小菜蛾鱼尼丁受体基因克隆及序列分析

利用RT-PCR和RACE技术克隆了小菜蛾(Plutella xylostella)鱼尼丁受体(Px-RyR)基因,其核苷酸序列全长15 748bp,5′非编码区267bp,3′端非编码区109bp,开放阅读区全长为15 372bp(GenBank登录号:JF927788),编码5 123个氨基酸残基。估测其蛋白分子量为579.39ku,等电点为5.45。该基因编码氨基酸序列和其他鳞翅目昆虫RyR氨基酸序列比对相似性较高(92%),与哺乳动物3种亚型RyRs的相似性为45%～47%。此外,二级结构预测,其C-末端存在6个跨膜区域;且Px-RyR中存在一个出现4次重复,长度为89～95个氨基酸的RyR结构域,平均相似性为33%。     

昆虫鱼尼丁受体及以其为靶标的杀虫剂的研究进展

植物保护领域以昆虫鱼尼丁受体(ryanodine receptor,RyR)为靶标的杀虫剂的研发取得了突破性进展。对近年来RyR在分子结构、功能调节,以及对以RyR为靶标的杀虫剂的作用机制方面的研究进展进行了综述。昆虫RyR与哺乳动物RyR仅有约47%的同源性,因而是一个有效的杀虫剂靶标。昆虫RyR克隆与表达技术的成熟为新型杀虫剂的开发和作用机制的研究提供了有力的工具。昆虫RyR单通道、配体结合和免疫学特性的研究补充了RyR的电生理学数据。近年来开发的新型RyR杀虫剂通过激活害虫鱼尼丁敏感的细胞内钙离子释放通道来达到杀虫的效果。     

小麦蓝矮植原体免疫膜蛋白(Imp)的跨膜区影响其在大肠杆菌中的表达

根据跨膜区预测结果,设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1,ImpF1/ImpR2,Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段,即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x,并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证,只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达,结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。根据跨膜区预测结果,设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1,ImpF1/ImpR2,Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段,即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x,并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证,只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达,结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。     

以昆虫鱼尼丁受体为靶标的化合物活性筛选方法研究进展

昆虫鱼尼丁受体(ryanodine receptor,RyR)是目前新型杀虫剂创制中备受关注的靶标之一。在以RyR为靶标的杀虫剂氟虫酰胺和氯虫酰胺商品化后,大量基于该靶标的新型化合物被设计并合成出来。为加速研发和快速准确筛选以昆虫RyR为靶标的药剂,主要以氟虫酰胺和氯虫酰胺的活性筛选研究为例,对作用于RyR化合物的各种活体筛选方法和离体筛选方法进行了综述。其中活体筛选方法主要包括:点滴法、浸叶法、浸卵法、浸虫法、注射法和喷雾法;离体筛选方法主要包括MTT法、膜片钳技术、荧光技术、膜片钳技术与荧光技术结合以及计算机模拟筛选。通过对各种方法的分析比较,认为离体筛选方法快速、准确,在新型RyR杀虫剂活性的筛选研究中将进一步得到应用。     

双委夜蛾非典型嗅觉受体Orco的克隆、分子特征及表达

为了解新发现的农业害虫双委夜蛾Athetis dissimilis(Hampson)非典型嗅觉受体Orco的分子特征与表达,通过分析转录组数据并利用RT-PCR技术克隆了双委夜蛾Orco基因,并采用实时定量PCR技术研究了该基因的组织表达谱。结果显示,双委夜蛾非典型嗅觉受体Orco基因开放阅读框全长1 422 bp,编码473个氨基酸,经氨基酸结构预测具有7个跨膜区,N端在膜内,C端在膜外;该基因编码的氨基酸序列与其它昆虫Orco序列相似性极高,因此将该基因命名为Adis Orco(Gen Bank登录号:KR632987),此氨基酸序列C末端第6跨膜区和第7跨膜区之间以及第7跨膜区的序列保守性最高;PCR结果显示,Adis Orco主要在成虫触角中表达,且在雄蛾触角中的表达量是雌蛾触角中的4.2倍,在足、翅、下唇须和喙等组织中也有少量表达。     

亚洲与太平洋区域植物保护委员会简介

亚洲和太平洋区域植物保护委员会(以下简称亚太地区植保委员会)是当前国际上被承认的9个区域性植保组织之一(注1),是联合国粮农组织直接管理的三个区域性植保组织中的一个(注2)。其前身是“东南亚和太平洋区域植物保护委员会”。它是1956年根据当时该区域10个国家所签订的     

亚太区域植物保护合作进入新阶段

<亚洲及太平洋区域植物保护协定>(以下简称协定)是亚太区域国家植物保护领域重要的多边区域合作协定,于1956年7月2日正式生效.作为成员国,全面了解区域植保组织及其协定的发展进程,积极参与相关活动,有利于推动我国与区域植保组织的合作,发展我国的植保事业.     

烟碱乙酰胆碱受体及其与新烟碱类相互作用的研究进展

对烟碱乙酰胆碱受体(nAChRs)的结构与功能、配体结合部位、门控机理以及与新烟碱类的相互作用进行了综述,并对nAChRs亚基基因突变和敲除对新烟碱类和多杀菌素敏感性的影响进行了讨论。nAChRs在脊椎动物和昆虫的胆碱能突触的快速神经传递中起着重要作用,其在昆虫中仅存在于中枢神经系统(CNS)中,而在脊椎动物中同时存在于CNS和神经肌肉连接处。nAChRs是新烟碱类杀虫剂、多杀菌素和杀螟丹的作用靶标。肌肉和CNS中的nAChRs是一个由两个α和三个非α(β,γ和δ)亚基组成的异数五聚体,该受体主要有三部分:一个在细胞外发现的区域(胞外区)、一个位于膜内的区域(跨膜区),另一个是位于细胞内的区域(胞质区)。每个亚基(从N-C端)都具有一个包含乙酰胆碱(ACh)结合部位的细胞外结构域;4个跨膜结构域(M1~4),其中M2的大部分氨基酸位于离子通道的内壁;一个胞质噜扑(loop)和一个胞外C端。通道门位于孔道内的疏水区。ACh结合部位位于天然和功能受体的两个亚基的界面,是由一个亚基的3个噜扑(A-C)和另一个亚基的3个噜扑(D-F)构成。每当受体与ACh(或其他激动剂)分子结合时,M2 α螺旋体的构象发生改变,使通道开启,处于阳离子传导状态,直至一个或两个激动剂分子从结合口袋解离,通道才关闭。如果激动剂一直存在,并反复结合,则通道处于脱敏状态。nAChRs与新烟碱类的各种选择性作用取决于新烟碱类的结构以及nAChRs的亚基组成。     

新疆博尔塔拉蒙古自治州区域交通分析与建设

对博尔塔拉蒙古自治州(以下简称博州)地区公路、铁路两种主要交通方式现状进行概述,分析、探究存在于交通方式中的问题,针对部分问题寻找具体的交通网建设想法,并建议提前做好管道建设准备,为博州区域交通网管的规划建设提供参考,同时也能促进博州地区各种交通方式相互补充、协调,从而发挥各类交通运输的优势,促进博州区域经济更快发展。     

黄河中上游部分地区土地覆盖动态变化研究

作为美国国家科学基金项目"中国生态恢复综合影响评价"的部分研究结果,对项目选定的四个评价区之一的黄河中上游地区的研究区域进行土地利用和覆盖变化研究,并以此评价退耕还林效果。利用覆盖研究区域的卫星遥感图像进行解译,以便获取研究区域土地利用和土地覆盖变化(LUCC)的历史数据,特别是大于25度坡度的LUCC以及退耕前后的LUCC数据,通过这些解译数据评价研究区域的退耕还林效果。解译后的数据显示退耕还林效果良好。选定24景跨20年(1978年至2007年)的Landsat遥感卫星图像数据,通过解译追踪研究区域LUCC历史演变,来判断退耕还林的执行效果。     

Anthranilic diamides: A new class of insecticides with a novel mode of action, ryanodine receptor activation

Development of insecticides with unique modes of action is necessary to combat widespread insecticide resistance. A new class of insecticides has been discovered, the anthranilic diamides, that provides exceptional control through action on a novel target, the ryanodine receptor. Anthranilic diamides potently activate this receptor, releasing stored calcium from the sarcoendoplasmic reticulum causing impaired regulation of muscle contraction. Expression of a recombinant Drosophila ryanodine receptor in a lepidopteran cell line confers sensitivity to anthranilic diamides similar to that observed with native receptors. Ligand-binding studies with radiolabeled ryanodine and radiolabeled anthranilic diamide in Periplaneta americana reveal a single, saturable binding site for this chemistry distinct from that of ryanodine. Further, calcium mobilization studies using mammalian cell lines indicate anthranilic diamides exhibit >500-fold differential selectivity toward insect, over mammalian, receptors. Consequently, anthranilic diamides offer a novel pharmacological tool for calcium signaling research in addition to a unique alternative to existing pest-management strategies.     

Development of a high‐throughput real‐time PCR assay for the detection of the R81T mutation in the nicotinic acetylcholine receptor of neonicotinoid‐resistant Myzus persicae

BACKGROUND: Myzus persicae is a globally important aphid pest that is mainly controlled through the application of chemical insecticides. Recently, a clone of M. persicae exhibiting control‐compromising levels of resistance to neonicotinoid insecticides was described. The resistance of this clone was associated with reduced affinity of imidacloprid for the target site (the nicotinic acetylcholine receptor) as a result of mutation of a key amino acid residue (R81T) in the loop D region of a nAChR β1 subunit. The potent levels of resistance conferred by this mechanism are cause for considerable concern, and the frequency and distribution of the mutation in worldwide populations of M. persicae require careful monitoring. In this study, a high‐throughput assay has been developed that allows detection of the mutation in individual aphids. RESULTS: A real‐time TaqMan assay to detect the R81T substitution was developed that proved to be sensitive and specific in tests of analytical sensitivity and in a blind genotyping trial of DNA extracted from individual aphids comprising the three possible genotypes. The assay was then used to examine the frequency of the R81T mutation in aphids collected and stored in ethanol from peach orchards in southern France. The R81T frequency varied from 33 to 100% in seven populations from the department of Gard, France. CONCLUSIONS: This study describes a rapid and sensitive assay that very effectively detects the R81T mutation in individual aphids. The results also have practical significance for the control of M. persicae in southern France and provide contemporary data to inform current resistance management strategies. Copyright © 2012 Society of Chemical Industry     

五种琥珀酸脱氢酶抑制剂类杀菌剂与灰葡萄孢琥珀酸脱氢酶的结合模式及抗性机制分析

为探究琥珀酸脱氢酶抑制剂 (SDHI) 类杀菌剂与灰葡萄孢Botrytis cinerea琥珀酸脱氢酶 (SDH) (以下简称BcSDH) 的结合方式,阐明BcSDH对SDHI类杀菌剂产生抗性的结构生物学机制,通过同源建模构建了BcSDH的三维模型,通过分子对接预测了5种SDHI (异丙噻菌胺、氟吡菌酰胺、氟唑菌酰胺、吡噻菌胺和啶酰菌胺) 与野生型和突变型BcSDH的亲和力及结合模式之间的变化,分析其抗药性机制,对相关突变位点进行保守性预测,并分析突变类型。结果表明:5种SDHI与BcSDH 具有较强的亲和力,其中酸部分插入BcSDH活性腔底,胺部分在活性腔口,能够形成牢固的疏水作用、氢键、卤键、π-π堆积作用和π-阳离子等相互作用。B-P225F氨基酸残基突变 (以下简称突变) 会造成活性腔口变窄,使得SDHI酸部分不能进入活性腔;B-P225L突变会造成异丙噻菌胺、氟吡菌酰胺和吡噻菌胺与靶标蛋白的结合模式发生变化,亲和力降低;B-H272R突变后,活性腔底变窄,与SDHI的亲和力下降。另外,保守性分析结果表明,B-P225和B-H272均位于BcSDH的保守区域,B-P225F、B-H272R和B-H272L突变可能为随机突变。因此推测BcSDH的B-P225F和B-H272R突变可能是引起灰葡萄孢对5种杀菌剂产生抗性的主要原因,也可能是引起SDHI类杀菌剂之间交互抗性的主要原因之一;B-P225L突变可能降低灰葡萄孢对部分杀菌剂的敏感性,而不是引起BcSDH对SDHI类杀菌剂产生交互抗性的主要原因。在实际生产中,应采取合理有效的抗性监测治理策略来延缓灰葡萄孢对SDHI类杀菌剂抗性的产生,在SDHI分子设计时也应考虑该位点氨基酸残基突变,避免产生交互抗性。     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

Investigating nicotinic acetylcholine receptor expression in neonicotinoid resistant Myzus persicae FRC

The peach–potato aphid Myzus persicae is a pest of many commercial crops due to its polyphagous nature of feeding and has a well-documented history of acquiring resistance to insecticides. In 2009 a strain (M. persicae FRC) emerged in southern France with a point mutation (R81T) at the nicotinic acetylcholine receptor (nAChR), the target site for neonicotinoids such as imidacloprid. This point mutation was associated with the loss of the high affinity imidacloprid binding site (pM Kd), with the single remaining binding site (low nM Kd) highly overexpressed compared to laboratory controls (Bass et al., 2011 [1]). Here we report that after 2 years of continuous selection in the glass house environment with neonicotinoids, the total level of IMD-sensitive nAChRs (low nM Kd) in M. persicae FRC is now comparable to laboratory controls (pM and low nM Kd). Interestingly, despite this large reduction in IMD-sensitive nAChRs, this was not associated with any significant alteration in NNIC-lethality. Additionally, sustained absence of neonicotinoid-selection did not alter nAChR protein levels. We suggest that alterations in nAChR protein expression level described in the original characterisation of the field-isolated M. persicae FRC is unlikely to have been a direct consequence of the R81T mutation. Rather, we speculate that nAChR expression in aphids is likely influenced by as yet unknown conditions in the natural field environment that are absent in the laboratory setting.     

Biocides, tributyltin and triphenyltin, as possible inhibitors of the human sulfotransferase involved in the estrogen homeostasis

This work using purified recombinant human estrogen sulfotransferase (hSULT1E1) aimed to investigate the mechanism of the inhibition of estrogen sulfation by organotin compounds. Tributyltin (TBT) inhibited the sulfation of estrone (E1) and 17 β-estradiol (E2) by hSULT1E1 competitively, with IC₅₀ values of, respectively, 3 and 12 μM. The sulfation of E1 and E2 was also inhibited competitively by triphenyltin (TPT), with IC₅₀ of, respectively, 10 and 5 μM. These data strongly suggested that His¹⁰⁷ residue might act as a ligand to establish a coordination bond with organotins at estrogen binding site in hSULT1E1. A more surprising finding was that TBT competed with 3′-phosphoadenosine 5′-phosphosulfate (PAPS), indicating that TBT may coordinate with certain amino acid residue such as Lys⁴⁷ at the PAPS binding site of hSULT1E1. Taken together, these data provided clear evidence that TBT and TPT have a capacity to disrupt endocrine-mediated events by inhibiting hSULT1E1 involved in the metabolism of sex steroids.     

Identification of the Rdl mutation in laboratory and field strains of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae)

In many insect species, resistance to cyclodiene insecticides is caused by amino acid substitutions at a single residue (A302) within the M2 transmembrane region of the gamma-aminobutyric acid (GABA) receptor sub-unit termed Rdl (resistance to dieldrin). These mutations (A302S and A302G) have also been shown to confer varying levels of cross-resistance to fipronil, a phenylpyrazole insecticide with a similar mode of action to cyclodienes. To investigate the possible occurrence of these mutations in the cat flea, Ctenocephalides felis (Bouché), a 176-bp fragment of the cat flea Rdl gene, encompassing the mutation site, was PCR amplified and sequenced from nine laboratory flea strains. The A302S mutation was found in eight of the nine strains analysed, although the relative frequency of the mutant allele varied between strains. Only one strain (R6) was found to be homozygous for the S302 allele in all the individuals tested, and this correlated with previous reports of low-level fipronil resistance in this strain. A PCR-based diagnostic assay, capable of screening individual fleas for this mutation, was developed and used to survey a range of fleas collected at random from veterinary clinics in the UK and USA. The A302S mutation was present at a high frequency in these domestic pet populations.     

An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides

BACKGROUND: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self‐splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. RESULTS: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5′–3′) 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above‐mentioned 1166 bp intron, a self‐splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. CONCLUSION: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron. Copyright © 2010 Society of Chemical Industry     

Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate insecticides

Interspecific comparisons of bioassay and biochemical data suggest two major patterns of target site resistance to carbamates and organophosphates. Pattern I resistance, which is generally more effective for carbamates, has been shown in two sub-species of mosquitoes to be due to a particular Gly-Ser mutation in the oxyanion hole within the active site of one of their two acetylcholinesterase enzymes. Intriguingly, different substitutions at the equivalent site confer organophosphate hydrolytic ability on other esterases responsible for metabolic resistance in some other species. In the case of the aphid, Myzus persicae, Pattern I resistance is due to a Ser-Phe mutation in the vicinity of the acyl pocket of acetylcholinesterase. Pattern II resistance is at least as effective for organophosphates as it is for carbamates and may even be specific to organophosphates in some cases. Molecular studies on this pattern of resistance in three higher Diptera show that it is due to changes that constrict the acetylcholinesterase active site gorge and limit binding of the insecticide to the catalytic residues at the base of the gorge. One case of Pattern II resistance in the mosquito, Culex tritaeniorhynchus, involves the same site near the acyl pocket of acetylcholinesterase, albeit a different substitution, as that involved in Pattern I resistance in M. persicae.     

The story of a new insecticidal chemistry class: the diamides

This paper describes the story of the invention of the diamides, a novel chemical class of insecticides. It starts with the pioneering work by Nihon Nohyaku researchers, who developed a herbicide lead with weak insecticidal activity to flubendiamide, a highly potent lepidoptericide. The journey continues with Nissan's isoxazolines and the invention of the anthranilamides by DuPont, culminating in the discovery of the blockbuster chlorantraniliprole and its analogue cyantraniliprole. The next steps are Syngenta's sulfoximines and bicyclic anthranilamides, Ishihara Sangyo Kaisha's cyclopropylamides, Sumitomo's hydrazides, Bayer's pyrazoles and tetrazoles, BASF's sulfoximines and more recent contributions from Chinese agrochemical companies and academic institutions. The diamides affect calcium homeostasis by binding to ryanodine receptors and releasing calcium from the intracellular stores. Investigations at Nihon Nohyaku, DuPont and Bayer on the action of the diamides on ryanodine receptors will be briefly reported.