, Gao GZ, et al. Differences in the high-temperature tolerance of Aphis craccivora (Hemiptera: Aphididae) on cotton and soybean: implications for ecological niche switching among hosts [J]. Appl Entomol Zool, 2017, 52(1): 9-18.
[3]	Pavithran S, Murugan M, Mannu J, et al. Identification of salivary proteins of the cowpea aphid Aphis craccivora by transcriptome and LC-MS/MS analyses [J]. Insect Biochem Mol Biol, 2024, 165: 104060.
[4]	Fouad EA, Abou-Yousef HM, Abdallah IS, et al. Resistance monitoring and enzyme activity in three field populations of cowpea aphid (Aphis craccivora) from Egypt [J]. Crop Prot, 2016, 81: 163-167.
[5]	Yang YX, Duan AL, Zhang C, et al. Overexpression of ATP-binding cassette transporters ABCG10, ABCH3 and ABCH4 in Aphis craccivora (Koch) facilitates its tolerance to imidacloprid [J]. Pestic Biochem Physiol, 2022, 186: 105170.
[6]	Kandasamy R, London D, Stam L, et al. Afidopyropen: New and potent modulator of insect transient receptor potential channels [J]. Insect Biochem Mol Biol, 2017, 84: 32-39.
[7]	Gerwick BC, Sparks TC. Natural products for pest control: an analysis of their role, value and future [J]. Pest Manag Sci, 2014, 70(8): 1169-1185.
[8]	Koch RL, da Silva Queiroz O, Aita RC, et al. Efficacy of afidopyropen against soybean aphid (Hemiptera: Aphididae) and toxicity to natural enemies [J]. Pest Manag Sci, 2020, 76(1): 375-383.
[9]	Wang R, Gao BL, Che WN, et al. First report of field resistance to afidopyropen, the novel pyropene insecticide, on Bemisia tabaci Mediterranean (Q biotype) from China [J]. Agronomy, 2022, 12(3): 724.
[10]	Li R, Cheng SH, Liang PZ, et al. Status of the resistance of Aphis gossypii glover, 1877 (Hemiptera: Aphididae) to afidopyropen originating from microbial secondary metabolites in China [J]. Toxins, 2022, 14(11): 750.
[11]	Casida JE. Neonicotinoids and other insect nicotinic receptor competitive modulators: progress and prospects [J]. Annu Rev Entomol, 2018, 63: 125-144.
[12]	Zhang LP, Lu H, Guo K, et al. Insecticide resistance status and detoxification enzymes of wheat aphids Sitobion avenae and Rhopalosiphum padi [J]. Sci China Life Sci, 2017, 60(8): 927-930.
[13]	Liu NN. Insecticide resistance in mosquitoes: impact, mechanisms, and research directions [J]. Annu Rev Entomol, 2015, 60: 537-559.
[14]	Ketterman AJ, Saisawang C, Wongsantichon J. Insect glutathione transferases [J]. Drug Metab Rev, 2011, 43(2): 253-265.
[15]	Gao HL, Lin XM, Yang BJ, et al. The roles of GSTs in fipronil resistance in Nilaparvata lugens: Over-expression and expression induction [J]. Pestic Biochem Physiol, 2021, 177: 104880.
[16]	Zhang BZ, Jiang YT, Cui LL, et al. Overexpression of SmUGGT1 confers imidacloprid resistance to Sitobion miscanthi (takahashi) [J]. J Agric Food Chem, 2024, 72(32): 17824-17833.
[17]	Liu JP, Liu Y, Wang W, et al. Characterizing three heat shock protein 70 genes of Aphis gossypii and their expression in response to temperature and insecticide stress [J]. J Agric Food Chem, 2025, 73(5): 2842-2852.
[18]	Yang YX, Wang AY, Fu XC, et al. microRNA-mediated detoxification network conferring afidopyropen tolerance in Aphis craccivora [J]. Pestic Biochem Physiol, 2025, 213: 106485.
[19]	Silva AX, Jander G, Samaniego H, et al. Insecticide resistance mechanisms in the green peach aphid Myzus persicae (Hemiptera: Aphididae) I: a transcriptomic survey [J]. PLoS One, 2012, 7(6): e36366.
[20]	Field LM, Bass C, Davies TGE, et al. Aphid genomics and its contribution to understanding aphids as crop pests [M]//Aphids as crop pests. UK: CABI, 2017: 37-49.
[21]	Ding WJ, Guo LZ, Xue YN, et al. Life parameters and physiological reactions of cotton aphids Aphis gossypii (Hemiptera: Aphididae) to sublethal concentrations of afidopyropen [J]. Agronomy, 2024, 14(2): 258.
[22]	Zhang JQ, Ge PT, Li DQ, et al. Two homologous carboxylesterase genes from Locusta migratoria with different tissue expression patterns and roles in insecticide detoxification [J]. J Insect Physiol, 2015, 77: 1-8.
[23]	Gonis E, Fraichard S, Chertemps T, et al. Expression patterns of Drosophila melanogaster glutathione transferases [J]. Insects, 2022, 13(7): 612.
[24]	Zhu YC, Guo ZB, Chen MS, et al. Major putative pesticide receptors, detoxification enzymes, and transcriptional profile of the midgut of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae) [J]. J Invertebr Pathol, 2011, 106(2): 296-307.
[25]	Maiwald F, Haas J, Hertlein G, et al. Expression profile of the entire detoxification gene inventory of the western honeybee, Apis mellifera across life stages [J]. Pestic Biochem Physiol, 2023, 192: 105410.
[26]	Xu ZB, Zou XP, Zhang N, et al. Detoxification of insecticides, allechemicals and heavy metals by glutathione S-transferase SlGSTE1 in the gut of Spodoptera litura [J]. Insect Sci, 2015, 22(4): 503-511.
[27]	Yang BJ, Lin XM, Yu N, et al. Contribution of glutathione S-transferases to imidacloprid resistance in Nilaparvata lugens [J]. J Agric Food Chem, 2020, 68(52): 15403-15408.
[28]	Li XX, Shi HY, Gao XW, et al. Characterization of UDP-glucuronosyltransferase genes and their possible roles in multi-insecticide resistance in Plutella xylostella (L.) [J]. Pest Manag Sci, 2018, 74(3): 695-704.
[29]	Du TH, Fu BL, Wei XG, et al. Knockdown of UGT352A5 decreases the thiamethoxam resistance in Bemisia tabaci (Hemiptera: Gennadius) [J]. Int J Biol Macromol, 2021, 186: 100-108.
[30]	Nair PMG, Choi J. Identification, characterization and expression profiles of Chironomus riparius glutathione S-transferase (GST) genes in response to cadmium and silver nanoparticles exposure [J]. Aquat Toxicol, 2011, 101(3-4): 550-560.
[31]	Yang YX, Wang AY, Zhang Y, et al. Activating pathway of three metabolic detoxification phases via down-regulated endogenous microRNAs, modulates triflumezopyrim tolerance in the small brown planthopper, Laodelphax striatellus (Fallén) [J]. Int J Biol Macromol, 2022, 222: 2439-2451.
[32]	Bass C, Carvalho RA, Oliphant L, et al. Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens [J]. Insect Mol Biol, 2011, 20(6): 763-773.
[33]	Pym A, Umina PA, Reidy-Crofts J, et al. Overexpression of UDP-glucuronosyltransferase and cytochrome P450 enzymes confers resistance to sulfoxaflor in field populations of the aphid, Myzus persicae [J]. Insect Biochem Mol Biol, 2022, 143: 103743.
[34]	Balakrishnan B, Su S, Wang K, et al. Identification, expression, and regulation of an omega class glutathione S-transferase in Rhopalosiphum padi (L.) (Hemiptera: Aphididae) under insecticide stress [J]. Front Physiol, 2018, 9: 427.
[35]	Liu L, Tang HC, Huang BJ, et al. A novel Theta-class glutathione S-transferase gene in Rhopalosiphum padi (L.) (Hemiptera: Aphididae): Identification, recombinant protein expression and function analyses of RpGSTT1 [J]. J Asia Pac Entomol, 2022, 25(2): 101915.
[36]	Zhang YX, Yang BJ, Yu N, et al. Insecticide resistance associated overexpression of two sigma GST genes assists Nilaparvata lugens to remedy oxidative stress from feeding on resistant rice variety [J]. Pestic Biochem Physiol, 2022, 188: 105230.
[37]	Fu XC, Xue C, Wang X, et al. Two detoxification enzyme genes, CYP6DA2 and CarFE4, mediate the susceptibility to afidopyropen in Semiaphis heraclei d [J]. Front Physiol, 2024, 15: 1478869.
[38]	Pan YO, Tian FY, Wei X, et al. Thiamethoxam resistance in Aphis gossypii glover relies on multiple UDP-glucuronosyltransferases [J]. Front Physiol, 2018, 9: 322.
[39]	Du TH, Xue H, Zhou XM, et al. The UDP-glycosyltransferase UGT352A3 contributes to the detoxification of thiamethoxam and imidacloprid in resistant whitefly [J]. Pestic Biochem Physiol, 2025, 208: 106321.
[40]	Tao F, Si FL, Hong R, et al. Glutathione S-transferase (GST) genes and their function associated with pyrethroid resistance in the malaria vector Anopheles sinensis [J]. Pest Manag Sci, 2022, 78(10): 4127-4139.