飞蝗LmGSTS2的酶学特性及其对马拉硫磷、 p,p’-DDT的代谢分析

doi:10.3864/j.issn.0578-1752.2019.08.009

摘要/Abstract

摘要：

【目的】将飞蝗（Locusta migratoria）谷胱甘肽硫转移酶sigma2（glutathione-S-transferases sigma2,LmGSTS2）在大肠杆菌中原核表达后进行纯化,研究LmGSTS2的酶学特性,并分析其对马拉硫磷和p,p’-DDT的代谢作用。结合飞蝗体内LmGSTs2的RNA干扰及杀虫剂生物学测定,评估LmGSTS2对杀虫剂的代谢解毒能力,为飞蝗抗性治理及合理施药提供理论依据。【方法】将LmGSTS2在BL21(DE3)中表达,通过Ni-NTA亲和层析对LmGSTS2进行纯化,以CDNB为底物检测LmGSTS2在不同温度和pH条件下的酶活性变化。在最适条件下（pH=7,27℃）,用纯化的LmGSTS2蛋白与马拉硫磷和p,p’-DDT进行孵育,通过超高效液相色谱（UPLC）评估LmGSTS2对马拉硫磷和p,p’-DDT的代谢解毒能力。进一步将3 μg dsLmGSTs2注入2龄飞蝗若虫体内,24 h后检测目的基因LmGSTs2的沉默效率,并结合杀虫剂生测试验分析飞蝗对杀虫剂敏感度的变化。【结果】将LmGSTS2在大肠杆菌中诱导表达,经SDS-PAGE检测后,发现与两个对照组pET28a/BL21(DE3)和未诱导的pET28a/BL21(DE3)-LmGSTS2相比,诱导后的pET28a/BL21(DE3)-LmGSTS2总蛋白在25 kD左右出现与目标蛋白大小相符的单一条带,表明LmGSTS2成功表达。对纯化后LmGSTS2蛋白的酶学特性研究结果表明,其最适反应pH范围为6—8,在pH=7时酶活性达到顶峰;最适反应温度范围为25—30℃,27℃时活性最高。在最适条件下,LmGSTS2分别与马拉硫磷和p,p’-DDT进行孵育。UPLC结果显示,与3个对照组相比（GSH+insecticide、active LmGSTS2+insecticide及inactive LmGSTS2+GSH+ insecticide）,LmGSTS2组马拉硫磷色谱峰面积分别降低83.6%、84.0%和84.6%,差异显著（P<0.05）,而p,p’-DDT色谱峰面积与对照组相比无显著变化（P>0.05）,说明LmGSTS2可对马拉硫磷进行代谢,对p,p’-DDT无代谢作用。进一步通过RNA干扰结合杀虫剂生测在飞蝗体内检测LmGSTS2蛋白对马拉硫磷的代谢解毒作用。将靶标基因的双链RNA注入2龄飞蝗若虫体内,24 h后可使飞蝗体内LmGSTs2表达量抑制96%。飞蝗对马拉硫磷的敏感度检测发现,与对照组相比,LmGSTs2基因沉默后飞蝗对马拉硫磷的敏感度增加,死亡率由29.9%上升至45.2%,表明LmGSTS2参与了马拉硫磷在体内的代谢解毒过程。【结论】将LmGSTS2在体外进行表达纯化,并以CDNB为底物检测到该酶最适反应条件为pH=7,27℃左右;对马拉硫磷的体内和体外检测结果显示,LmGSTS2参与马拉硫磷在飞蝗体内的代谢解毒过程;对p,p’-DDT的体外研究结果显示该酶不参与p,p’-DDT的代谢。

关键词: 飞蝗, 杀虫剂, 谷胱甘肽硫转移酶, 超高效液相色谱, RNA干扰

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)

马雯,刘娇,张学尧,申国华,秦雪梅,张建琴. 飞蝗LmGSTS2的酶学特性及其对马拉硫磷、 p,p’-DDT的代谢分析[J]. 中国农业科学, 2019, 52(8): 1389-1399.

MA Wen,LIU Jiao,ZHANG XueYao,SHEN GuoHua,QIN XueMei,ZHANG JianQin. Enzymatic Characteristics and Metabolic Analysis to Malathion and p,p’-DDT of LmGSTS2 from Locusta migratoria[J]. Scientia Agricultura Sinica, 2019, 52(8): 1389-1399.

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杀虫剂 Insecticide	流动相 Mobile phase (A%)	洗脱时间 Elution time (min)	检测波长 Detection wavelength (nm)	上样量 Sample application (μL)	柱温 Temperature (℃)
马拉硫磷Malathion	80	8	210	2	35
p,p’-DDT	92	10	232	2	35

引物 Primer	引物序列Sequence of primer (5′-3′)
dsLmGSTs2F	taatacgactcactatagggGAAGAAGACATGGCAGTCTC
dsLmGSTs2R	taatacgactcactatagggCTGGTATTTCAAGTACAGTACGT
RT-LmGSTs2F	ATGGCGCCAAAATTCAAGTTAC
RT-LmGSTs2R	TCACAGAGGCCACTGCTGTAGA

	A	B	C	D
马拉硫磷峰面积 Peak area of malathion	34.98±4.20	213.40±31.80*	218.47±15.19*	226.76±19.16*
p,p’-DDT峰面积 Peak area of p,p’-DDT	177.80±14.88	169.34±8.95	193.07±7.19	178.57±13.63