特异腐质霉角质酶-OMP25融合蛋白在大肠杆菌中的高效表达

【目的】通过探究特异腐质霉角质酶-OMP25融合蛋白(HiC-OMP25)在不同大肠杆菌(Escherichia coli)菌株中的表达情况、底物降解情况、热稳定性及宿主菌细胞膜通透性与细胞表面疏水性,揭示表达HiC-OMP25时不同宿主菌的差异性,并进一步提高HiC-OMP25在大肠杆菌中的表达量。【方法】分别在E.coli BL21(DE3)及E.coli C43(DE3)中表达HiC-OMP25,并测定其对对硝基苯丁酸酯(4-nitrophenol butyrate,pNPB)、聚丙烯酸乙酯(polyethyl acrylate,PEA)的降解效果、50℃稳定性;测定表达HiC-OMP25时宿主菌的细胞膜通透性及细胞表面疏水性变化;共表达伴侣蛋白提高HiC-OMP25在E.coli C43(DE3)中的表达量。【结果】HiC-OMP25在E.coli BL21(DE3)与E.coli C43(DE3)中均成功表达并降解pNPB,但前者对PEA的降解效果及50 ℃稳定性均低于后者。同时,表达HiC-OMP25显著增强了E.coli BL21(DE3)的细胞膜通透性及细胞表面疏水性。HiC-OMP25与巯基氧化酶(Erv1p)、二硫键异构酶(DsbC)在E.coli C43(DE3)中共表达时,其表达量为原始菌株的2.14倍,且对pNPB及PEA均有良好的降解效果。【结论】异源表达时,HiC-OMP25在E.coli C43(DE3)中正确折叠,而在E.coli BL21(DE3)中未完全正确折叠;通过共表达伴侣蛋白提高了HiC-OMP25在E.coli C43(DE3)中的表达量,为以后HiC-OMP25的工业化生产及应用奠定了基础。     

酵母己糖转运蛋白Hxt7启动子突变体的构建与表达(英)

葡萄糖在酵母细胞中的转运是通过一个庞大的己糖转运子家族实现的.Hxt7为一种高亲和性转运蛋白，在这个家族中的表达丰度最高.它的表达为低浓度葡萄糖所诱导.为了研究葡萄糖转运对细胞生长和葡萄糖阻遏的控制作用，提高对糖酵解通路和糖代谢过程的可调控性，该研究对HXT7启动子进行逐步删除，并将含启动子区域长短不同的HXT7基因整合转入酿酒酵母己糖转运子缺失菌株hxtΔ(hxt1～hxt7, gal2缺失)的基因组，构建一组仅含HXT7且表达水平不一的酵母启动子突变株.将HXT7在突变株中的表达与在野生菌株（MC996A）中的表达进行比较，对它们在含50 mmol/L葡萄糖培养基中的生长速率、葡萄糖消耗性能、HXT7 mRNA及蛋白质表达水平等进行了实时测定.结果表明，当葡萄糖浓度低于5 mmol/L时，HXT7的表达量最高.高浓度葡萄糖环境下，HXT7在突变株中的表达高于在野生株中的表达，显示出不完全性葡萄糖阻遏效应.HXT7的表达水平与启动子长短和基因拷贝数有关.位于－495至－346之间的149 bp HXT7启动子区域对HXT7的表达至关重要.启动子短于346 bp，HXT7基本不表达.     

水稻条斑病细菌hrp基因诱导表达系统的建立

水稻条斑病细菌(Xanthomonas oryzae pv.oryzicola,Xooc)决定在非寄主植物上激发过敏反应(hypersensitive response)和在寄主水稻上具致病性(pathogenicity)的hrp基因簇是诱导表达的。为研究hrp基因的功能,利用hpa1和hrpX基因的启动子与gfp基因进行融合,构建了hrp基因诱导表达系统。绿色荧光蛋白表达揭示,Xooc的hrp基因在营养丰富的NB培养基上不能有效表达,在hrp诱导培养基XOM3上可有效表达。以hrpX和hrpG突变体为参照,RT-PCR研究结果提示,Xooc野生型菌株hpa1基因在NB上不能有效表达,在XOM3培养基上可有效表达。相应地,hrpX突变体中hpa1基因不能被诱导表达,而在hrpG突变体中hpa1基因转录表达水平低于野生菌。研究结果还证实,水稻悬浮细胞能高效诱导Xooc的hrp基因表达。Xooc hrp基因诱导表达系统的建立为研究hrp基因功能、发掘T3SS效应分子以及开展Xooc致病性研究奠定了基础。     

肿瘤中neu基因的过量表达及其抑制

neu基因编码一种和表皮生长因子受体同源的磷酸蛋白,具有酪氨酸激酶的活性.近年来在多种人类肿瘤中发现neu基因的扩增和(或)过量表达.一些蛋白质因子或化学药物可以在转录水平阻遏neu基因的过量表达或者降低其产物p185^neu的酪氨酸激酶活性,抑制具有neu基因过量表达的癌细胞的转移和增殖.     

龙眼ERF家族成员鉴定及其在体胚发生早期的表达

为了解龙眼ERF家族的基本性质以及在体胚发生早期的表达规律,该研究对龙眼基因组鉴定出108个ERF家族成员进行基本理化性质分析与进化树构建,并结合龙眼转录组及miRNA、lncRNA数据库对DlERF家族成员与lncRNA、miRNA之间的关系预测与表达模式分析。结果显示：(1)理化性质及进化树分析表明,AP2/ERF结构域在龙眼中相对最为保守,DlERF对龙眼的抗胁迫能力以及对病菌的防御能力可能起着重要作用。(2)RNA Seq中的表达量分析可知,95个ERF基因在转录组中检测到表达,在愈伤组织(EC)、不完全胚性紧实结构(ICpEC)与球形胚(GE)阶段中分别存在31、11与53个ERF基因高表达。(3)qRT PCR结果显示,在龙眼体胚发生早期显著表达5个ERF基因中,Dlo_008317.1ERF15 2、Dlo_022310.1ERF1 1与Dlo_009939.1ERF98在GE阶段表达量显著高于EC与ICpEC阶段,Dlo_009070.1ERF106 3与Dlo_022634.1ERF22 1则分别在EC与ICpEC阶段高表达。(4)DlERF家族成员与相关lncRNA、miRNA表达量分析显示,LTCONS_00013739对靶基因Dlo_008317.1ERF15 2为正调控关系;Dlo_miR413与Dlo_miR1510a共同靶向Dlo_009070.1ERF106 3,从EC到GE阶段均表现出显著负调控关系,而Dlo_miR413与Dlo_008317.1ERF15 2的表达量表现出正相关,推测相比于Dlo_008317.1ERF15 2,Dlo_miR413更倾向于调控Dlo_009070.1ERF106 3;调控Dlo_009939.1ERF98相关的Dlo_miR408与Dlo_miR774b,从表达趋势来看,Dlo_miR774b在龙眼体胚发生早期过程能够负调控Dlo_009939.1ERF98;Dlo_miR399c与Dlo_022310.1ERF1 1在EC到GE阶段可能存在负调控关系。(5)不同梯度浓度的乙烯处理使得DlERF基因表达显著下调。这些发现提供了有关龙眼体胚发生早期DlERF的重要见解,从而为将来对龙眼体胚发生早期过程中DlERF的功能分析奠定了基础。     

不同视觉经验中zif268基因的表达模式

zif268基因编码一转录因子ZIF268. 在发育的视皮层中,zif268基因的表达模式受发育的调节. 在具有正常视觉经验的视皮层中,zif268基因有较高水平的表达. 视觉废置后,视皮层内该基因的表达水平显著降低. 通过视觉刺激可显著增强该基因的表达. 有关zif268基因表达模式的研究对于阐明该基因在哺乳动物视觉系统中的生理功能起到借鉴的作用.     

梅花2个SVP基因的克隆与表达分析

SVP (SHORT VEGETATIVE PHASE)是MADS box家族一员,它能够整合多条开花途径的开花信号,调节植物由营养生长向生殖生长的转变。为了解梅花(Prunus mume Sieb. et Zucc.)成花转变过程的分子机理,该研究采用RT PCR方法从梅花‘长蕊绿萼’中克隆到2个SVP的同源基因,分别命名为PmSVP1和PmSVP2,并采用荧光定量PCR对2个基因的表达模式进行分析。序列分析表明,PmSVP1和PmSVP2的编码区长度分别为687 和672 bp,分别编码228和223氨基酸。系统进化结果显示,PmSVP1与拟南芥AtSVP以及一些木本植物SVP同源基因聚为一组,PmSVP2与马铃薯、乳浆大戟等草本植物中的SVP同源基因聚为一组。实时荧光定量分析表明,在成年梅花中,2个PmSVP基因主要在茎、叶和叶芽等营养器官中表达,且都在叶中表达量最高。在1月龄幼苗中,PmSVP1基因在根、茎、叶中都有表达,PmSVP2基因则没有任何表达;在梅花花芽分化过程中,PmSVP1和PmSVP2基因的表达量均呈现下调表达的趋势。研究推测,PmSVP1和PmSVP2基因可能参与调控梅花从营养生长向生殖生长的转变。     

拟南芥AtNHX2启动子的克隆及表达模式分析(英)

AtNHX2基因是拟南芥NHX基因家族的一员,编码了一种液泡膜中的Na⁺/H⁺反向运输体并对拟南芥的耐盐能力起着重要的作用.采用PCR扩增的方法克隆了拟南芥AtNHX2基因启始密码子上游约2.8 kb的DNA片段,并将其克隆到植物表达载体pCAMBIA1301-1中,通过基因枪轰击洋葱表皮瞬时表达的方法,初步检测启动子的活性.将重组质粒pCAMBIA1301-1/AtNHX2 promoter转化拟南芥并筛选纯合子.AtNHX2 promoter-GUS分析显示AtNHX2在所有的组织中均有表达,包括根尖.在保卫细胞中检测到了强烈的GUS表达,这一结果表明,AtNHX2对特殊细胞的pH调控和K⁺自身稳定方面起着重要的作用.AtNHX2启动子的活性可被NaCl抑制,并且抑制的强度和NaCl的浓度成正相关. 300 mmol/L KCl处理可增强启动子的活性,说明NaCl和KCl是在转录水平上调控AtNHX2的表达.在老叶中GUS活性比在新叶中GUS活性强,这说明了AtNHX2优先将有毒的离子积累在老叶中,从而有利于植物的正常发育.在根毛细胞中也观测到了强烈的GUS活性,这就暗示了AtNHX2在扩大的液泡中储存Na⁺.     

Sex steroids and their actions on the birdsong system

It is probably not surprising to most of us that the endocrine system plays a significant role in controlling the singing behavior of birds. We are familiar with the song of birds as a conspicuous acoustic feature of our environment during the avian breeding season. We often witness song when it is produced by birds (males) that are aggressively establishing and defending territories and that are advertising to available females. Thus, it is easy to imagine that song is likely to be stimulated by gonadal hormones. However, the ways in which gonadal sex steroids influence the various parts of the brain at various stages of the bird's life to influence song are complex and far from being completely understood. In this review, I will highlight some of the significant discoveries that have contributed to our view that the songbird brain is a significant and dynamic target of sex steroids. I will also describe what we have learned about properties of the endocrine system and the brain and how they each contribute to making androgens or estrogens available to particular parts of the songbird brain. Finally, I will describe some new research directions that may help answer some unresolved issues about hormonal effects on the songbird brain. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 619–631, 1997     

Neuro-evolutionary patterning of sociality

Evolutionary shifts in species-typical group size ('sociality') probably reflect natural selection on motivational processes such as social arousal, approach-avoidance, reward, stress/anxiety and dominance. Using four songbird species that differ selectively in sociality (one territorial, one modestly gregarious, and two highly gregarious species), we here examined immediate early gene (IEG) responses of relevant brain regions following exposure to a same-sex conspecific. The paradigm limited behavioural performance, thus species differences should reflect divergence in motivational and/or perceptual processes. Within the extended medial amygdala (which is involved in appetitive approach, social arousal and avoidance), we observed species differences in IEG response that are negatively graded in relation to sociality. In addition, brain areas that are involved in social stress and dominance-related behaviour (ventrolateral septum, anterior hypothalamus and lateral subdivision of the ventromedial hypothalamus) exhibited IEG responses that dichotomously distinguish the territorial species from the three gregarious species. The IEG responses of areas involved in reward (nucleus accumbens and ventral pallidum) and general stress processes (e.g. paraventricular hypothalamus, lateral bed nucleus of the stria terminalis and most areas of the lateral septum) do not correlate with sociality, indicating that social evolution has been accompanied by selection on a relatively discrete suite of motivational systems.     

Identification and characterization of an Egr ortholog as a neural immediate early gene in the European honeybee (Apis mellifera L.)

To date, there are only few reports of immediate early genes (IEGs) available in insects. Aiming at identifying a conserved IEG in insects, we characterized an Egr homolog of the honeybee (AmEgr: Apis mellifera Egr). AmEgr was transiently induced in whole worker brains after seizure induction. In situ hybridization for AmEgr indicated that neural activity of a certain mushroom body (a higher brain center) neuron subtype, which is the same as that we previously identified using another non-coding IEG, termed kakusei, is more enhanced in forager brains. These findings suggest that Egr can be utilized as an IEG in insects.     

Adult neurogenesis: From canaries to the clinic

Neuronal precursor cells persist in the adult vertebrate forebrain, residing primarily in the ventricular/subventricular zone (SZ). In vivo, SZ precursors yield progeny which may die or give rise to glia. Yet they may also generate neurons, which are recruited to restricted regions such as the avian telencephalon and mammalian olfactory bulb. The survival of neurons arising from adult progenitors is dictated by both the availability of a permissive pathway for migration and the environment into which migration occurs. In the songbird higher vocal center (HVC), both humoral and contact-mediated signals modulate the migration and survival of new neurons, through an orchestrated set of hormonally regulated paracrine interactions. New neurons of the songbird brain depart the SZ to enter the brain parenchyma by migrating upon radial guide fibers, which emanate from cell bodies in the ventricular epithelium. The radial guide cells coderive with new neurons from a common progenitor, which is widespread throughout the songbird SZ. Neural precursors are also widely distributed in the adult mammalian SZ, although it is unclear whether avian and mammalian progenitor cells are homologous: Whereas neuronal recruitment persists throughout much of the songbird forebrain, in mammals it is limited to the olfactory bulb. In humans, the adult SZ appears to largely cease neurogenesis in vivo, although it, too, can produce neurons in vitro. In both rats and humans, the differentiation and survival of neurons arising from the postnatal SZ may be regulated by access to postmitotic trophic factors. Indeed, serial application of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) has allowed the generation and maintenance of neurons from the adult human SZ. This suggests the feasibility of inducing neurogenesis in the human brain, both in situ and through implanted progenitors. In this regard, using cell-specific neural promoters coupled to fluorescent reporters, defined progenitor phenotypes may now be isolated by fluorescence-activated cell sorting. Together, these findings give hope that structural brain repair through induced neurogenesis and neurogenic implants will soon be a clinical reality. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 267–286, 1998     

Complex Responses of Songbirds to Soil Acidification of Managed Beech Forests in Central Europe

Abstract Many forest ecosystems have been extensively degraded by acidifying pollutants over the last decades. There is also widespread concern about population decline of a number of songbirds due to acidification-driven calcium limitation. We hypothesize that the carrying capacity of forests decreases with reduced soil calcium content. We analyzed songbird assemblage/habitat relationships by territory mapping at 30 sites in a total of five managed beech forests. The sites harbored 38 songbird species. A canonical correspondence analysis showed that soil acidification, represented by soil calcium content, had a significant influence on the species assemblages of the 14 most frequent songbirds. The vertical complexity of the canopy additionally influenced the assemblages. Multiple regressions revealed that the density of the territories of song thrush (Turdus philomelos) and nuthatch (Sitta europaea) were positively correlated to calcium content. Blackbirds (Turdus merula) seemed to compensate for the negative effect of acidification by selecting breeding sites close to forest edges to forage in less acidified agricultural habitats. In contrast, the territorial density of robin (Erithacus rubecula), chaffinch (Phylloscopus collybita) and coal tit (Parus ater) increased with increasing soil acidification. Contrary to our hypothesis, the overall carrying capacity of songbird was not reduced in forests with acidified soils. However, the nesting success of song thrush and blackbird, as determined by the number of fledglings, was higher in the forest with the highest calcium content when compared to the forest with the lowest calcium content. We conclude that some species are severely affected by acidification, whereas others seem to have evolved successful strategies to meet their calcium demand even in acidified habitats. Electronic supplementary material: The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cross-reactions between human and animal plasma proteins

Summary Cross-reactions between human plasma proteins and their homologues in primate blood were investigated systematically. From the three groups of proteins distinguished earlier [2] two have been especially examined; these findings are reported and discussed in the present communication. The Immunological Evolution Group (IEG) I, comprising IgA (-chain), IgD (-chain) and inter--trypsininhibitor, cross-reacts with pongid plasma only, IEG IIa, i.e. IgM (-chain), ₂-glycoproteins II and III and cholinesterase, does so with the pongid and cercopithecoid plasmas tested; IEG IIb, including acid _l-glycoprotein, _2HS-glycoprotein, _l-trypsininhibitor, haptoglobin and hemopexin, cross-reacts with pongid, cercopithecoid and cebus (platyrrhinian) plasma and finally IEG IIc, consisting of transferrin and Gc-globulin, does so with all primate plasmas tested, including prosimians. All the proteins named do not cross-react however with non-primate proteins as do those of IEG III. It is concluded, that the determinants reacting in the primate proteins increase in their evolutionary ages from IEG I over IEG IIa, IIb to IIc in the same way as the last common ancestors of man and the crossreacting species increase.

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Zusammenfassung Die Kreuzreaktionen zwischen menschlichen Plasmaeiweißen und ihren Homologen im Blut von subhumanen Primaten wurden systematisch untersucht. Von den drei früher voneinander abgetrennten Gruppen [2] wurden zwei für die vorliegende Versuchsreihe herausgegriffen; eine von ihnen konnte weiter unterteilt werden. Die erhobenen Befunde werden berichtet und diskutiert. Die Immunologische Evolutions-Gruppe (IEG) I, die IgA (-Kette), IgD (-Kette) und den Inter--Trypsininhibitor umfaßt, zeight Kreuzreaktionen nur mit den Plasmen von Pongiden. Die IEG IIa, zu der IgM (-Kette), die ₂ II und III und Cholinesterase gehören, kreuzreagiert mit den entsprechenden Plasmaproteinen der geprüften Pongiden und Cercopithecoidea, die IEG IIb—das sind saueres _l-Glycoprotein, _2HS-Glycoprotein, _l-Trypsininhibitor, Haptoglobin und Haemopexin — mit Pongiden, Cercopithecoidea und Cebus (Platyrrhini) und endlich die IEG IIc, die sich aus Transferrin und dem Gc-Globulin zusammensetzt, mit allen geprüften Primatenplasmen einschließlich denen von Prosimiern. Alle hier genannten Plasmaeiweiße zeigen jedoch keine Kreuzreaktionen mit Proteinen von Nichtprimaten, wie dies bei der IEG III der Fall ist.Aus den vorliegenden Befunden wird der Schluß gezogen, daß die Determinanten der Primatenproteine, die jeweils reagieren, in ihrem phylogenetischen Alter von IEG Iüber IEG IIa, IIb, IIc im gleichen Maße ansteigen, wie die letzten gemeinsamen Vorfahren von Mensch und den kreuzreagierenden Arten.

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(Chief: Prof. Dr. E. Krah)     

Linking songbird nest predation to seedling density: Sugar maple masting as a resource pulse in a forest food web

The ecological literature presents considerable evidence for top‐down forcing on the maintenance of species diversity. Yet, in temperate forests, bottom‐up forces often exert a strong influence on ecosystem functioning. Here, we report on the indirect influence of a pulsed resource, sugar maple (Acer saccharum) seed production, on nest survival in a migratory songbird. We hypothesized that seed production in year t would determine daily nest survival rate in year t + 1 through its effects on seed‐eating rodents. We used the density of sugar maple seedlings (with cotyledons) in year t + 1 as a proxy for seed production in year t and predicted that it would be inversely related to songbird nest survival the same year. We estimated the density of sugar maple seedlings, eastern chipmunk (Tamias striatus) activity, and daily nest survival rate in the ovenbird (Seiurus aurocapilla) over four successive years in a northern hardwood forest of New Brunswick, Canada. Seedling density varied by two orders of magnitude between years, whereas an index of chipmunk activity changed by an order of magnitude. Both variables were positively correlated and negatively correlated to daily nest survival rate. A logistic‐exposure model including only seedling density received the greatest level of support (lowest AIC_c). Previous studies have reported the effect of sugar maple masting on seed‐eating rodent populations, but the strong link we report between seedling density and songbird nest survival is novel. A nocturnal seed‐eating nest predator, deer mouse (Peromyscus maniculatus), was not considered in our models, which may explain why chipmunk was not the best predictor of daily nest survival rate. The trophic linkages we observed are remarkably strong for a temperate forest ecosystem and might become more prevalent in northeastern North America, at least on calcium‐rich soils, with the loss of large‐diameter beech trees as a result of beech bark disease.     

Managing for multiple species: greater sage-grouse and sagebrush songbirds

Human activity has altered 33–50% of Earth's surface, including temperate grasslands and sagebrush rangelands, resulting in a loss of biodiversity. By promoting habitat for sensitive or wide-ranging species, less exigent species may be protected in an umbrella effect. The greater sage-grouse (Centrocercus urophasianus; sage-grouse) has been proposed as an umbrella for other sagebrush-obligate species because it has an extensive range that overlaps with many other species, it is sensitive to anthropogenic activity, it requires resources over large landscapes, and its habitat needs are known. The efficacy of the umbrella concept, however, is often assumed and rarely tested. Therefore, we surveyed sage-grouse pellet occurrence and sagebrush-associated songbird abundance in northwest Colorado, USA, to determine the amount of habitat overlap between sage-grouse and 4 songbirds (Brewer's sparrow [Spizella breweri], sage thrasher [Oreoscoptes montanus], sagebrush sparrow [Artemisiospiza nevadensis]), and green-tailed towhee [Pipilo chlorurus]). During May and June 2013–2015, we conducted standard point count breeding surveys for songbirds and counted sage-grouse pellets within 300 10-m radius plots. We modeled songbird abundance and sage-grouse pellet occurrence with multi-scaled environmental features, such as sagebrush cover and bare ground. To evaluate sage-grouse as an umbrella for sagebrush-associated passerines, we determined the correlation between probability of sage-grouse pellet occurrence and model-predicted songbird densities per sampling plot. We then classified the sage-grouse probability of occurrence as high (probability >0.5) and low (probability ≤0.5) and mapped model-predicted surfaces for each species in our study area. We determined average songbird density in areas of high and low probability of sage-grouse occurrence. Sagebrush cover at intermediate scales was an important predictor for all species, and ground cover was important for all species except sage thrashers. Areas with a higher probability of sage-grouse occurrence also contained higher densities of Brewer's sparrows, green-tailed towhees, and sage thrashers, but predicted sagebrush sparrow densities were lower in these areas. In northwest Colorado, sage-grouse may be an effective umbrella for Brewer's sparrows, green-tailed towhees, and sage thrashers, but sage-grouse habitat does not appear to capture areas that support high sagebrush sparrow densities. A multi-species focus may be the best management and conservation strategy for several species of concern, especially those with conflicting habitat requirements. © The Wildlife Society, 2019