有机磷和拟除虫菊酯抗性棉铃虫靶标抗性的分子机理(英文)

对有机磷和拟除虫菊酯抗性 (R)棉铃虫靶标抗性的分子机理 ,即乙酰胆碱酯酶 (AChE)和钠通道敏感度降低进行了研究。根据AChE的动力学常数表明 ,R品系AChE的活性和Vmax值分别是S品系的 1 0 9和 1 2 3倍 ,但R品系的AChE的Km 值仅是S品系的 0 6 7倍。R品系AChE对DDVP和马拉硫磷的Ki值分别是S品系的 0 4 4和 0 55。这表明AChE发生了质的变化。还应用PCR技术对抗性棉铃虫的击倒抗性 (kdr)进行了鉴定 ,克隆了钠通道的IIS6序列、IIS5和IIS6连接片段以及II和III连接片段 ,测序后比较了R和S品系以及其它昆虫的同源性 ,结果在氨基酸水平未发现有任何差异 ,这表明该抗性棉铃虫品系不涉及kdr。     

Genetics of Indoxacarb resistance in Helicoverpa armigera (Hubner)

The present investigation was done with the aim of studying the genetics of Indoxacarb resistance. Selection of Helicoverpa armigera (Hubner) with Indoxacarb was done for eight generations to develop resistance. Reciprocal crosses between resistant and susceptible populations were made to understand the population genetics of Indoxacarb resistance in H. armigera . Generation-wise selection with Indoxacarb was evaluated for resistance development in H. armigera . The LC₅₀ of Indoxacarb was 2.81 p.p.m. for the first selected generation, and it increased to 272.55 p.p.m. after eight selected generations, which is a 1238.86-fold resistance compared to the susceptible strain. The estimated realized heritability (h²) after eight generations of selection with Indoxacarb was 0.45. The number of generations required for a tenfold increase in LC₅₀ (1/R) was estimated to be 2.59. The response to Indoxacarb selection in H. armigera was 0.39, the estimated selection differential (S) was 0.87, and the phenotypic standard deviation (σp) was 0.03. Reciprocal crosses between Indoxacarb resistant and susceptible strains revealed that the inheritance of Indoxacarb resistance was autosomal: neither maternal effect nor linkage was evident. The values of D_LC (0.10 and 0.09) indicated completely recessive inheritance of Indoxacarb resistance.     

Acetylcholinesterase mutation in an insecticide-resistant population of the codling moth Cydia pomonella (L.)

Two strains of Cydia pomonella (L.) (Lepidoptera: Tortricidae) were selected in the lab by exposure to increasing concentrations of diflubenzuron (Rdfb strain) or azinphos-methyl (Raz strain). Insecticide bioassays showed that the adults of the Rdfb strain exhibited a 2.6-fold and a 7.7-fold resistance ratio to azinphos-methyl and carbaryl, respectively compared to a susceptible strain (S) whereas the adults of the Raz strain exhibited a 6.7-fold resistance ratio to azinphos-methyl and a 130-fold resistance ratio to carbaryl. In the Raz strain, a target site resistance mechanism was suggested by the inhibition of acetylcholinesterase (AChE) activity. In fact the ki values did not discriminate the S and Rdfb strains, while the Raz strain exhibited a 1.7-fold and a 14-fold increase in ki value compared to the S strain for azinphos-methyl oxon and carbaryl, respectively. To verify this hypothesis, two cloned AChE cDNAs sequences (named cydpom-ace2 e cydpom-ace1) were compared between the susceptible and the resistant strains. No difference in the deduced amino acid sequence was found in cydpom-ace2 (orthologous to the Drosophila melanogaster AChE). In the putative cydpom-ace1 (paralogous to the Drosophila AChE), a single amino acid substitution F399V was exclusively present in the Raz strain. The F399 lined the active site of the enzyme and the F399V substitution likely could influence the accessibility of different types of inhibitors to the catalytic site of the insensitive cydpom-ace1.     

Contributions of traT and iss genes to the serum resistance phenotype of plasmid ColV2-K94

Abstract A genetic determinant for serum resistance, designated iss , has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT , on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT⁺ Iss⁺) contained the kanamycin (Km) resistance transposon Tn 903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT⁺), pWS16 (TraT⁺) and pWS18 (TraT⁺ Iss⁺) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.     

Identification of the Cationic Amino Acid Transporter (System y+) of the Rat Blood-Brain Barrier

Abstract: Cationic amino acids are transported from blood into brain by a saturable carrier at the blood-brain barrier (BBB). The transport properties of this carrier were examined in the rat using an in situ brain perfusion technique. Influx into brain via this system was found to be sodium independent and followed Michaelis-Men-ten kinetics with half-saturation constants (K_m) of 50–100 μM and maximal transport rates of 22–26 nmol/min/g for L-lysine, L-arginine, and L-ornithine. The kinetic properties matched that of System y⁺, the sodium-independent cationic amino acid transporter, the cDNA for which has been cloned from the mouse. To determine if the cloned receptor is expressed at the BBB, we assayed RNA from rat cerebral microvessels and choroid plexus for the presence of the cloned transporter mRNA by RNase protection. The mRNA was present in both cerebral microvessels and choroid plexus and was enriched in microvessels 38-fold as compared with whole brain. The results indicate that System y⁺ is present at the BBB and that its mRNA is more densely expressed at cerebral microvessels than in whole brain.     

Increasing tolerance to Cry1Ac cotton from cotton bollworm, Helicoverpa armigera, was confirmed in Bt cotton farming area of China

Abstract.  1. Changes in the frequency of Cry1Ac resistance genes and shifts in tolerance of cotton bollworm, Helicoverpa armigera , to the Cry1Ac toxin were assessed using bioassays of F₁ and F₂ offspring of isofemale lines from Anci County of Hebei Province (a multiple-crop system including corn, soybean, peanut, and Bt cotton) and Xiajin County of Shandong Province (an intensive Bt cotton planting area) in Northern China during 2002–2005.
2. A conservative analysis of the overall results indicated that there was a small increase in the frequency of major, non-recessive resistance genes over time.
3. The relative average development ratings [RADR – growth rate of a line on a Bt diet in proportion to the growth rate on a non-Bt (NBT) diet] of the bollworm larvae in F₁ tests increased significantly from year to year, indicating a gradual trend towards higher tolerance to Cry1Ac in the field populations.
4. There were also significant positive correlations between RADR of the lines in the F₁ generation and the RADR of their F₂ offspring, indicating that the tolerance was genetically based.
5. Quantitative genetic simulation analysis showed that resistance of H . armigera to Bt cotton in Xiajin could evolve to a high level in 11–15 years if no effective resistance management measures are carried out.     

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field

An obligately anaerobic spirochete designated strain SEBR 4228^T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration growth range 1.0–10%) at 37°C (growth temperature range 20–40°C) and pH of 7.0–7.2 (pH growth range pH 5.5–8.0). Strain SEBR 4228^T grew on carbohydrates (glucose, fructose, ribose, d -xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H₂S. Glucose was oxidised to lactate, acetate, CO₂ and H₂S in the presence of thiosulfate but in its absence lactate, ethanol, CO₂ and H₂ were produced. Fumarate was fermented to acetate and succinate. The G+C content of strain SEBR 4228^T was 50%. Strain SEBR 4228^T was spiral shaped measuring 5–30 by 0.3–0.5 μm and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228^T possessed features typical of the members of the genus Spirochaeta . 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228^T is a new species, which we have designated Spirochaeta smaragdinae . The type strain is SEBR 4228^T (= DSM 11293).     

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.     

Seasonal and geographical toxicity of Indoxacarb against Helicoverpa armigera and influence of different host plants against Indoxacarb in India, 2005–2007

Indoxacarb, an oxadiazine insecticide, was evaluated for its effectiveness against Helicoverpa armigera collected from selected locations in India. Determination of Indoxacarb efficacy was done using a log-dose probit (LDP) bioassay against third instars collected from cotton ( Gossypium arborium ) fields near Akola, India. Monthly levels of toxicity of Indoxacarb were determined from July 2005 to March 2007. The maximum tolerance level of Indoxacarb was reported for the Amaravati strain (5.09 p.p.m.) and the minimum tolerance level for the Fatehbad strain (0.22 p.p.m.). Seasonal monitoring of Indoxacarb toxicity revealed an increased trend in tolerance from July 2005 to February 2006, which decreased from March 2006. The LC₅₀ of Indoxacarb was 2.71 p.p.m. in July 2005 and 17.14 p.p.m. in February 2006. During 2006–2007, the LC₅₀ was 3.84 p.p.m. at the start of the season and in March 2007 it was 13.51 p.p.m. The minimum LC₅₀ of Indoxacarb was reported for H. armigera larvae fed on Legasca spp. (1.62 p.p.m.) and the maximum LC₅₀ was reported for H. armigera reared on chickpea ( Cicer arietium ) (8.45 p.p.m.). LC₅₀ of 2.73 and 4.56 p.p.m. were reported for H. armigera fed on cotton ( Gossypium arborium ) and pigeonpea ( Cajanus cajan ), respectively.     

Homoserine kinase from the phototrophic bacterium Rhodospirillum rubrum is not sensitive to feedback inhibition by l-threonine

Abstract Homoserine kinase (EC 2.7.1.39), one of the enzymes of L -threonine synthesis, was purified 200-fold from the phototrophic bacterium Rhodospirillum rubrum strain S1 by salt precipitation, hydrophobic interaction chromatography and gel filtration. The enzyme had a M _r of about 145000 and was active with L -homoserine ( K _m= 3 mM) and ATP ( K _m= 0.44 mM). In contrast to the kinase from the enteric bacterium, Escherichia coli , the R. rubrum enzyme was neither stabilized nor inhibited by L -threonine. Of 18 amino acids and metabolites tested (including L -allo-threonine, D -allo-threonine, DL -homocysteine, o -phosphoserine and L -norleucine), none was found to be inhibitory.     

河北转Bt基因棉田棉铃虫对杀虫剂的抗性及相关酶活性的变化

为明确河北省推广种植植转Bt基因抗虫棉（简称Bt棉）后, 棉铃虫Helicoverpa armigera (Hübner)对常用杀虫剂的抗药性水平及其生化机理, 2011-2012年采用点滴法对保定南郊、 沧州南皮、 邢台巨鹿3个地区的田间种群以及敏感种群进行了室内毒力测定, 并采用生化分析法对4个种群相关的羧酸酯酶（carboxylesterase, CarE）、 谷胱甘肽S 转移酶(glutathione S-transferases, GSTs)和乙酰胆碱酯酶(acetylcholinesterase, AChE)的活性进行了研究。结果表明: 3个田间种群对高效氯氰菊酯和氰戊菊酯处于中至高抗水平, 抗性倍数为20.02~73.70倍; 对灭多威处于低至中抗水平, 抗性倍数为6.27~11.84倍; 对高效氯氟氰菊酯(抗性倍数: 1.07~4.20倍), 辛硫磷、 毒死蜱和马拉硫磷(抗性倍数: 1.00~2.69倍), 以及氯虫苯甲酰胺（抗性倍数: 2.00~3.67倍）均处于敏感水平。3个田间种群的CarE, GSTs和AChE活性分别是敏感种群的1.06~1.23, 1.20~1.63和1.15~1.23倍, 这可能与其对高效氯氰菊酯、 氰戊菊酯和灭多威产生的抗性有关。     

棉铃虫AChE不同分子型对抑制剂的敏感度及其与抗药性的关系(英文)

在分离到的棉铃虫AChE五种不同的分子型中 ,2 .1s和 8.7sAChE抗性品系对毒扁豆碱的敏感度明显低于敏感品系 ,成虫头部I50 值分别相差 1 86.3和 84.8倍 ,幼虫I50 值分别相差 1 0 1 0 倍和 1 0 5 倍。幼虫 5.3sAChE对毒扁豆碱的敏感度抗性品系和敏感品系相差达 1 2 3倍 ,而成虫则没有差异。研究结果表明 2 .1s、5.3s和 8.7sAChE敏感度降低可能是造成棉铃虫对有机磷和氨基甲酸酯类杀虫药剂产生抗性的主要原因     

Inheritance of resistance and cross resistance pattern in indoxacarb-resistant diamondback moth Plutella xylostella L.

Leaf-dip assay of Plutella xylostella against indoxacarb showed that the concentration that produced 50% mortality (LC₅₀) of indoxacarb ranged from 20.1 to 11.9 ppm, with highest in Nasik and lowest levels in Coimbatore strains. In selection studies, the LC₅₀ of indoxacarb was 18.5 ppm at generation 1 (G1), which increased to 31.3-fold (167.8 ppm) resistance after ten exposed generations (G10) as compared to unexposed. The LC₅₀ of quinalphos was 74.4 ppm, which increased to 10.0-fold (631.5 ppm) resistance after G10. The LC₅₀ of cypermethrin resistant strain resulted in an 11.5-fold increase in resistance after G10. In P. xylostella , heritability (h²) after ten generations of selection was estimated at 0.4. The number of generations required for tenfold increase in LC₅₀ (1/R) were 6.7. The response to indoxacarb selection in P. xylostella was 0.2 and the selection differential was estimated as 0.4. The phenotypic standard deviation was 0.2. Reciprocal crosses between indoxacarb-resistant and susceptible strains showed that the inheritance of indoxacarb resistance was autosomal. The degree of heritability (D_LC) (0.4, 0.4) indicated incomplete recessive inheritance of indoxacarb resistance.     

Comparative pharmacology and cloning of two novel arachnid sodium channels: Exploring the adaptive insensitivity of scorpion to its toxins

Scorpion toxins have been found lacking effect on Na(+) current of its own sodium channel, whereas the molecular mechanism remains mystery. In this study, the binding affinity of pharmacologically distinct scorpion toxins was found much weaker to scorpion (Buthus martensii) nerve synaptosomes than to spider (Ornithoctonus huwena) ones. The sodium channel cDNA from these two species were further cloned. The deduced proteins contain 1871 and 1987 amino acids respectively. Several key amino acid substitutions, i.e., A1610V, I1611L and S1617K, are found in IVS3-S4 constituting receptor site-3, and for receptor site-4, two residues (Leu-Pro) are inserted near IIS4 of scorpion sodium channel.     

Isolation of a gene encoding cysteine synthase from Flavobacterium K3–15

Abstract The cysteine synthase gene ( cysK ) from Flavobacterium K_3–15 was cloned and sequenced. The gene exhibits 30–50% identity to known cysteine synthases on both the DNA and the amino acid levels. The pyridoxal phosphate binding site of the enzyme is part of a conserved motif comprising seven amino acids (SIKDRIA). The lys31 residue of the flavobacterial enzyme is conserved in all known cysteine synthases. The cysK gene from Flavobacterium K_3–15 was heterologously expressed and the gene product identified by immunoblotting and determination of the enzyme activity.     

Toxicological and biochemical characterizations of malathion sensitivity in two field populations of Oxya chinensis （Orthoptera： Acridoidea）

We evaluate comparative toxicity of malathion in the two populations of the grasshopper Oxya chinensis, collected from Daixian and Fanshi of Shanxi province, China. General esterases and acetylcholinesterase （ACHE） from the two populations were characterized and compared. LD50 of the Daixian population （7.58 μg/g body weight） was 2.02-fold higher than that of the Fanshi population （3.75μg/g body weight）. General esterase-specific activities in the Daixian population were 1.91,130 and 1.85-fold higher than those in the Fanshi population, when α-NA, α-NB and β-NA were used as a substrate, respectively. Kinetic studies of general esterase showed that Vmax values of general esterases hydrolyzing α-NA,α-NB and β-NA in the Daixian population were 2.15-, 1.12-, and 1.47-fold, respectively, higher than those in the Fanshi population. The AChE activity of the Fanshi population was 1.54-fold higher than that of the Daixian population. Kinetic analysis of AChE showed that significant differences were presented between the two populations in the Km values; and the Vmax value in the Fanshi population was higher than that in the Daixian population. Inhibition studies of AChE indicated that AChE from the Daixian population was 2.56-, 2.80-, and 2.29-fold less sensitive to inhibition by paraoxon, chlorpyrifos-oxon, and demeton-S-methyl, respectively, than that from the Fanshi population. These biochemical characterizations of general esterases and AChE were consistent with malathion bioassay in the two populations. It is inferred that the reduced sensitivity of altered AChE and increased general esterase activities play an important role in the differences of insusceptibility of Oxya chinensis to malathion between the two populations.     

Membrane Currents Induced in Xenopus Oocytes by the C-Terminal Fragment of the β-Amyloid Precursor Protein

Abstract: There is mounting evidence that at least some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the β-amyloid precursor protein (βAPP). Most research has focused on the amyloid β protein (Aβ), which has been shown to possess ion channel activity. However, the possible role of other cleaved products of the βAPP is less clear. We have investigated the ability of various products of βAPP to induce membrane ion currents by applying them to Xenopus oocytes, a model system used extensively for investigating electrophysiological aspects of cellular, including neuronal, signalling. We focussed on the 105-amino-acid C-terminal fragment (CT₁₀₅) (containing the full sequence Aβ), which has previously been found to be toxic to cells, although little is known about its mode of action. We have found that CT₁₀₅ is exceedingly potent, with a threshold concentration of 100–200 n M , in inducing nonselective ion currents when applied from either outside or inside the oocyte and is more effective than either βAPP or the Aβ fragments, β_25–35 or β_1–40. The ion channel activity of CT₁₀₅ was concentration dependent and blocked by a monoclonal antibody to Aβ. These results suggest the possible involvement of CT₁₀₅ in inducing the neural toxicity characteristic of AD.     

Synthesis on nontoxic, antibody-binding Escherichia coli heat-stable enterotoxin (STa) peptides

Abstract Modified Escherichia coli heat-stable enterotoxin (ST_a) peptides have been synthesized to identify structures that retain the immunological properties of native ST_a but lack toxicity. Two synthetic peptides, corresponding to the 15 C-terminal amino acid residues of ST_a (ST_h) except for the replacement of one or two Cys residues by Ala, had ≥ 35 000-fold reduced toxicity in infant mice despite almost intact activity as compared to native ST_a to inhibit monoclonal anti-ST antibody binding to solid phase ST_a. Three shorter peptides of 6–8 amino acid residues also did not manifest any toxic activity and showed significant but much reduced reactivity with anti-ST_a antibodies.     

Genetic variability and heritability of drought-induced abscisic acid accumulation in spring wheat

Abstract. Twenty cultivars of spring wheat were examined for variation in abscisic acid (ABA) accumulation following partial dehydration of excised leaves. A 3-fold range of ABA concentration was obtained.
A cross between two cultivars which differed in drought-induced ABA accumulation was used to study the heritability of ABA accumulation and to develop lines differing in their capacity to accumulate ABA. Broad sense heritability was 0–32 between the F₂ and F₃ generations and 0–70 between the F₃ and F₄ generations. Apparent homozygosity for ABA accumulation was achieved in several selections at the F₄. The possible significance for drought resistance of differences in capacity to accumulate ABA is discussed.