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Elevated oxidative detoxification is a major mechanism responsible for pyrethroid resistance in Helicoverpa armigera from Asia. Constitutive overexpression of CYP9A12 and CYP9A14 was associated with pyrethroid resistance in the YGF strain of H. armigera. CYP9A12 and CYP9A14 were functionally expressed in the W(R) strain of yeast (Saccharomyces cerevisiae) transformed with a plasmid shuttle vector pYES2. The cell lysates prepared from yeast transformed with CYP9A12 and CYP9A14, respectively, exhibited considerable O-demethylation activities against two model substrates p-nitroanisole (0.59 and 0.42 nmol p-nitrophenol min−1 mg protein−1) and methoxyresorufin (2.98 and 5.41 pmol resorufin min−1 mg protein−1), and clearance activity against the pyrethroid esfenvalerate (8.18 and 4.29 pmol esfenvalerate min−1 mg protein−1). These results provide important evidence on the role of CYP9A12 and CYP9A14 in conferring pyrethroid resistance in H. armigera, and also demonstrate that the yeast expression system can provide necessary redox environment for insect P450s to metabolize xenobiotics. 相似文献
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黏虫是我国作物上最重要的害虫之一。细胞色素P450能够参与昆虫外源物质代谢。本研究采用RACE技术克隆了一条编码黏虫P450基因的cDNA序列,并通过Real-time PCR技术,检测了4种外源物质对该基因表达的诱导效应。该基因被国际P450命名委员会命名为CYP9A113,GenBank登录号为KY436739。利用2.5%高效氯氟氰菊酯乳油的LD_(50)处理黏虫3 h,LD_(10)、LD_(30)和LD_(50)处理12 h和24 h,可诱导表达CYP9A113基因;20%氯虫苯甲酰胺悬浮剂的LD_(10)处理黏虫12、24和48 h,LD_(30)和LD_(50)处理24 h,CYP9A113基因表达呈诱导效应;0.1和0.5 mg/mL香豆素处理6、12、24和48 h,CYP9A113基因表达均呈诱导效应;0.1和0.5 mg/mL吲哚-3-甲醇处理3、6、12、24和48 h,CYP9A113基因表达均呈诱导效应。 相似文献
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甜菜夜蛾细胞色素P450(CYP9A11)与3种杀虫剂的结合机理研究 总被引：1，自引：0，他引：1
细胞色素P450(以下简称CYP)与昆虫的抗药性密切相关。本研究运用AutoDock分子对接技术和分子力学泊松-波尔兹曼表面积法(molecular mechanics Poisson-Boltzmann surface area,MM-PBSA)结合自由能计算方法,分析了甜菜夜蛾CYP9A11与3种杀虫剂结合的作用位点、作用力类型和大小。结果表明:CYP9A11与毒死蜱结合形成两个氢键,有8个氨基酸残基参与形成疏水作用力,二者结合自由能为-3659.80 kJ/mol;CYP9A11与灭多威结合形成5个氢键,有3个氨基酸残基形成疏水作用力,结合自由能为-470.92 kJ/mol;CYP9A11中有7个氨基酸残基与氯氰菊酯结合形成疏水作用力,结合自由能为-473.44 kJ/mol。范德华力是CYP9A11与毒死蜱结合的主要驱动力,极性溶剂化能是CYP9A11与氯氰菊酯和灭多威结合的主要驱动力,这些结果为阐明甜菜夜蛾CYP9A11与3种杀虫剂的结合机理提供了参考。 相似文献
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Tomohide Uno Satoru Kaji Tatsushi Goto Hiromasa Imaishi Masahiko Nakamura Kengo Kanamaru Hiroshi Yamagata Yoshio Kaminishi Takao Itakura 《Pesticide biochemistry and physiology》2011,101(2):93-102
Through the use of a number of bioconversion experiments we demonstrated that P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica) metabolized a number of herbicides and the drug phenacetin. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolites were extracted and analyzed by high-performance liquid chromatography. Proteins CYP1A9 and CYP1C1 metabolized 50 nmol of the drug phenacetin to yield 12.1 and 1.1 nmol of product (acetaminophen), respectively. Further incubation of CYP1A9 with 50 nmol of the herbicides chlorotoluron, diuron, linuron, simazine, or atrazine yielded 16.5, 18.5, 7.3, 1.6, or 0.8 nmol of product, respectively. CYP1C1 also metabolized linuron, diuron, and simazine yield 5.4, 4.6, or 0.7 nmol of product, respectively. Next, polyclonal antibody was isolated by immunizing with two conjugated-peptides (amino acid residues 272–290 and 294–310) of CYP1A9. This antibody did not recognize human CYP1A2 or CYP1C1. Western blotting using the antibody revealed one band in the livers of Japanese eel and tilapia (Oreochromis niloticus). Theses results suggest that CYP1A9 and CYP1C1 metabolize herbicides, and that CYP1A9 is an useful biomarker of contamination when detected with this antibody. 相似文献
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紫茎泽兰CYP75基因cDNA片段的克隆与鉴定 总被引：6，自引：0，他引：6
根据菊科植物P450基因CYP75 B5(GenEMBL AF313489)和C YP75 B6(GenEMBL AF313488)核酸序列同源区设计引物,用RT-PCR方法从紫茎泽兰植株中获得一大小为378bp的细胞色素P450基因片段。经克隆、测序及氨基酸序列同源性比较,发现由该片段推导出的氨基酸序列与C YP75 B5和C YP75 B6的氨基酸序列分别具有79.5%和85.6%的同源性,与C YP76 B1、C YP81 B1 v1、C YP81 E1 v2同源性分别为40.8%、35.2%、35.0%,与C YP73 A家族的同源性在28.9%～31.4%;所绘制的系谱树与同源性分析结果一致。因此,初步确定该序列为C YP75家族中某一成员的结构基因片段。 相似文献
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Ana Maria Coimbra António Figueiredo-Fernandes Maria Armanda Reis-Henriques 《Pesticide biochemistry and physiology》2007,89(3):230-236
Endosulfan is a worldwide used insecticide suspected to be highly toxic to aquatic organisms, including fish. Most of the available studies have focused in water exposures, although this pollutant can be transferred through food chain. Therefore, in the present study, the effects of Endosulfan on tilapia (Oreochromis niloticus), when administered through the diet. Fish were fed 21 days with diets containing 1 and 0.5 μg g−1 of Endosulfan, after which qualitative histological liver analysis showed that Endosulfan induced hepatocyte destruction, vessel endothelium rupture and increased melanomacrophages aggregates. To test lower environmentally relevant doses of Endosulfan could induce hepatic damage, as well as other negative effects, such as altered phase I metabolism and plasma thyroid hormone levels. Hence, tilapia were orally exposed to 0.1 and 0.001 μg g−1 for 35 days. Low environmentally realistic doses of Endosulfan were still able to induce liver histopathological damage such as increased hepatocyte vacuolization and increased eosinophil granular cell aggregates. Liver cytochrome P450 1A activity, evaluated through ethoxyresorufin-o-deethylase (EROD), was enhanced in tilapia exposed to 0.001 μg g−1, whereas the highest dose had no measurable effects in this enzyme activity. Fish exposed to 0.1 μg g−1 of Endosulfan had depressed T4 plasma levels. Overall, the results of the present study further demonstrate the toxic effects of Endosulfan in tilapia when administered in the diet at environmentally relevant concentrations, which indicates that in the field food chain transfer may also be an importance source of this pollutant. 相似文献
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由葡萄座腔菌科Botryosphaeriaceae真菌引起的葡萄溃疡病Botryosphaeria dieback是葡萄上的主要枝干病害,严重影响葡萄产量和品质。鉴定和分析参与葡萄与葡萄溃疡病菌互作的基因,有助于揭示和阐明葡萄抗溃疡病的信号通路。本研究根据葡萄溃疡病菌侵染后的葡萄转录组数据信息,利用逆转录聚合酶链式反应技术(RTPCR)克隆了一个受葡萄溃疡病菌诱导上调表达的乙醛脱氢酶基因VvALDH10A9(Vitis vinifera aldehyde dehydrogenase 10A9)。系统进化树分析表明,该基因编码的蛋白与拟南芥AtALDH10A8亲缘关系最近。利用实时荧光定量PCR分析VvALDH10A9的表达结果表明:VvALDH10A9的表达具有组织特异性,在茎、叶和花中表达量最高,在根中表达量相对较低;葡萄溃疡病菌侵染后,VvALDH10A9在葡萄抗病品种中表达变化不明显,而在葡萄感病品种中该基因显著上调表达。利用原核蛋白表达系统诱导合成的VvALDH10A9蛋白,经纯化后可降解乙醛,表明VvALDH10A9蛋白具有乙醛脱氢酶活性。 相似文献
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昆虫体内的细胞色素P450酶系统在昆虫解毒过程中发挥着重要作用。本次试验通过转录组测序获得一条新的黏虫P450基因，经国际委员会命名为CYP9A134（登录号为MT990973）。该序列全长为1801 bp，开放序列长度为1596 bp，编码531个氨基酸，分子质量为61.42 kDa，等电点为4.90。在黏虫幼虫阶段用2.5%高效氯氟氰菊酯和20%氯虫苯甲酰胺诱导时该基因表达量会有不同程度上升，最高的可分别达对照组的2.6倍和6.5倍。RNA干扰后，该基因表达量最低下降了70%，以上2种杀虫剂LD30杀虫效果分别提高了13%和25%；在黏虫成虫阶段用20%氯虫苯甲酰胺LD30诱导时，该基因表达量最高可达7.8倍。RNA干扰后，该基因表达量最低下降了61%，LD30剂量的氯虫苯甲酰胺杀虫效果提高26%。结果表明，该基因可能在杀虫剂诱导的解毒代谢过程中发挥重要作用。 相似文献
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小菜蛾Plutella xylostella是为害十字花科作物的世界性重要害虫,对氯虫苯甲酰胺产生了严重抗性。已经明确小菜蛾CYP6BG1的过量表达与其对氯虫苯甲酰胺的抗性相关。本文进一步通过昆虫杆状病毒表达系统在Sf9细胞中表达了小菜蛾CYP6BG1和NADPH-细胞色素P450还原酶(CPR)蛋白,并检测Sf9细胞表达CYP6BG1蛋白后对氯虫苯甲酰胺的敏感变化及氯虫苯甲酰胺经CYP6BG1蛋白代谢后对小菜蛾幼虫毒力的变化。结果显示:Sf9细胞中与CPR共表达的CYP6BG1具有7-乙氧基香豆素O-脱乙基酶活性,且细胞对氯虫苯甲酰胺的敏感性显著下降。将表达的蛋白与不同浓度的氯虫苯甲酰胺孵育24 h后,对3龄小菜蛾幼虫致死率显著低于对照组。本研究结果间接表明CYP6BG1能够降解氯虫苯甲酰胺,从而增强小菜蛾对氯虫苯甲酰胺的抗性。 相似文献
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Plant cell cultures in which the appropriate P450 cDNA is introduced are expected to metabolise certain pesticides in large quantities. Two species of human P450 (CYP1A1 and CYP1A2) were introduced into tobacco cells (Nicotiana tabacum L) by Agrobacterium-mediated transformation. The transgenic plant cell cultures were selected by combination of kanamycin-resistance, 7-ethoxycoumarin O-de-ethylase activity, PCR and Western blot analysis. For metabolism studies, 14C-labelled atrazine was used as a model substance. The metabolites de-ethylatrazine and de-isopropylatrazine were found in the control culture as well as in the transgenic culture, whereas the non-phytotoxic metabolite de-ethyl-de-isopropylatrazine was found only in the transgenic cell cultures. The results showed that both foreign enzymes CYP1A1 and CYP1A2 catalyse N-dealkylation of atrazine. However, CYP1A2 exhibited a higher conversion rate than CYP1A1. In a time-course study the enzyme CYP1A2 catalysed predominantly N-de-ethylation followed by de-isopropylation. The extent of metabolism was considerably higher than in non-transformed cell cultures. The transgenic cell cultures can therefore be suitable tools for the production of large quantities of primary oxidised pesticide metabolites. 相似文献
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为阐明稻瘟菌Magnaporthe oryzae甾醇14α-脱甲基酶（sterol 14α-demethylase，CYP51）与抑制剂的互作机制，首先通过分子生物学软件预测并分析稻瘟菌CYP51蛋白的跨膜域、二级结构以及氨基酸序列保守性，对CYP51蛋白的氮端跨膜域序列进行截除处理并以此构建蛋白表达质粒；其次对表达质粒进行原核表达，并使用亲和层析、酶切、透析和凝胶过滤层析等多种蛋白纯化手段得到目的蛋白；最后利用坐滴法对CYP51蛋白与抑制剂的复合体的结晶进行筛选。结果表明，稻瘟菌CYP51A和CYP51B蛋白均有两段跨膜域，在不破坏CYP51蛋白底物和抑制剂结合区域的前提下，将稻瘟菌CYP51A蛋白氮端1~100位氨基酸以及稻瘟菌CYP51B蛋白氮端1~110位氨基酸截除，构建蛋白表达质粒pET28a-His6-MBP-TEV-CYP51A和pET28a-MBP-TEV-CYP51B-His6。经原核表达与多种纯化方法成功获得质量佳且纯度高的稻瘟菌CYP51A和CYP51B-His6单体蛋白，并分别与氯氟醚菌唑、烯唑醇及戊唑醇等抑制剂孵育获得复合体，稻瘟菌CYP51B蛋白与烯唑醇复合体在0.1 mol/L酒石酸钾钠、0.1 mol/LTris-HCl （pH 8.5）、0.4 mol/L水合硫酸镁结晶条件下长出晶体。 相似文献
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甘蓝夜蛾CYP9A90基因的克隆及溴氰菊酯对其诱导表达 总被引：3，自引：0，他引：3
P450 CYP9A家族基因与昆虫对拟除虫菊酯类杀虫剂的抗性相关。为证实溴氰菊酯对甘蓝夜蛾CYP9A基因的诱导效果,本研究采用RT-PCR和RACE技术克隆获得甘蓝夜蛾CYP9A基因,real-time PCR检测该基因在甘蓝夜蛾不同组织中的表达差异及溴氰菊酯处理甘蓝夜蛾5龄幼虫不同时间后该基因相对表达量变化,研究结果可为甘蓝夜蛾对溴氰菊酯的抗性治理提供依据。结果表明:克隆得到甘蓝夜蛾CYP9A基因cDNA全长序列,该序列包含1 828bp,包括1个125bp的5′非编码区,1个104bp的3′非编码区和1个1 599bp的开放阅读框,编码532个氨基酸,分子量约为61.1kDa,等电点为8.84,GenBank登录号为KR676343,被国际P450命名委员会命名为CYP9A90。Real-time PCR分析结果表明,该基因在甘蓝夜蛾5龄幼虫6个组织中表达情况不同,其中在脂肪体中表达量最高。低剂量溴氰菊酯作用后不同时间点CYP9A90mRNA总体呈现诱导表达趋势。 相似文献
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Genetic linkage of hscp (heliothis sodium channel protein) and CYP6B10 was discovered in Heliothis virescens. The hscp gene encodes the sodium channel target of pyrethroid insecticides and cytochrome P450 genes encode important enzymes involved in detoxication of various pesticides. Previously, two mechanisms, nerve insensitivity due to sodium channel and synergism by propynyl aryl ethers, were observed in pyrethroid-resistant H virescens and were not separated by repeated back-crossing. We hypothesized genetic linkage of target site insensitivity and monooxygenase-mediated detoxication. Single nucleotide polymorphisms were discovered in IIS6 of hscp; Hpy of hscp and CYP6B10. Segregation of these and other markers was tested in backcrosses. We observed cosegregation of hscp to CYP6B10, but both genes assorted independently of y, ye and sex. Genes y and ye assorted independently of each other. This was the first observation of linkage between genes controlling detoxication and sodium ion channel insensitivity in a species known to express high levels of pyrethroid resistance. Linkage was not likely because this species has 31 chromosomes; therefore, we will investigate the possibility of a resistance cassette. We expect similar linkage in other noctuid pests. 相似文献