Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64).     

基于转录组测序解析草地贪夜蛾对溴氰虫酰胺的解毒代谢分子机制

为阐明草地贪夜蛾Spodoptera frugiperda对溴氰虫酰胺的解毒代谢分子机制,通过LC₅₀的溴氰虫酰胺诱导草地贪夜蛾3龄幼虫后,利用酶活测定和转录组测序鉴定解毒代谢相关基因,并采用实时荧光定量PCR技术对细胞色素P450单加氧酶(cytochrome P450 monooxygenase,P450)基因进行验证分析。结果表明,经LC₅₀的溴氰虫酰胺处理后,草地贪夜蛾3龄幼虫体内3种解毒代谢酶活性较对照均有所升高,但仅P450活性较对照显著升高,而谷胱甘肽S-转移酶和羧酸酯酶与对照无显著差异。经LC₅₀的溴氰虫酰胺处理后草地贪夜蛾3龄幼虫转录组中共筛选到1 408个差异表达基因,其中上调表达的基因有935个,下调表达的基因有473个。药物代谢-细胞色素P450通路、药物代谢-其他酶通路及细胞色素P450对异生物质的代谢通路中有超过20个基因存在差异表达。在草地贪夜蛾转录组中筛选鉴定到121个P450基因,其中,属于CYP2、CYP3、CYP4以及Mito家簇的基因分别有9、45、58和9个,而经LC_5...     

Isolation and functional characterization in yeast of CYP72A18, a rice cytochrome P450 that catalyzes (ω-1)-hydroxylation of the herbicide pelargonic acid

Cytochrome P450 proteins play important roles in plant herbicide selectivity. Here, we demonstrate metabolism of the herbicide pelargonic acid by CYP72A18, a novel cytochrome P450 isolated from the rice Oryza sativa L. cv. Nipponbare. The CYP72A18 cDNA was cloned from rice and heterologously expressed in Saccharomyces cerevisiae AH22 cells from the alcohol dehydrogenase (ADH1) promoter. Microsomes isolated from recombinant yeast cells contained the CYP72A18, which was found to catalyze the (ω-1)-hydroxylation of the herbicide pelargonic acid. We also show that (ω-1)-hydroxypelargonic acid has reduced herbicide activity against rice seedlings. Based on these results, we suggest that CYP72A18 participates in the detoxification of the herbicide pelargonic acid in rice plants.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

异色瓢虫与七星瓢虫细胞色素P450、抗菌肽和溶菌酶基因的鉴定及表达分析

为探究异色瓢虫Harmonia axyridis环境竞争力的来源,以七星瓢虫Coccinella septempunctata为对照,采用同源比对法对两者细胞色素P450（cytochrome P450,CYP450）、抗菌肽和溶菌酶基因进行鉴定,并对差异表达基因进行分析和荧光定量PCR验证。结果显示,在异色瓢虫中共发现89个CYP450基因、25个抗菌肽基因和18个溶菌酶基因;在七星瓢虫中共发现127个CYP450基因、17个抗菌肽基因和9个溶菌酶基因。异色瓢虫编码抗菌肽和溶菌酶的基因较七星瓢虫存在明显的扩增,而CYP450基因的扩增少于七星瓢虫,表明抗菌肽家族在异色瓢虫环境竞争过程中有着更重要的作用;通过转录组的差异表达分析以及荧光定量PCR验证,发现大部分抗菌肽和溶菌酶基因在脂肪体中高表达,据此推测脂肪体为2种瓢虫抗菌肽等免疫因子的主要合成场所。     

Functional expression of Helicoverpa armigera CYP9A12 and CYP9A14 in Saccharomyces cerevisiae

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.     

Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.     

亚致死剂量氟啶虫胺腈对灰飞虱细胞色素P450的影响

为明确氟啶虫胺腈对灰飞虱Laodelphax striatellus细胞色素P450的影响,评估其抗药性风险,采用点滴法、酶活力分析法和实时荧光定量PCR法分别测定了氟啶虫胺腈对灰飞虱的毒性及对其细胞色素P450酶活力和P450基因表达量的影响。结果表明,氟啶虫胺腈对灰飞虱的致死中量LD_(50)随着虫龄的增加而升高,1~5龄若虫的LD_(50)为0.10~0.94 ng/头,雌、雄成虫的LD_(50)分别为1.09 ng/头和1.07 ng/头。氟啶虫胺腈对4龄若虫的亚致死剂量LD_(10)、LD_(30)和LD_(50)分别为0.17、0.41、0.76 ng/头,处理灰飞虱4龄若虫后可将其体内P450酶活力分别显著提高1.60、1.97和1.22倍;而各处理响应的P450基因的种类和数量不同,但相对表达量均受到诱导;CYP4DE1、CYP426A1、CYP303A1、CYP4C、CYP6FK1和CYP4C71v2的相对表达量表现出时间效应,表达量高峰在处理后24 h或48 h。表明不同亚致死剂量的氟啶虫胺腈在特定时间点可提高相应的细胞色素P450基因表达量,从而使酶蛋白量增加,可能导致灰飞虱体内细胞色素P450酶活力上升。     

稻曲病气象风险评估及适宜度等级预测技术

为了弄清适宜稻曲病发生发展的气象条件以及对应的气象适宜度等级,提高水稻气象型病害防控能力,本文利用江苏省稻曲病大田调查资料,结合历史气象资料,通过评估发病敏感时段和致病风险,以及分析江苏省稻曲病最大成灾风险度出现日期以及空间分布情况,指出8月下旬为稻曲病高发期,对于发病风险高且程度重的沿淮、沿江以及江淮之间地区需要加强病害防御。在此基础上,综合考虑不同连续致病天数对稻曲病流行的影响,利用最优化技术,构建了综合稻曲病指数,并划分气象条件适宜度等级。通过单站和多站检验发现该指数对"中等、大流行"发病实况的判定准确率极高,但容易高估"轻度流行"的程度。     

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。     

四种P450基因调控小菜蛾对氯虫苯甲酰胺的抗性

为研究CYP6家族P450基因在小菜蛾Plutella xylostella代谢氯虫苯甲酰胺中的作用,利用浸叶法测定不同小菜蛾种群3龄幼虫对氯虫苯甲酰的抗性水平,采用实时荧光定量PCR(quantitative realtime PCR,q RT-PCR)方法分析CYP1v3、CYP1v4、CYP6B6和CYP6f这4种P450基因在小菜蛾不同抗性种群体内的表达差异及杀虫剂的短期诱导效应,并通过RNA干扰技术沉默4种P450基因后分析小菜蛾对氯虫苯甲酰胺的敏感性。结果显示,4种P450基因在中等抗性水平小菜蛾种群体内高表达;氯虫苯甲酰胺处理可诱导4种基因显著上调表达。分别沉默4种P450基因后,处理组小菜蛾的P450酶活力显著下降37.60%～54.82%,且处理组小菜蛾的死亡率显著高于对照组;同时沉默4种基因处理组的小菜蛾P450酶活力显著低于其他各处理组。表明4种P450基因可能同时参于小菜蛾对氯虫苯甲酰胺的代谢。     

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.     

Effect of celangulin V on detoxification enzymes in Mythimna separata and Agrotis ypsilon

Celangulin V (CA-V), a β-dihydroagrofuran sesquiterpene polyol ester, is extracted from the root bark of Chinese bittersweet, Celastrus augulatus Maxim. It exhibited selective toxicity against different insects. By CO-difference spectral and biochemical method, the effects of CA-V on two kinds of detoxification enzymes, cytochrome P450 (P450 and NADPH-cytochrome P450 reductase) and glutathione S-transferase, were investigated in oriental armyworm, Mythimna separata and black cutworm, Agrotis ypsilon. CA-V showed higher induction against P450 of M. separata than that of A. ypsilon. Treated by CA-V, the maximum absorption of M. separata increased 1.2 and 0.8 nm than the control, respectively. Meanwhile, compared with the control, the P450 content and NADPH-P450 reductase activity in treated M. separata larvae increased 1.46-, 2.26- and 1.26-, 2.56-fold, respectively. But in treated A. ypsilon larvae, they all increased a little more than those of control. So far as M. separata and A. ypsilon, whether there is exposure of CA-V or not, the P450 content and GST activity in A. ypsilon were obviously higher than those in M. separata. It suggested that the content or activity difference of these two kinds of detoxification enzymes may have important roles in the selective toxicity of CA-V in M. separata and A. ypsilon.     

Human metabolism of atrazine

Atrazine (ATZ) metabolism by human liver microsomes (HLM), cytochrome P450 (CYP) isoforms, and human liver (HL) S9 fractions, was investigated using HPLC/PDA and LC/MS/MS. CYP-dependent metabolites from pooled HLM are desethylatrazine (DEA), desisopropylatrazine (DIA), 1-hydroxyisopropylatrazine (HIATZ), and 2-hydroxyethyl atrazine (HEATZ). DEA and DIA were major metabolites in pooled HLM. CYP1A2 and 2C19, respectively, were major isoforms for DEA and DIA production. CYP3A4, while less active, is generally at high concentrations, produces both DEA and DIA and is significant. The percent total normalized rates (%TNR) for CYP1A2 and 3A4 in pooled HLM were 63% and 24% for DEA, and 35% and 56% for DIA production. Single donor HLM samples, showed correlations for CYP1A2 (r = 0.92) and 3A4 (r = 0.81) for DEA and DIA production, while variations in production of DEA and DIA were 8.5- and 6.0-fold, respectively. Pooled S9 fractions also mediate glutathione conjugation of atrazine, DEA and DIA.