甲基二磺隆及吡唑解草酯对不同品种小麦ALS酶的影响

采用温室盆栽茎叶喷雾处理,研究了3%甲基二磺隆油悬浮剂以及安全剂吡唑解草酯对不同品种小麦的耐药性及其对靶标酶乙酰乳酸合成酶(ALS)的影响。结果表明,甲基二磺隆对敏18号小麦株高抑制程度明显高于抗6号。离体ALS酶含量和活力测定结果表明,抗6号ALS含量高于敏18号,在200和400 μmol/L的吡唑解草酯溶液中黑暗培养7 d,抗6号小麦ALS比活力比对照分别增加1.29和1.58倍,敏18号比对照分别增加0.25和0.15倍。抗6号小麦品种对甲基二磺隆耐药性较强。小麦自身ALS含量和比活力差异以及安全剂吡唑解草酯对它们的诱导差异可能是小麦品种对甲基二磺隆耐药性不同的原因之一。     

抗阿维菌素小菜蛾的细胞色素P450酶系研究

通过对不同发育时期敏感和抗阿维菌素小菜蛾品系细胞色素P450含量的测定,以及使用不同模式底物对P450单加氧酶活性的比较研究发现:除成虫期外,不同发育时期抗性品系小菜蛾中P450和细胞色素b5的含量都高于敏感品系;抗性品系还原型辅酶Ⅱ(NADPH)-细胞色素P450还原酶活性是敏感品系的1.97倍;同时发现抗性品系中甲氧试卤灵-O-脱甲基酶(MROD)、乙氧试卤灵-O-脱乙基酶(EROD)、乙氧基香豆素-O-脱乙基酶(ECOD)以及对硝基苯甲醚-O-脱甲基酶(PNOD)的活性均明显高于敏感品系,分别为敏感品系的9.41、4.15、1.67和2.94倍。研究结果表明,细胞色素P450含量和单加氧酶活性的增高是小菜蛾对阿维菌素产生抗性的一个重要机制。     

环氧虫啶对苜蓿蚜的毒力及对其体内解毒酶活性的影响

为研究桥环新烟碱类化合物对苜蓿蚜的影响,以吡虫啉为对照药剂,采用带虫浸叶法测定了环氧虫啶等桥环新烟碱类化合物对苜蓿蚜的毒力及对其体内解毒酶活性的影响。结果表明:以环氧虫啶为代表的七元桥环新烟碱类化合物对苜蓿蚜具有较好的杀虫活性,其中,环氧虫啶的LC50值为3.454 mg/L,高于八元桥环新烟碱化合物。酶抑制剂顺丁烯二乙酯（DEM）和胡椒基丁醚（PBO）对环氧虫啶均具有显著的增效作用,增效比分别为4.02和3.22;而对照药剂吡虫啉仅PBO对其具有明显增效作用。与空白对照组相比,经LC50浓度环氧虫啶和吡虫啉处理后,存活苜蓿蚜体内谷胱甘肽S-转移酶（GSTs）和细胞色素P450s活力均显著升高（P<0.05）,其中,环氧虫啶处理组GSTs和P450s活力分别达到（2.730±0.012）和（0.239±0.009）μmol/（mg pro.·min）,诱导能力弱于吡虫啉;而苜蓿蚜体内羧酸酯酶（CarE）活性则无明显变化。研究显示,在苜蓿蚜对环氧虫啶的解毒代谢过程中,GSTs和细胞色素P450s可能发挥着主要作用。     

细胞色素P450s酶系催化除草剂代谢的研究进展

综述了细胞色素P450s酶系催化的单加氧反应机理,细胞色素P450s酶系在酰胺类、三氮苯类、磺酰脲类、脲类、苯氧羧酸类等除草剂的活性或降解代谢中的催化反应。讨论了研究细胞色素P450s酶系代谢作用在除草剂选择性、抗药性机理,抗除草剂作物的培育以及除草剂安全剂的解毒机理等方面的意义。     

聚乙二醇8000对棉铃虫微粒体蛋白沉淀作用的研究

为建立棉铃虫Helicoverpa armigera微粒体P450的分离纯化方案,比较了不同浓度下聚乙二醇8000(PEG8000)对棉铃虫幼虫中肠和脂肪体微粒体蛋白的沉淀作用。结果表明,终浓度为8.0×104 mg/L的PEG8000可以使中肠和脂肪体微粒体蛋白的78%沉淀下来,其中包含的细胞色素P450分别占中肠和脂肪体P450总量的28%和34%。SDS-PAGE显示,中肠微粒体经8.0×104 mg/L的PEG8000沉淀后,被沉淀蛋白的分子质量主要集中在14.1 ~ 40 kD和66 ~97 kD范围内。     

细胞色素P450s的纯化与鉴定

综述了细胞色素 P450s纯化和鉴定技术的研究进展。纯化技术包括色谱、电泳和基因克隆技术 ;鉴定方法有光谱测定、酶活测定、电泳分析和蛋白质印迹测定。     

RNAi介导的棉铃虫细胞色素P450酶系几种组分基因沉默对高效氯氰菊酯毒力的影响

为进一步明确细胞色素P450 CYP6B7、细胞色素b5(cytochrome b5,Cyt-b5)和细胞色素P450还原酶(cytochrome P450 reductase,CPR)在棉铃虫Helicoverpa armigera (Hübner)对拟除虫菊酯类药剂抗性中的作用,通过RNA干扰技术沉默了抗氰戊菊酯棉铃虫中3种组分的基因,并测定基因沉默后高效氯氰菊酯对棉铃虫的毒力变化.结果显示,经单个CYP6B7或CYP6 B7与CPR、Cyt-b5基因的双链RNA注射处理后12 ～ 48 h,棉铃虫幼虫死亡率在19.4％ ～ 92.5％之间,明显高于空白对照处理的10.47％ ～ 62.87％;高效氯氰菊酯对基因沉默后幼虫的LD50值随着时间的延长而逐渐下降,12～48 h的LD50值在0.97～2.97 μg/头之间,而空白对照处理为2.27 ～ 3.57μg/头.研究表明,细胞色素P450 CYP6B7,或CYP6B7与CPR、Cyt-b5基因的沉默,可提高高效氯氰菊酯对抗性棉铃虫的毒力,进一步证明了CYP6B7、CPR和Cyt-b5在棉铃虫对菊酯类药剂抗性中起着重要作用.     

多功能氧化酶系(MFO)与棉铃虫抗药性关系初步研究

本文在增效作用测定基础上 ,研究比较了多功能氧化酶系 (MFO)的三个组分 (细胞色素 P4 50、细胞色素 b5、细胞色素 c还原酶 )在相对敏感棉铃虫 (HDS)与抗氰戊菊酯棉铃虫 (KQR)中的含量、活性及其组织分布规律。增效作用测定结果表明 ,PBO及 SV1对氰戊菊酯具有显著的增效作用 ,增效倍数分别达 2 53.99和71.4 4倍 ;初步表明 MFO在 KQR种群棉铃虫的抗药性中起着重要作用 ;随后的生化研究表明 :KQR种群棉铃虫中肠、脂肪体及体壁的三个组分含量均显著或极显著高于 HDS种群棉铃虫相应部位的含量 ,其中细胞色素 P4 50在上述三个部位中的含量分别是 HDS种群棉铃虫的 1.76、3.12、4 .15倍 ,细胞色素 b5含量分别是HDS种群棉铃虫的 1.57、6 .2 8、2 .39倍 ,细胞色素 c还原酶活性分别是 HDS种群棉铃虫的 1.4 0、1.88、1.6 4倍 ,说明多功能氧化酶系与棉铃虫的抗药性密切相关。     

苹果轮纹病菌对吡唑醚菌酯的敏感性及Cytb基因序列分析

为了明确苹果轮纹病菌Botryosphaeria dothidea对吡唑醚菌酯的敏感性和吡唑醚菌酯的靶标基因序列, 采用菌丝生长速率法测定了水杨肟酸对B. dothidea 菌丝生长的作用, 探讨了添加或不添加水杨肟酸的情况下病原菌对吡唑醚菌酯敏感性的变化; 并测定了不同产区的80株B. dothidea 对吡唑醚菌酯的敏感性以及440株B. dothidea 对吡唑醚菌酯的抗性; 然后, 扩增并分析了具有不同敏感性的菌株的细胞色素b基因 (Cytb) 序列?结果表明:不同浓度水杨肟酸对菌丝生长的抑制作用不同?添加40 μg/mL水杨肟酸不影响吡唑醚菌酯的EC50?菌株的敏感性频率符合近似正态分布, 各产区菌株对吡唑醚菌酯的敏感性没有显著差异, 吡唑醚菌酯平均EC50为(2.95±2.11) μg/mL, 没有检测到抗性菌株?靶标基因序列分析表明, Cytb基因在F129?G137和G143位密码子上没有产生点突变, 首次发现在143位密码子后有内含子插入?     

基于文献计量学的昆虫细胞色素P450研究发展态势分析

利用文献计量法和关键词词频法,对1999年至2012年间,国内外在昆虫细胞色素P450研究领域的研究成果进行梳理和研究,结果显示:昆虫细胞色素P450文献数量在此期间呈现出明显的增长趋势,说明昆虫细胞色素P450日益得到学者的广泛关注。关键词词频法说明对模式昆虫果蝇P450的研究依然是热点,而卫生害虫、农业害虫以及经济昆虫P450的研究已逐渐成为新的研究热点;而研究方向上正向昆虫P450调控表达的理论研究及其实践应用拓展。     

The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.)

Herbicide resistance or tolerance in weeds mediated by cytochrome P450 monooxygenase is a considerable problem. However, cytochrome P450 mediated resistance or tolerance in weeds was less studied. Thus, in this work, the role of the cytochrome P450 monooxygenase in the different responses of Poa annua and Alopecurus aequalis to fenoxaprop-P-ethyl was studied. We found that the effect of fenoxaprop-P-ethyl could be synergized by piperonyl butoxide (PBO) in P. annua, but not by malathion. After being treated with fenoxaprop-P-ethyl (containing mefenpyr-diethyl), the contents of cytochrome P450 and cytochrome b₅ in P. annua increased significantly compared to plants treated with mefenpyr-diethyl only or untreated plants. However, the increase was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of ρ-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-deethylase (EROD), ethoxycoumarin oxidase (ECOD) and NADPH-dependent cytochrome P450 reductase mediated by cytochrome P450 monooxygenase increased in P. annua after treatment with fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450 reductase. Besides this, cytochrome P450 monooxygenase activity toward fenoxaprop-P-ethyl in P. annua increased significantly compared to untreated or treated with mefenpyr-diethyl plants and treated or untreated A. aequalis. Cytochrome P450 monooxygenase may play an important role in the different responses to fenoxaprop-P-ethyl in P. annua and A. aequalis.     

绿磺隆、醚苯磺隆及其体外代谢物对乙酰乳酸合成酶的离体抑制作用

利用异源表达于酵母细胞中的小麦细胞色素P450cDNA(CYP71C6v1)研究了磺酰脲类除草剂绿磺隆、醚苯磺隆的代谢作用。结果表明,代谢产物5-羟基绿磺隆和5-羟基醚苯磺隆能够抑制乙酰乳酸合成酶(ALS酶)活性,且代谢产物与母体化合物绿磺隆、醚苯磺隆抑制ALS酶活性的IC50值差异小,但是代谢产物在茎叶喷雾小麦和菜豆时,均未表现出活性。绿磺隆及其代谢产物抑制小麦ALS酶活性的IC50值分别为7.1×10-9和7.9×10-9mol/L,抑制菜豆ALS酶活性的IC50分别为3.6×10-9和4.1×10-9mol/L;醚苯磺隆及其代谢产物抑制小麦ALS酶活性的IC50分别为4.6×10-9和5.3×10-9mol/L,抑制菜豆ALS酶活性的IC50分别为4.7×10-9和4.9×10-9mol/L。结果表明,在磺酰脲类分子苯环5位上进行结构改造,有可能得到高活性的化合物。     

Expression of a wheat cytochrome P450 monooxygenase in yeast and its inhibition by glyphosate

Glyphosate is a non-selective herbicide which acts by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase. Wheat cytochrome P450 monooxygenase specifically catalyzes the metabolism of some sulfonylurea herbicides such as chlorsulfuron and triasulfuron. Here we report that glyphosate is an inhibitor of a wheat cytochrome (CYP71C6v1), the cDNA of which was amplified by RT-PCR and heterologously expressed in yeast. The microsomal fractions derived from this strain had a Soret peak at 502 nm in the reduced carbon monoxide difference spectrum, which is a typical spectral characteristic. The addition of glyphosate to the microsomal protein resulted in a Type II spectrum indicative of binding via the nitrogen group to haem of cytochrome P450 as a sixth ligand. A spectral dissociation constant, K(s) of 70 micromol litre(-1) was observed and an IC50 of 11 micromol litre(-1) was found for glyphosate inhibition of CYP71C6v1 P450 activity.     

Phase 1 and 2 Metabolism in Freshly Isolated Hepatocytes and Subcellular Fractions from Rat,Mouse, Chicken and Ox Livers

In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148·2(±75·7) and 20·6(±11·5) pmol mg^-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6β-hydroxytestosterone (6β-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16α-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2α-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664·2(±203·5)) compared with rat, chicken or ox (320·4(±64·0), 341·5(±13·9) and 256·3(±109·9) nmol min^-1 mg^-1 cytosolic protein, respectively). © 1997 SCI.     

Expression of a wheat cytochrome P450 monooxygenase cDNA in yeast catalyzes the metabolism of sulfonylurea herbicides

A wheat cytochrome P450 cDNA (CYP71C6v1) was cloned by RT-PCR and heterologously expressed in yeast. The microsomal fractions derived from this strain could catalyze the metabolism of some sulfonylurea herbicides such as chlorsulfuron, triasulfuron, metsulfuron-metyl, bensulfuron-metyl, and tribenuron-metyl, but not sulfonylurea herbicides such as thifensulfuron and pyrazosulfuron. Kinetic parameters K_m for chlorsulfuron and triasulfuron were 57 (±15) μM and 38 (±16) μM in vitro, respectively. Analysis of the metabolites demonstrated that the CYP71C6v1 functioned as a 5-phenyl ring hydroxylase when chlorsulfuron and triasulfuron were the substrates.     

In-vivo effects of 2,4-D and atrazine on cytochrome P-450 and insecticide toxicity in southern armyworm (Spodoptera eridania) larvae

After feeding 2,4-D or atrazine in a diet to southern armyworm (Spodoptera eridania Cram.) larvae for three days, the effect on total content and activities of cytochrome P450 and on insecticide toxicity were determined. Both 2,4-D and atrazine induced cytochrome P450-catalyzed aldrin epoxidation (AE) and methoxyresorufin O-demethylatin (MROD). The 2,4-D was a more potent inducer for total cytochrome P450 content, whereas atrazine disproportionately increased AE. Both compounds increased MROD significantly. The apparent kinetic characteristics of AE indicates that 2,4-D and atrazine induced similar P450 isozymes (K_m 8.78 and 7.80 μM, respectively), which may differ from the constitutive isozyme (K_m 3.14 μM). The 2,4-D-induced cytochrome P450 contributed to decreased carbaryl and permethrin toxicity, whereas the atrazine-induced cytochrome P450 caused decreased parathion and permethrin toxicity. The carbaryl toxicity correlated directly with 2,4-D-induced total P450 content and activities but not with atrazine-induced changes. The 2,4-D and atrazine also induced nonspecific esterase activity which may contribute to permethrin detoxification.     

Synergistic and antagonistic effects of atrazine on the toxicity of organophosphorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera: Chironomidae)

The acute toxicities of two organophosphorodithioate (dimethoate and disulfoton) and two organophosphorothioate (omethoate and demeton-S-methyl) insecticides were evaluated individually and in binary combination with the herbicide atrazine using fourth-instar larvae of the aquatic midge, Chironomus tentans. Atrazine alone up to 1000 μg/L did not show significant toxicity to the midges in a 48-h bioassay. However, atrazine concentrations as low as 1 μg/L in combination with dimethoate at EC₂₅ (concentration to affect 25% of tested midges), 100 μg/L in combination with disulfoton (EC₂₅), and 10 μg/L in combination with demeton-S-methyl (EC₂₅) significantly enhanced the toxicity of each organophosphate insecticide. In contrast, atrazine concentrations of 10 μg/L and above in combination with omethoate (EC₂₅) significantly decreased the toxicity of the insecticide. Biochemical analysis indicated that increased toxicity of dimethoate, disulfoton, and demeton-S-methyl in binary combination with atrazine correlated to the increased inhibition of acetylcholinesterase. Furthermore, cytochrome P450-dependent O-deethylation activity in the midges exposed to atrazine at 1000 μg/L was 1.5-fold higher than that in the control midges. Thus, atrazine appeared to induce cytochrome P450 monooxygenases in the midges. Elevated cytochrome P450 monooxygenase activity may increase the toxicities of dimethoate, disulfoton, and demeton-S-methyl by enhancing the oxidative activation of dimethoate into omethoate, and disulfoton and demeton-S-methyl into their sulfoxide analogs with increased anticholinesterase activity. In contrast, atrazine reduced the toxicity of omethoate possibly by enhancing the oxidative metabolic detoxification since omethoate does not require oxidative activation.