Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (8): 1389-1399.doi: 10.3864/j.issn.0578-1752.2019.08.009

• PLANT PROTECTION • Previous Articles     Next Articles

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
  • Received:2018-11-23 Accepted:2018-12-27 Online:2019-04-16 Published:2019-04-26
  • Contact: XueMei QIN,JianQin ZHANG E-mail:qinxm@sxu.edu.cn;jiangqinzh3@sxu.edu.cn

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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)

Table 1

The detection conditions of UPLC for insecticides"

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

Table 2

The sequences of dsRNA and RT-qPCR primers of LmGSTs2"

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

Fig. 1

Expression and purification of LmGSTS2 M：10—180 kD蛋白分子量标准 10-180 kD protein molecular weight standards。A：LmGSTS2的表达 Expression of LmGSTS2。1：BL21(DE3);2：pET28a/BL21(DE3);3：未诱导pET28a/BL21(DE3)-LmGSTS2 pET28a/BL21(DE3)-LmGSTS2 before induction;4：诱导后的pET28a/BL21(DE3)-LmGSTS2 pET28a/BL21(DE3)-LmGSTS2 after induction。B：LmGSTS2的纯化 Purification of LmGSTS2。 1：未纯化的LmGSTS2总蛋白Total protein of LmGSTS2 before purification;2：过柱穿透液 Flow through fraction;3—12：不同浓度咪唑缓冲液洗脱组分Elution components with different concentrations of imidazole buffer"

Fig. 2

Activity changes of LmGSTS2 under different conditions"

Fig. 3

Chromatograms of malathion metabolized by LmGSTS2 A: Active LmGSTS2+GSH+malathion; B: GSH+malathion; C: Active LmGSTS2+ malathion; D: Inactive LmGSTS2+GSH+malathion"

Table 3

Chromatographic peak area of insecticides in different groups (mAu×min)"

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

Fig. 4

Chromatograms of p,p’-DDT metabolized by LmGSTS2 A: Active LmGSTS2+GSH+ p,p’-DDT; B: GSH+ p,p’-DDT; C: Active LmGSTS2+ p,p’-DDT; D: Inactive LmGSTS2+GSH+ p,p’-DDT"

Fig. 5

Silencing efficiency and L. migratoria sensitivity to malathion after LmGSTs2 expression suppression A：dsLmGSTs2沉默效率 Silencing efficiency of dsLmGSTs2。**: P<0.01 (t-test);B：飞蝗对马拉硫磷敏感度分析 Sensitivity of L. migratoria to malathion。*: P<0.05 (t-test)"

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