中國農業科學 ?? 2019, Vol. 52 ?? Issue (8): 1389-1399.doi: 10.3864/j.issn.0578-1752.2019.08.009

? 植物保護 ? 上一篇    下一篇

飛蝗LmGSTS2的酶學特性及其對馬拉硫磷、 p,p’-DDT的代謝分析

馬雯1,劉嬌2,張學堯2,申國華3,秦雪梅1(),張建琴1()   

  1. 1 山西大學中醫藥現代研究中心,太原 030006
    2 山西大學應用生物學研究所,太原 030006
    3 山西省食品藥品檢驗所,太原 030001
  • 收稿日期:2018-11-23 接受日期:2018-12-27 出版日期:2019-04-16 發布日期:2019-04-26
  • 通訊作者: 秦雪梅,張建琴 E-mail:[email protected];[email protected]
  • 作者簡介:馬雯,E-mail:[email protected]
  • 基金資助:
    國家自然科學基金國際合作項目(30810103907);山西省應用基礎研究項目(201601D202058);2016省級配套國家科研項目(226546001);地產中藥功效物質研究與利用山西省重點實驗室(201605D111004)

Enzymatic Characteristics and Metabolic Analysis to Malathion and p,p’-DDT of LmGSTS2 from Locusta migratoria

MA Wen1,LIU Jiao2,ZHANG XueYao2,SHEN GuoHua3,QIN XueMei1(),ZHANG JianQin1()   

  1. 1 Modern Research Center For Traditional Chinese Medicine, Shanxi University, Taiyuan 030006
    2 Institute of Applied Biology, Shanxi University, Taiyuan 030006
    3 Shanxi Provincial Institute for Food and Drug Control, Taiyuan 030001

摘要:

【目的】將飛蝗(Locusta migratoria)谷胱甘肽硫轉移酶sigma2(glutathione-S-transferases sigma2,LmGSTS2)在大腸桿菌中原核表達后進行純化,研究LmGSTS2的酶學特性,并分析其對馬拉硫磷和p,p’-DDT的代謝作用。結合飛蝗體內LmGSTs2的RNA干擾及殺蟲劑生物學測定,評估LmGSTS2對殺蟲劑的代謝解毒能力,為飛蝗抗性治理及合理施藥提供理論依據。【方法】將LmGSTS2在BL21(DE3)中表達,通過Ni-NTA親和層析對LmGSTS2進行純化,以CDNB為底物檢測LmGSTS2在不同溫度和pH條件下的酶活性變化。在最適條件下(pH=7,27℃),用純化的LmGSTS2蛋白與馬拉硫磷和p,p’-DDT進行孵育,通過超高效液相色譜(UPLC)評估LmGSTS2對馬拉硫磷和p,p’-DDT的代謝解毒能力。進一步將3 μg dsLmGSTs2注入2齡飛蝗若蟲體內,24 h后檢測目的基因LmGSTs2的沉默效率,并結合殺蟲劑生測試驗分析飛蝗對殺蟲劑敏感度的變化。【結果】將LmGSTS2在大腸桿菌中誘導表達,經SDS-PAGE檢測后,發現與兩個對照組pET28a/BL21(DE3)和未誘導的pET28a/BL21(DE3)-LmGSTS2相比,誘導后的pET28a/BL21(DE3)-LmGSTS2總蛋白在25 kD左右出現與目標蛋白大小相符的單一條帶,表明LmGSTS2成功表達。對純化后LmGSTS2蛋白的酶學特性研究結果表明,其最適反應pH范圍為6—8,在pH=7時酶活性達到頂峰;最適反應溫度范圍為25—30℃,27℃時活性最高。在最適條件下,LmGSTS2分別與馬拉硫磷和p,p’-DDT進行孵育。UPLC結果顯示,與3個對照組相比(GSH+insecticide、active LmGSTS2+insecticide及inactive LmGSTS2+GSH+ insecticide),LmGSTS2組馬拉硫磷色譜峰面積分別降低83.6%、84.0%和84.6%,差異顯著(P<0.05),而p,p’-DDT色譜峰面積與對照組相比無顯著變化(P>0.05),說明LmGSTS2可對馬拉硫磷進行代謝,對p,p’-DDT無代謝作用。進一步通過RNA干擾結合殺蟲劑生測在飛蝗體內檢測LmGSTS2蛋白對馬拉硫磷的代謝解毒作用。將靶標基因的雙鏈RNA注入2齡飛蝗若蟲體內,24 h后可使飛蝗體內LmGSTs2表達量抑制96%。飛蝗對馬拉硫磷的敏感度檢測發現,與對照組相比,LmGSTs2基因沉默后飛蝗對馬拉硫磷的敏感度增加,死亡率由29.9%上升至45.2%,表明LmGSTS2參與了馬拉硫磷在體內的代謝解毒過程。【結論】將LmGSTS2在體外進行表達純化,并以CDNB為底物檢測到該酶最適反應條件為pH=7,27℃左右;對馬拉硫磷的體內和體外檢測結果顯示,LmGSTS2參與馬拉硫磷在飛蝗體內的代謝解毒過程;對p,p’-DDT的體外研究結果顯示該酶不參與p,p’-DDT的代謝。

關鍵詞: 飛蝗, 殺蟲劑, 谷胱甘肽硫轉移酶, 超高效液相色譜, RNA干擾

Abstract:

【Objective】 Glutathione S-transferase sigma2 (LmGSTS2) from Locusta migratoria was expressed in Escherichia coli and purified in order to analyze the enzymatic characteristics. The objective of this research was to study the effect of LmGSTS2 on malathion and p,p’-DDT metabolism. The detoxification ability of LmGSTS2 was assessed by using LmGSTs2 RNA interference (RNAi) and insecticide bioassay. It will provide a theoretical basis for management of locust resistance and rational insecticide application.【Method】LmGSTS2 was expressed in BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. The activities of LmGSTS2 under different conditions (temperature and pH) were detected using CDNB as substrate. Under the optimal conditions (pH 7, 27℃), malathion and p,p’-DDT were incubated with purified LmGSTS2 protein. The metabolic detoxification ability of LmGSTS2 to malathion and p,p’-DDT was evaluated by ultra performance liquid chromatography (UPLC). Furthermore, 3 μg of dsLmGSTs2 was injected into 2nd instar nymph, RNAi efficiency of LmGSTs2 was tested at 24 h after dsLmGSTs2 injection and the sensitivity of L. migratoria to malathion was analyzed at 24 h after malathion exposure. 【Result】 LmGSTs2 was induced to express in E. coli. After SDS-PAGE detection, it was found that an extra band around 25 kD in the total protein of pET28a/BL21 (DE3)-LmGSTS2 after induction compared with pET28a/BL21(DE3) and uninduced pET28a/BL21(DE3)-LmGSTS2, which is regarded as the target protein size, indicating that LmGSTS2 was successfully expressed in the bacteria. The results of the study on the enzymatic characteristics of the purified LmGSTS2 protein showed that the optimum reaction pH was 6-8, the enzyme activity reached the peak at pH=7, the optimum reaction temperature was 25-30℃, and the activity was the highest at 27℃. LmGSTS2 was exposed to malathion and p,p’-DDT respectively at pH 7, 27℃. The results of UPLC showed that the peak area of malathion after incubation with LmGSTS2 decreased by 83.6%, 84.0% and 84.6%, respectively, compared with GSH+insecticide, active LmGSTS2+insecticide and inactive LmGSTS2+GSH+insecticide (P<0.05). However, there was no significant change in the peak area of p,p’-DDT compared with the control group (P>0.05), indicating that LmGSTS2 could metabolize malathion, but had no effect on the metabolism of p,p’-DDT. The role of LmGSTS2 in the detoxification process of malathion was further verified by RNA interference. The dsRNA of the target gene was injected into the 2nd instar nymph. After 24 h, the mRNA expression of LmGSTs2 was inhibited by 96%. The sensitivity test showed that compared with the control group, the sensitivity of L. migratoria to malathion increased after gene silencing, and the mortality increased from 29.9% to 45.2%, indicating that LmGSTS2 was involved in the detoxification process of malathion in L. migratoria. 【Conclusion】 LmGSTS2 was expressed and purified in vitro, and the optimal reaction condition of the enzyme was pH=7, 27℃ using CDNB as substrate. In vivo and in vitro assays for malathion metabolism showed that LmGSTS2 was involved in the metabolic detoxification in L. migratoria. In vitro assay for p,p’-DDT metabolism showed that LmGSTS2 was not involved in the metabolism of p,p’-DDT.

Key words: Locusta migratoria, insecticide, glutathione-S-transferases, ultra performance liquid chromatography (UPLC), RNA interference (RNAi)

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