MBF2转录调控小菜蛾谷胱甘肽S-转移酶代谢氯虫苯甲酰胺的功能

doi:10.3864/j.issn.0578-1752.2023.04.006

摘要/Abstract

摘要：

【目的】探究转录调控因子MBF2 （multiprotein bridging factor 2）调控小菜蛾（Plutella xylostella）体内谷胱甘肽S-转移酶（glutathione S-transferase，GST）的功能，及其在小菜蛾抗氯虫苯甲酰胺中的作用，为阐明小菜蛾对氯虫苯甲酰胺的解毒代谢机制提供理论依据。【方法】采用浸叶法测定敏感（SS）、广东连州（LZ）、云南通海（TH）3个小菜蛾种群对氯虫苯甲酰胺的抗性水平，酶动力学法测定3个种群的GST活性；使用实时荧光定量PCR（RT-qPCR）分析MBF2在不同抗性种群小菜蛾中的表达差异；采用氯虫苯甲酰胺LC₅₀诱导12 h，分析其对小菜蛾MBF2的诱导表达作用；应用RNA干扰（RNA interference，RNAi）技术研究MBF2 对GST基因的调控作用，并测定沉默MBF2后小菜蛾的GST活性；结合沉默后小菜蛾对药剂的敏感性变化，验证MBF2在小菜蛾抗氯虫苯甲酰胺中的作用。【结果】两个抗性种群（TH和LZ）相较SS种群分别达到了中抗及高抗的水平，TH和LZ抗性倍数分别为54.27和289.58；TH和LZ小菜蛾种群体内GST活性均显著高于SS种群；与SS种群相比，MBF2在抗性种群小菜蛾体内显著上调表达，在中抗和高抗种群中的表达量分别为敏感种群4.6和9.4倍。小菜蛾短期暴露于LC₅₀浓度氯虫苯甲酰胺中，敏感、中抗及高抗种群均在8 h内MBF2表达量显著上调，最高表达量升至对照的10倍。沉默MBF2 24 h，小菜蛾9个GST基因表达量显著降低，其中GSTZ1及GSTU1的下调量均达90%以上；处理组（dsMBF2）GST活性相较于阴性对照（dsGFP）降低57.76%；75 mg·L^-1氯虫苯甲酰胺处理后，处理组较对照组死亡率提升23.34%。【结论】MBF2可能通过调控GST的转录表达，提高小菜蛾对氯虫苯甲酰胺的代谢能力，从而参与小菜蛾对氯虫苯甲酰胺的解毒作用。研究结果可为小菜蛾抗药性机理的深入解析及新型药剂的研发提供依据。

关键词: 小菜蛾, 转录调控, 谷胱甘肽S-转移酶, 氯虫苯甲酰胺, 抗药性, MBF2

Abstract:

【Objective】The objective of this study is to explore the regulation function of transcription regulation factor MBF2 (multiprotein bridging factor 2) on glutathione S-transferase (GST) in Plutella xylostella and its role in resistance to chlorantraniliprole, and to provide a theoretical basis for clarifying the metabolic mechanism of P. xylostella to detoxify chlorantraniliprole. 【Method】The resistance level of P. xylostella to chlorantraniliprole in three populations, susceptible (SS), Lianzhou (LZ) and Tonghai (TH), was determined by leaf dipping method and the GST activity of three populations was measured by enzyme kinetic method. Real-time quantitative PCR (RT-qPCR) was used to analyze the expression difference of MBF2 in different P. xylostella populations. After induction for 12 h under the LC₅₀ of chlorantraniliprole, the induced expression level of MBF2 in different populations was analyzed. The regulatory role of MBF2 on specific GST genes was confirmed by RNA interference (RNAi) and the GST activity of MBF2-silenced P. xylostella was analyzed. Susceptibility variations of MBF2-silenced P. xylostella populations to chlorantraniliprole were conducted by leaf dipping method, which support that MBF2 influenced the resistance of P. xylostella to chlorantraniliprole. 【Result】The TH population was moderately resistant and LZ population was highly resistant to chlorantraniliprole when respectively compared to susceptible population (SS), with the resistance ratio of 54.27 and 289.58. The GST activity of TH and LZ populations was significantly higher than that of SS population. Compared with SS population, MBF2 was significantly up-regulated in resistant populations, and the expression level of MBF2 in the moderately resistant and highly resistant populations was 4.6 and 9.4 times that of susceptible population, respectively. When P. xylostella was exposed to chlorantraniliprole at LC₅₀ for a short time, the expression levels of MBF2 in susceptible, moderately resistant and highly resistant populations were significantly increased within 8 hours, and the highest expression was 10 times that of the control. After RNAi treatment for 24 hours, the expression levels of 9 GST genes in MBF2-silenced P. xylostella were significantly decreased, and GSTZ1 as well as GSTU1 decreased by more than 90%. Compared with the negative control (dsGFP), the GST activity in the treatment group (dsMBF2) decreased by 57.76%. After treatment with 75 mg·L^-1 chlorantraniliprole, the mortality in the treatment group increased by 23.34% compared with the control group. 【Conclusion】It is speculated that MBF2 improves the metabolism of chlorantraniliprole by regulating the transcription of GSTs, thereby participating in the detoxification effect of P. xylostella to chlorantraniliprole. The results can provide experimental basis for further analysis of P. xylostella resistance mechanisms to insecticides and research as well as development of new insecticides.

Key words: Plutella xylostella, transcriptional regulation, glutathione S-transferase (GST), chlorantraniliprole, insecticide resistance, MBF2

葛天成, 尹飞, 胡琼波, 彭争科, 李振宇. MBF2转录调控小菜蛾谷胱甘肽S-转移酶代谢氯虫苯甲酰胺的功能[J]. 中国农业科学, 2023, 56(4): 665-673.

GE TianCheng, YIN Fei, HU QiongBo, PENG ZhengKe, LI ZhenYu. Function of MBF2 Transcriptionally Regulating Glutathione S-transferase Metabolizing Chlorantraniliprole in Plutella xylostella[J]. Scientia Agricultura Sinica, 2023, 56(4): 665-673.

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种群 Population	斜率 Slope (mean±SE)	LC₅₀95%置信区间 Confidence interval (mg·L^-1)	相关系数 Relevant coefficient	χ² (df)	抗性倍数 Resistance ratio
SS	2.60±0.34	0.26 (0.19-0.38)	0.995	0.59 (3)	-
TH	2.72±0.49	14.11 (10.23-18.74)	0.932	5.74 (3)	54.27
LZ	2.86±0.52	75.29 (55.50-108.41)	0.981	0.82 (3)	289.58

种群 Population	酶活性 Enzymatic activity (nmol·min^-1·mg^-1)
SS	134.83±7.50c
TH	217.79±24.34b
LZ	295.71±12.13a