细胞色素P450介导的果蝇抗药性研究进展

细胞色素P450 (cytochrome P450, CYP450)超基因家族是由一些数量多而功能复杂的血红蛋白酶基因所组成,该代谢酶系作为一种几乎地球上所有需氧生物都存在的重要生存策略,可以调控多种内源物质及外源化合物的代谢,参与了众多重要的生命过程,代谢解毒作用是该酶系重要功能之一。细胞色素P450的代谢解毒作用受药物影响,机体通过改变基因表达量,实现增强代谢解毒,加快机体对于有害物质的代谢,从而使得机体对有害环境产生一定的适应性,进而使得机体产生耐药性或抗药性。本研究说明果蝇细胞色素P450介导的杀虫剂类药物代谢机制及代谢抗性的特点等方面的研究,对明确果蝇的抗药性机制研究具有参考意义。     

东亚飞蝗谷胱甘肽S-转移酶RNA干扰效率研究

东亚飞蝗Locusta migratoria manilensis(Meyen)是我国主要的农业害虫之一,已发现东亚飞蝗对某些农药产生了抗性,其抗性机制可能与谷胱甘肽硫转移酶(GST)代谢解毒相关.本研究利用特异性引物合成东亚飞蝗GST 4个不同家族基因的双链RNA(dsRNA),将dsRNA注射到东亚飞蝗幼虫体内,采...     

CPR和P450基因在昆虫抗药性中的作用研究进展

P450酶系在昆虫代谢农药中有重要作用，NADPH-细胞色素P450还原酶（NADPH-cytochrome P450 reductase，CPR）和细胞色素P450（P450）在该酶系起核心作用。昆虫具有P450超基因家族，但只有一个单一的CPR基因，CPR是昆虫所有参与农药代谢的P450酶的唯一电子供体，其影响P450活性。P450基因的高水平表达在害虫抗药性中具有重要作用，P450基因介导的昆虫抗药性是最重要的代谢抗性类型。不同P450基因的高表达的调控机制不同，引起P450基因过量表达的原因可能有P450基因的编码区突变、顺式作用元件和反式作用因子变化、基因扩增等。细胞色素P450介导的抗药性存在一定程度的进化可塑性，即同种昆虫不同种群对相同的农药产生抗药性时，导致抗性产生的P450基因不同；同一昆虫品系在某种农药的抗性选择压力下，影响抗性的P450基因的种类和表达特性会随着持续的农药选择而发生变化。最近的研究显示，CPR的变异和昆虫抗药性相关，但是昆虫CPR基因介导抗药性的机制还缺乏深入研究。全面阐释P450酶系介导昆虫抗药性的机制、建立基于P450基因表达量变化与CPR突变的抗性分子标记，对于害虫抗药性治理具有重要意义。     

昆虫抗药性机理：行为和生理改变及解毒代谢增强（英文）

杀虫剂抗性是指“生物的一个品系发展了对该生物正常种群中大多数个体具有致死作用剂量的杀虫药剂的能力”。行为改变、生理学上的变化或代谢解毒等抗性机制能够降低毒物到达靶标的有效剂量。行为抗性是指减少昆虫与毒物接触或使昆虫能够存活于对大多数对正常个体致死（或有害）的环境中的任何行为。生理学改变的机制包括杀虫剂对表皮的穿透性降低、增加对药剂阻隔（sequestration）或储存和加速杀虫剂的排泄。细胞色素P450、水解酶和谷胱甘肽S-转移酶是杀虫药剂代谢解毒的主要3大酶系。细胞色素P450是一个超基因家族,是生物体内对外源性和内源性化合物解毒代谢或活化最重要的酶系。在许多害虫中发现P450介导的解毒代谢增加导致了对杀虫药剂抗性的增加。谷胱甘肽S-转移酶是可溶性的 二聚体蛋白,与代谢解毒、大量内源性和外源性化合物的排泄有关,许多昆虫中证明其抗药性与该酶活性增加有关。水解酶实际上是一组异源的酶类,其对抗药性的作用包括通过基因扩增增加酶量,作为结合蛋白隔离杀虫药剂或通过增加酶的活性加强对药剂的水解作用。     

棉酚和烟碱对棉铃虫的生长和细胞色素P—450单加氧酶活性的影响

以人工饲料添加测定了0．5％的棉酚和烟碱对棉铃虫的生长和细胞色素P－450单加氧酶（简称P－450酶系）活性的影响。研究结果显示,在测定浓度下,高龄棉铃虫短期取食含棉酚和烟碱的人工饲料后,对幼虫的生长没有显著影响,由此表明,棉铃虫对其主要寄主植物中的次生物质棉酚和烟碱具有很好的适应能力。与此同时,棉铃虫中肠微粒体P－450酶系的蛋白组成和酶活性发生了不同的变化,有升有降,有的没有变化。棉铃虫可能通过调整P－450酶系的各种蛋白含量和酶的活力水平,来适应对植物次生物质的代谢解毒的需要。另外,棉铃虫取食棉酚和烟碱后,细胞色素B5含量均显著提高,而细胞色素P－450含量均显著降低,细胞色素B5在棉铃虫对棉酚和烟碱的解毒代谢中可能发挥着更为重要的作用。     

柞蚕细胞色素P-450和酰胺酶的研究

柞蚕(Antheraea pernyi G.)在我国蚕业生产中占有重要地位,防治其病虫害主要靠农药。因此研究蚕体内与农药代谢有关的酶类是有意义的。 微粒体多功能氧化酶系(MFO)起着代谢杀虫剂的中心作用,并与杀虫剂的选择毒性,抗性以及增效现象密切相关。细胞色素P-450(下简称P-450)是MFO的关键成分,     

细胞色素P450介导抗性的进化可塑性

细胞色素P450是超基因家族,由其介导的杀虫剂代谢解毒的增强是昆虫产生抗药性的普遍而主要的机制。近年的研究表明,细胞色素P450介导的代谢抗性表现出一定程度的进化可塑性:即使是同种昆虫的不同种群在相同种类杀虫剂的胁迫下,进化选择出的抗性相关的细胞色素P450也有所不同,抗性的产生也可以是几种不同细胞色素P450协同作用或控制P450表达的调控因子的不同。     

细胞色素P450酶系与除草剂代谢

细胞色素P450是广泛存在于动物、植物和微生物体内的一类具有混合功能的血红素氧化酶系。它不但能够催化苯丙烷类、萜类化合物和脂肪酸等内源性物质的生物合成 ,而且参与许多外源性物质包括除草剂等的生物氧化。综述了代谢除草剂的细菌、哺乳动物和植物细胞色素P450酶系 ,概述了细胞色素P450酶系参与除草剂代谢的作用方式 :脱烷基化作用、环甲基化羟基化作用和芳环的羟基化作用等。这些细胞色素P450酶系在培育除草剂抗性作物、生物安全和生物修复方面表现出了巨大的潜能     

一个致倦库蚊杀虫剂敏感品系的筛选

化学防治是控制蚊虫传播疾病的主要方法, 抗性监测表明我国蚊虫已对有机磷、 有机氯、 氨基甲酸酯和拟除虫菊酯类杀虫剂产生了不同程度的抗性。蚊虫抗药性的分子机制主要包括靶标抗性和三大解毒酶家族带来的代谢抗性。筛选对杀虫剂敏感的品系是抗性监测和抗性机理研究必不可少的材料。本研究通过从一个致倦库蚊Culex pipiens quinquefasciatus野生种群筛选无乙酰胆碱酯酶G119S突变且具有低活性羧酸酯酶、 P450单加氧酶和谷胱甘肽-S-转移酶的单雌系, 建立了一个对杀虫剂敏感的致倦库蚊品系。该品系的羧酸酯酶活性是敏感品系S-lab的2.5倍, P450单加氧酶和谷胱甘肽-S-转移酶的活性与S-lab相当。生物测定表明, 与S-lab相比, 该品系对有机磷杀虫剂有低于2倍的抗性, 对氨基甲酸酯和拟除虫菊酯类杀虫剂没有抗性, 可以作为相对敏感品系用于抗性监测。     

昆虫谷胱甘肽S-转移酶的多样性及其介导的抗药性

谷胱甘肽S-转移酶(GSTs)是一类广泛分布于生物体的多功能解毒酶系,参与许多内外源有毒物质的代谢。昆虫GSTs目前主要分为6个已知亚族,其中Delta和Epsion是昆虫特异的亚族,已鉴定的抗性相关基因主要分属于这两个亚族。作为重要的解毒酶,它主要参与昆虫对有机磷、拟除虫菊酯和有机氯等杀虫剂的抗性形成。本文主要对昆虫细胞质GSTs的分类、基因多样性及其在抗药性中的作用等相关研究进展进行综述。     

Determination of metabolic resistance mechanisms in pyrethroid‐resistant and fipronil‐tolerant brown dog ticks

Rhipicephalus sanguineus (Latreille) (Ixodida: Ixodidae) is a three‐host dog tick found worldwide that is able to complete its' entire lifecycle indoors. Options for the management of R. sanguineus are limited and its' control relies largely on only a few acaricidal active ingredients. Previous studies have confirmed permethrin resistance and fipronil tolerance in R. sanguineus populations, commonly conferred by metabolic detoxification or target site mutations. Herein, five strains of permethrin‐resistant and three strains of fipronil‐tolerant ticks were evaluated for metabolic resistance using synergists to block metabolic enzymes. Synergist studies were completed with triphenyl phosphate (TPP) for esterase inhibition, piperonyl butoxide (PBO) for cytochrome P450 inhibition, and diethyl maleate (DEM) for glutathione‐S‐transferase inhibition. Additionally, increased esterase activity was confirmed using gel electrophoresis. The most important metabolic detoxification mechanism in permethrin‐resistant ticks was increased esterase activity, followed by increased cytochrome P450 activity. The inhibition of metabolic enzymes did not have a marked impact on fipronil‐tolerant tick strains.     

丝氨酸蛋白酶抑制剂Serpin1对东亚飞蝗酶学免疫的影响

Serpins是东亚飞蝗Locusta migratoria manilensis体内具有免疫调节功能的一类丝氨酸蛋白酶抑制剂.前期研究发现Serpin1能够降低绿僵菌Metarhizium对蝗虫的杀虫效果,本研究旨在从酶学角度明确Serpin1蛋白抑制绿僵菌毒力的原因,进一步揭示Serpins的功能与作用机制.本实验采用饵剂饲喂的方法进一步明确Serpin1蛋白对绿僵菌侵染东亚飞蝗的抑制效果;测定绿僵菌侵染东亚飞蝗过程中,添加Serpin1蛋白对东亚飞蝗体内保护酶(超氧化物歧化酶SOD、过氧化物酶POD、酚氧化酶PO)、解毒酶(多功能氧化酶MFO、谷胱甘肽转移酶GSTs、乙酰胆碱酯酶AchE)共6种酶的影响,以明确Serpin1对东亚飞蝗酶学免疫的调节作用.结果表明,Serpin1能够显著降低绿僵菌对蝗虫的杀虫效果;将Serpin1与绿僵菌混合后处理东亚飞蝗,12 d后其死亡率为63.5％,显著低于绿僵菌单独处理(死亡率为80.6％).酶活测定结果显示,将绿僵菌IMI330189与Serpin1蛋白混合处理后,与绿僵菌处理组相比,东亚飞蝗体内保护酶SOD和PO的活力总体表现为上调,而POD的活力呈现降低的趋势;解毒酶MFO、GSTs的活性呈现升高趋势,AChE的活力呈现先升高后降低的趋势.上述结果表明,Serpin1蛋白能够增强东亚飞蝗体内解毒酶和保护酶的活性,提高东亚飞蝗的酶学免疫,增强对绿僵菌侵染的抵御能力,从而降低东亚飞蝗的死亡率.本研究为进一步揭示Serpins的功能提供了参考.     

Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibilities to three organophosphate (OP) insecticides (malathion, chlorpyrifos, and phoxim), responses to three metabolic synergists [triphenyl phosphate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM)], activities of major detoxification enzymes [general esterases (ESTs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s)], and sensitivity of the target enzyme acetylcholinesterase (AChE) were compared between a laboratory-susceptible strain (LS) and a field-resistant population (FR) of the oriental migratory locust, Locusta migratoria manilensis (Meyen). The FR was significantly resistant to malathion (57.5-fold), but marginally resistant to chlorpyrifos (5.4) and phoxim (2.9). The malathion resistance of the FR was significantly diminished by TPP (synergism ratio: 16.2) and DEM (3.3), but was unchanged by PBO. In contrast, none of these synergists significantly affected the toxicity of malathion in the LS. Biochemical studies indicated that EST and GST activities in the FR were 2.1- to 3.2-fold and 1.2- to 2.0-fold, respectively, higher than those in the LS, but there was no significant difference in P450 activity between the LS and FR. Furthermore, AChE from the FR showed 4.0-fold higher activity but was 3.2-, 2.2-, and 1.1-fold less sensitive to inhibition by malaoxon, chlorpyrifos-oxon, and phoxim, respectively, than that from the LS. All these results clearly indicated that the observed malathion resistance in the FR was conferred by multiple mechanisms, including increased detoxification by ESTs and GSTs, and increased activity and reduced sensitivity of AChE to OP inhibition.     

Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases

Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.     

Plant cytochrome P450-mediated herbicide metabolism

In the last two decades it has become apparent that enzymes of the P450 monooxygenase (P450) superfamily are responsible for the Phase I metabolism of numerous herbicides representing several classes of organic compounds. The majority of experimental evidence for P450 involvement in herbicide metabolism has been derived from in vitro studies in which the catalytic activity of plant microsomes towards herbicidal substrates was measured in the presence of various P450 inhibitors and activators. While the studies with microsomes elicited much appreciation for the pivotal roles of plant P450s in herbicide metabolism, detailed characterization of these enzymes only became possible after the isolation of genes encoding specific isoforms responsible for herbicide conversion. Several lines of evidence suggest that the development of herbicide resistance in weeds by enhanced detoxification is frequently associated with elevated levels of P450 activity. Enhanced detoxification-based herbicide resistance is particularly difficult to control, because it can involve resistance to multiple, chemically unrelated classes of herbicides. Continued research efforts are aimed at elucidating the role of P450s in the metabolic fates of herbicides in plants and the development of herbicide resistance in weeds. Recent advances made in the isolation and genetic manipulation of P450 enzymes have created new opportunities for their application in engineering herbicide tolerance and bioremediation.     

Biochemical characterization of hydrolytic and oxidative enzymes in insecticide resistant and susceptible strains of the German cockroach (Dictyoptera: Blattellidae).

We have identified resistance mechanisms in the German cockroach, Blattella germanica (L.), for propoxur and chlorpyrifos in strains of cockroaches that display multiresistance to several organophosphate and carbamate insecticides. The resistance mechanisms involve the combined effects of increased oxidative and hydrolytic metabolism and both strains are resistant to chlorpyrifos and propoxur. Experiments designed to test for similarity in metabolic enzymes suggest that, although the mechanisms involve similar processes, the enzymes responsible for insecticide detoxification are different in the two strains. Both resistant strains exhibited enhanced activity toward alpha-naphtholic esters relative to a standard susceptible strain; however, analysis of the progeny from resistant X susceptible crosses suggests that this general esterase activity is inherited differently than propoxur or chlorpyrifos resistance. Hybrids of the propoxur-resistant strain displayed the highest activity of all cockroaches tested, in contrast to hybrids of the chlorpyrifos-resistant strain, which were similar to the susceptible strain. Native gel electrophoresis of cytosolic preparations provided further evidence for differences in the pattern of hydrolytic enzymes and inheritance of resistance in the two strains. Analysis of components of the cytochrome P450-dependent monooxygenase system and activities toward model substrates indicate that the two resistance mechanisms also involve different oxidative processes. The propoxur-resistant strain displayed significantly higher levels of total cytochrome P450, but no other components were correlated with resistance. In contrast with the chlopyrifos-resistant strain, which was similar to the susceptible strain in all parameters measured, activity toward model substrates was higher in the propoxur-resistant strain than in any of the other strains and hybrids tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P450 MA expression in insecticide-resistant German cockroaches (Dictyoptera: Blattellidae).

Cytochrome P450 monooxygenases are detoxification enzymes commonly involved in insecticide resistance by insects. Recently, an overexpressed form of this enzyme, P450 MA, was purified from an insecticide-resistant strain of German cockroach, Blattella germanica (L.), and polyclonal antisera (anti-P450 MA) was produced. To test hypotheses that the overexpressed condition of P450 MA has evolved in > 1 geographic location and that P450 MA might be involved in insecticide resistance to specific insecticides, investigations were conducted using 4 insecticide-resistant and 1 susceptible German cockroach strains. In western blots that used anti-P450 MA antiserum as a probe, substantial differences in expression of P450 MA were observed. Strains showing the highest P450 MA expression had both the highest tolerance to the organophosphate insecticide chlorpyrifos and cytochrome P450-mediated demethylation activity. Results support the hypothesis that cytochrome P450 MA is potentially overexpressed in insecticide-resistant populations on a global scale.     

Metabolic imidacloprid resistance in the brown planthopper,Nilaparvata lugens,relies on multiple P450 enzymes

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance.