Analysis of the Schwanniomyces occidentalis SWA2 gene promoter in Saccharomyces cerevisiae

The effect of different carbon sources on the expression in Saccharomyces cerevisiae of the SWA2 alpha-amylase gene from Schwanniomyces occidentalis was studied from constructs containing its 5' region (-223 to +15), which were fused in-frame to the lacZ gene coding sequence. Maximal expression was achieved with the non-fermentable substrates ethanol and/or glycerol, whereas lower levels were found with maltose or galactose. In contrast, glucose repressed it, even in the presence of any of these other carbon sources. Deletion analyses of the -233 to -85 SWA2 promoter region permitted the identification of two fragments involved in both glucose repression and ethanol activation. A possible region required for cAMP regulation was localised. The SWA2 promoter contains a MIG1-binding GC box whose deletion caused a five-fold increase in the glucose-repressed reporter expression. Despite this, expression of the SWA2 promoter was not MIG1-dependent.     

Effects of sub-lethal cold stress on the Western Flower Thrips, Frankliniella occidentalis

In order to establish in a new geographical area, introduced insects must be able to survive any period of adverse conditions such as a temperate winter and be capable of subsequent development to adulthood and/or reproduction. However, this aspect of insect overwintering and cold tolerance has been poorly studied. At high latitudes, Frankliniella occidentalis is typically associated with artificially heated glasshouses, but has some ability to tolerate low temperatures and may survive winter field conditions for short periods, or for longer periods of time during mild winters. The effects of overwintering on the viability of survivors are, however, unknown. In this study, acute and chronic cold exposure regimes were imposed on first instar larvae and adult female Western Flower Thrips, after which the longevity, development and reproductive capacity of the survivors were monitored and compared to those of non-stressed individuals. Survival of cold exposure did not affect subsequent survivorship of immature or adult insects, though cold treated larvae took approximately two days longer to reach adulthood than untreated individuals (at 20°C, 18L:6D). Chill treatment of adult females significantly reduced their rate of reproduction (from 1.45 to 0.93 larvae day^-1), reproductive lifespan (from 13.3 to 9.2 days) and as a result, total reproductive output (from 20.4 to 10.8 larvae), compared to control females. Acute exposure resulted in non-significant decreases of the same parameters. The relevance of the above effects to overwintering of F. occidentalis is discussed.     

Indication of transthylakoid proton-fluxes in Aegopodium podagraria L. by light-induced changes of plasmalemma potential,chlorophyll fluorescence and light-scattering

The time course of the responses of chlorophyll fluorescence in leaves of Aegopodium podagraria to changes in irradiance does not necessarily show the time constant of thylakoid energization at energy fluence rates below 10–25 W·m^-2. In addition, other measures of thylakoid energization, such as lightscattering at 532 nm and the responses to saturating flashes, show that the related component disappears from these signals at low fluence rates, but not necessarily all together at the same fluence rate. However, this time constant still appears in the light-induced responses of the plasmalemma potential. This implies that the effect on the electrogenic proton pump in the plasmalemma is the most sensitive indicator of proton fluxes into the inner thylakoid space. These results are a further indication that energy-quenching is coupled ther indication that energy-quenching is coupled to transthylakoid proton fluxes via an intermediate, which is not active in Aegopodium podagraria at low irradiances.Abbreviations and symbols _i time constant - F chlorophyll fluorescence - I constant component of irradiance - I _v variable component of irradiance - S light-scattering - q _E high-energy state quenching of chlorophyll fluorescence - T transmittance at 532 nm - V plasmalemma potential     

Nucleotide sequence of the extracellular α-amylase gene in the yeast Schwanniomyces occidentalis ATCC 26077

The Schwanniomyces occidentalis (formerly castellii) ATCC 26077 (CBS 2863) alpha-amylase (AMY 26077) gene was cloned in Saccharomyces cerevisiae and sequenced. An open-reading frame encoding the AMY consists of 1536 base pairs and contains 512 amino-acid residues, which is almost the same in size as the AMY of Sch. occidentalis ATCC 26076 and CCRC 21164. The amino-acid sequence of AMY 26077 differed from that of ATCC 26076 alpha-amylase (AMY 26076) at two residues and from that of CCRC 21164 alpha-amylase (AMY 21164) at three residues. Comparison of the AMY 26077 gene with its homologues from two other strains (Sch. alluvius CBS 1153 and Sch. persoonii CBS 2169) using several restriction enzymes revealed that the AMY 26077 was very similar to AMY CBS 1153 but different from that of CBS 2169.     

Guanosine triphosphate-binding proteins on the plasmalemma of spinach leaf cells

The molecular mechanism of light perception through phytochrome is not well understood. This red-light photosensor has been implicated in various physiological processes, including the photoinduction of flowering. A few recent studies have shown that phytochrome initiates signal transduction chains via guanosine triphosphate (GTP)-binding proteins (G-proteins). We show here by different approaches that G-proteins exist in spinach (Spinacia oleracea L. cv. Nobel). Binding of GTP on the plasmalemma has been partially characterized and its possible regulation by red light examined by in-vitro assays. These experiments indicate a clear regulation of GTP binding by red light and also by Mastoparan. At least three G-proteins or protein subunits were found to be associated with the plasmalemma of leaf cells. The use of an antibody raised against an animal Gβ subunit confirmed the presence of heterotrimeric G-proteins. Separation of a crude membrane extract by free-flow electrophoresis also showed that some G-proteins could exist on the tonoplast.     

外源茉莉酸诱导菜豆对西花蓟马和南方小花蝽的行为反应

【目的】为明确茉莉酸诱导的菜豆对西花蓟马Frankliniella occidentalis和南方小花蝽Orius similis的行为反应。【方法】采用四臂嗅觉仪测定了西花蓟马和南方小花蝽对不同浓度茉莉酸诱导菜豆后的行为反应,并用气相色谱-质谱联用仪测定了不同浓度茉莉酸处理后菜豆挥发物的成分。【结果】不同浓度茉莉酸处理的菜豆植株对西花蓟马和南方小花蝽分别有不同程度的驱避和吸引作用,以1 mmol/L的茉莉酸处理植株对西花蓟马的驱避作用最强,0.1 mmol/L的茉莉酸处理植株对南方小花蝽的吸引作用最强。不同处理菜豆的挥发物在含量和成分上存在显著差异,(Z)-3-己烯丙酸酯、2-异丙基-甲氧基毗嗦只有在茉莉酸处理植株中检测到。结合不同浓度茉莉酸处理植株对西花蓟马和南方小花蝽的行为反应及菜豆挥发物含量的变化趋势,推测(E)-2-己烯醛对西花蓟马有驱避作用,(E)-2-乙酸叶醇酯对南方小花蝽具有引诱作用。【结论】茉莉酸处理菜豆后,植物挥发物种类和含量发生了变化,在增强菜豆植株抗虫性的同时,还可增强捕食性天敌南方小花蝽的搜索和捕食能力。     

Interaction of trap colour attractiveness with morphological variability of the thrips Frankliniella occidentalis (Pergande 1895)

In the latitude of Kiev, 80% of Frankliniella occidentalis (Pergande 1895) population was caught on the colour traps from 11 a.m. to 5 p.m. in summer and from 11 a.m. to 3 p.m. in winter. Meanwhile, 10% of the population did not react to the colour traps at all (“passive” individuals), whereas for 20% of the population it was impossible to determine the clear colour with attractive properties. The thrips attracted with blue and yellow color traps, had more massive and variable body parts to be connected with their flight. Their antennae and segment sizes were significantly smaller than compared to the control variant, which could be explained by eyesight domination over other organs of sensory orientation. The indices of heterogeneity of morphological features can be used as evaluation criteria for the adaptive variability and stability of the thrips population.     

Permeabilization of the plasmalemma and wall of soybean root cells to macromolecules

A technique has been developed that results in the reversible permeabilization of the cell wall and plasmalemma of soybean (Glycine max (L.) Merr.) root cells grown in suspension and callus culture. Cells in culture are treated with saponin (0.1 mg/ml) for 15 min at room temperature. They are then coincubated in separate experiments with fluorescent-derivatized dextrans (20–70 kDa) or fluorescein-conjugated goat anti-rabbit immunoglobulin G to ascertain the exclusion size of macromolecules capable of diffusing across the cell wall and plasmalemma into the cytoplasm. Following an incubation period of 30 min, it was observed by conventional and confocal fluorescence microscopy that all derivatized macromolecules tested (20–140 kDa) could be incorporated into the cytoplasm, but not into the vacuole. This procedure did not appear to affect cell viability adversely. A normal doubling time was observed for these cells following the permeabilization procedure.Abbreviations FDA fluorescein diacetate - FITC-20 kDa, FITC-40 kDa, FITC-70 kDa dextrans fluorescein-derivatized 20-kDa, 40-kDa, and 70-kDa dextrans - IgG immunoglobulin G - kDa kilodalton Paramjit K. Gharyal wishes to thank the Nitrogen Availability Program at Michigan State University for financial support. We also thank Edwin de Feijter of Meridian Instruments for technical assistance in performing the confocal measurements. This work was supported by a grant from the U.S. — Israel Binational Agricultural Research and Development Fund (BARD project No. US-1384-87).     

Influence of culture conditions on the cell yield and amylases biosynthesis in continuous culture by Schwanniomyces castellii

Schwanniomyces castellii excreted -amylase and amyloglucosidase into the medium in the presence of starch. The biosynthesis and the rate of excretion were influenced by dissolved oxygen (specially for -amylase), pH of the culture and dilution rate. The cell yield observed (0.59) remained constant up to D=0.35h^-1 with starch as substrate. But in the case of growth on glucose, the yield observed was equal to 0.62 up to a dilution rate of D=0.18 h^-1. Beyond this value Y x/s decreased and ethanol was produced. The onset of fermentation dependend partly on the nature of the substrate and not only on the environment in particular on the quantity of dissolved oxygen present.     

西花蓟马的鉴别及其与近缘种的区别

西花蓟马是我国2 0 0 3年在北京新发现的外来入侵害虫,因虫体很小鉴定困难。该文提供了西花蓟马详细的形态鉴别特征,同时给出了与西花蓟马相似的烟蓟马、花蓟马和佛罗里达花蓟马共4种花蓟马的鉴定检索表。     

温度对西花蓟马(Frankliniella occidentalis)生长发育和存活率的影响

在15、20、25℃和30℃,RH75％的条件下,分析了温度与西花蓟马发育速率的关系,并测定了西花蓟马的发育起点温度、有效积温和存活率。结果表明：在15～30℃范围内,西花蓟马各虫期的发育速率随温度的升高而加快;用直接最优法对发育起点温度和有效积温进行估算,求得西花蓟马的卵、若虫、预蛹和伪蛹及世代发育起点温度分别为5．70、7．04、4．49、6．84℃和6．23℃;世代的有效积温为219．73d·℃。西花蓟马的世代存活率在25℃时最高,为40．12％;30℃时西花蓟马的存活率最低,为17．80％。     

Membrane particles and gap junctions in the retinas of two species of cephalopods,Octopus ocellatus and Sepiella japonica

To study correlation between membrane structure and photoreceptor function, we compared the size and density of intramembrane particles (IMPs) in various membrane compartments of freeze-fractured retinas in a cuttle-fish, Sepiella japonica, and an octopus, Octopus ocellatus. Distribution of gap junctions in the retinas was also examined. Similar results were obtained in the two species. P-faces of both rhabdomeric microvillar membrane and non-rhabdomeric plasma membrane of the apical process were characterized by a random distribution of dense IMPs (ca. 5500-6500/microns2), which showed a unimodal size distribution with a mean diameter of ca. 10 nm. Unlike other invertebrate ocelli, the plasma membrane of the cell body in both the outer and inner segments had significantly denser P-face particles (ca. 7500-8000/microns2) than the rhabdomeric microvillar membrane. The size distribution of IMPs in each part of the membrane was also unimodal, but with a mean diameter of ca. 8 nm. In tangential fractures, each lamella of the myeloid body showed a patchwork of P-faces with irregularly arranged, dense particles and E-faces with orderly patterened granulation. Density and size distribution of the P-face particles in the myeloid membrane resembled those in the rhabdomeric microvillar membrane. The plasma membranes of the supporting cell and the gial cell had relatively sparse P-face particles (ca. 1500-3000/microns2). In addition to the previously reported gap junctions, which connected visual cell inner segments with each other, directly or via collaterals, small gap junctions were found between the visual cell axons and presumed efferent nerve fibres in the plexiform layer. Large-sized gap junctions provided mutual connections for both supporting cells and glial cells. In conclusion, IMPs of 10 nm in mean diameter in the microvillar and non-microvillar parts of the apical process plasma membrane and in the myeloid membrane represent the molecules or their clusters of two photopigments in the cephalopod visual cell, rhodopsin and retinochrome, respectively, and electrical transmission plays a role in visual cell-efferent nerve interactions.     

北京地区西花蓟马田间种群的抗药性监测

采用叶管药膜法在室内测定了2009—2010年北京海淀地区和昌平地区西花蓟马Frankliniella occidentalis(Pergande)田间种群对12种杀虫剂敏感性的年度变化。结果表明,北京昌平和海淀地区的西花蓟马对多数的药剂仍处于敏感状态,但对氯氟氰菊酯已产生近40倍的抗性,昌平种群对多杀菌素具有产生低水平抗性的趋势(抗性倍数为4倍)。推荐药剂包括多杀菌素、阿维菌素、甲氨基阿维菌素苯甲酸盐(甲维盐)、毒死蜱和溴虫腈,应注意与其它药剂的轮换使用,灭多威对西花蓟马的毒力水平最低,不推荐使用。     

Electrostatic surface properties of plasmalemma vesicles from oat and wheat roots. Ion binding and screening investigated by 9-aminoacridine fluorescence

Right-side-out and sealed plasmalemma vesicles were isolated from roots of spring wheat (Triticum aestivum L. cv. Drabant) and oat (Avena sativa L. cv. Brighton) by two-phase partition in a medium containing sucrose (0.25 mol l^-1). Oat root plasmalemma vesicles were discovered to contain a strongly fluorescent compound with an emission maximum at 418 nm. The surface potential of the membranes was monitored by 9-aminoacridine fluorescence and the effect of protein concentration, mannitol versus sucrose, absence of osmoticum, concentrations of salt, and titrations with chelators investigated. It is concluded that i) protein concentrations of less than 50 g ml^-1 for oat and 100 g ml^-1 for wheat plasmalemma vesicles should be used to avoid serious problems with non-linearity of response of 9-aminoacridine fluorescence, ii) mannitol can be used instead of sucrose as the osmoticum, iii) the vesicles were ruptured in the absence of osmoticum allowing us to monitor both sides of the membranes, iv) plasmalemma vesicles from oat roots are more negative than vesicles from wheat roots, and v) oat and wheat root plasmalemma vesicles are isolated with about the same amounts of bound Ca²⁺ and Mg²⁺. These bound divalent cations may not, however, reflect the in-vivo conditions since the tissues were homogenised in the presence of ethylenediaminetetraacetic acid.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - c1/2 value concentration at which half of the maximum effect is observed - Mops 3-(N-morpholino)propanesulfonic acid     

二斑叶螨的卵和丝网对西花蓟马生长发育和繁殖的影响

为探讨二斑叶螨Tetranychus urticae Koch的卵和丝网对西花蓟马Frankliniella occidentalis(Pergande)的影响,本文研究了西花蓟马分别取食健康菜豆叶片、带二斑叶螨卵的菜豆叶片、带卵+丝网的菜豆叶片3种不同情况下,西花蓟马生长发育、存活和繁殖情况,并获得了其生命参数。结果表明西花蓟马在带二斑叶螨卵的菜豆叶片上发育最快,其未成熟期为11.35 d,明显短于另外两个处理,在带有二斑叶螨卵和丝网的菜豆叶片上成虫获得率最低为63%,西花蓟马在不同处理之间的成虫寿命和繁殖力没有明显的差异;西花蓟马取食3种菜豆叶片的内禀增长率分别为0.1779、0.1827和0.1710。以上结果说明取食二斑叶螨的卵可缩短西花蓟马发育时间,对种群增长有利,二斑叶螨的丝网可降低西花蓟马的存活率。     

外源茉莉酸诱导的菜豆叶片生化抗性及其对西花蓟马体内保护酶和解毒酶活性的影响

为探讨外源茉莉酸诱导的菜豆叶片抗性及对西花蓟马体内酶活性的影响,在室内对菜豆植株分别喷施1、0.1、0.01和0.001 mmol·L^-1 4个浓度的茉莉酸,以健康植株为对照,分别于处理后1、5和10 d测定菜豆叶片营养物质及次生物质的含量.另在同样处理叶片上分别接西花蓟马2龄若虫,分析其体内保护酶和解毒酶活性的变化.结果表明: 不同浓度茉莉酸处理1 d后,菜豆叶片的蛋白质含量和健康植株没有明显差异,但在5和10 d时显著低于健康植株;菜豆叶片游离氨基酸含量在茉莉酸处理1 d后显著高于健康植株,之后逐渐降低;茉莉酸处理下菜豆叶片可溶性糖含量显著低于健康植株,并随着茉莉酸浓度的升高和处理时间的延长而进一步下降;叶绿素含量在处理1 d后显著降低,随着处理时间的增加逐渐升高.叶片单宁、黄酮和总酚的含量在不同浓度茉莉酸和处理时间下均显著高于对照.蓟马取食导致菜豆叶片生化物质含量的变化与外源茉莉酸诱导的相似.西花蓟马取食茉莉酸处理的菜豆植株24 h后,体内保护酶系(超氧化物歧化酶、过氧化氢酶、过氧化物酶)和解毒酶系(谷胱甘肽S-转移酶、羧酸酯酶、乙酰胆碱酯酶)均明显高于健康植株,但茉莉酸浓度与处理时间对其影响程度不同.取食虫害菜豆叶片后西花蓟马体内酶活性的变化与取食外源茉莉酸诱导的叶片相似.说明外源茉莉酸处理可诱导菜豆植株的抗性,西花蓟马取食处理后的菜豆叶片可产生明显的反防御来适应寄主植物的变化.     

High-level production of yeast (Schwanniomyces occidentalis) phytase in transgenic rice plants by a combination of signal sequence and codon modification of the phytase gene

This study was designed to produce yeast (Schwanniomyces occidentalis) phytase in rice with a view to future applications in the animal feed industry. To achieve high-level production, chimeric genes with the secretory signal sequence of the rice chitinase-3 gene were constructed using either the original full-length or N-truncated yeast phytase gene, or a modified gene whose codon usage was changed to be more similar to that of rice, and then introduced into rice (Oryza sativa L.). When the original phytase genes were used, the phytase activity in the leaves of transgenic rice was of the same level as in wild-type plants, whose mean value was 0.039 U/g fresh weight (g-FW) (1 U of activity was defined as 1 micromol P released per min at 37 degrees C). In contrast, the enzyme activity was increased markedly when codon-modified phytase genes were introduced: up to 4.6 U/g-FW of leaves for full-length codon-modified phytase, and 10.6 U/g-FW for truncated codon-modified phytase. A decrease in the optimum temperature and thermal stability was observed in the truncated heterologous enzyme, suggesting that the N-terminal region plays an important role in enzymatic properties. In contrast, the optimum temperature and pH of full-length heterologous phytase were indistinguishable from those of the benchmark yeast phytase, although the heterologous enzyme was less glycosylated. Full-length heterologous phytase in leaf extract showed extreme stability. These results indicate that codon modification, combined with the use of a secretory signal sequence, can be used to produce substantial amounts of yeast phytase, and possibly any phytases from various organisms, in an active and stable form.     

西花蓟马对多杀菌素的抗性汰选和遗传方式

在室内进行了西花蓟马Frankliniella occidentalis(Pergande)种群对多杀菌素的抗性筛选和抗性遗传方式分析。经过2年多的汰选,抗性水平相比较初始种群提高了30倍,与同时测定的敏感品系相比抗性达到80.8倍。抗性遗传方式分析结果表明,正、反交后代显性度分别为0.51和0.43,二者差异不显著,说明西花蓟马对多杀菌素的抗性为常染色体、不完全显性遗传;剂量对数死亡机率值曲线分析结果显示回交后代在死亡率50%处,自交后代在死亡率25%和75%处未出现明显平坡,回交和自交后代实测值卡方检验进一步证实抗性为多基因控制。     

Inhibition of plant cell membrane transport phenomena induced by zearalenone (F-2)

Zearalenone (F-2), an estrogenic factor produced by a number of Fusarium spp., stimulates a leakage of electrolytes, -cyanin and aminoacids from three plant tissues. F-2 inhibits rubidium uptake in roots of Zea mays L. and Beta vulgaris L. var. rubra. However the effect in the latter tissue is evident after long-term treatments with the toxin. Rubidium uptake is not affected in Solanum tuberosum L. var. Bintje. The toxin also causes inhibition of H⁺ extrusion, of root elongation, of ATPase activity of plasmalemma-enriched fractions and depolarization of transmembrane potentials of corn roots. The evidence presented supports the hypothesis that F-2 affects plasma membranes of several plant species.Abbreviations F-2 Zearalenone - PD Potential difference - FC Fusicoccin - DCCD Dicyclohexylcarbodiimide