梨形四膜虫衰老过程中DNA含量的变化

应用一种新型的细胞核内DNA含量测定方法──图像分析法,测定真核细胞梨形四膜虫衰老过程中DNA含量的变化.根据Beer-Lambert定律,以细胞核在不同生长期内的积分光密度的水平表示核内DNA含量的变化.该方法具有测量速度快,重复性好,操作简单,结果可靠等优点.实验结果表明:四膜虫在进入对数生长期时,DNA含量逐渐达到高峰,随着细胞逐渐老化,细胞分裂次数及核内DNA含量逐步减少.     

Comparison of axonemal proteins from two kinds of Tetrahymena. I. Different characteristics of dyneins in heat stability

Tetrahymena thermophila could still swim after incubation of the cell body at 40°C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment. Turbidity measurements revealed that axonemes of T. pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T. thermophilia still had the activity under the same conditions. In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena. Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment. Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T. thermophilia is more stable in maintaining native configuration than that of T. pyriformis against the heat treatment, although both treated dyneins keep three (α, β and γ) subunits. Analysis by peptide mapping demonstrated that β- and γ-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species. These results imply that dynein of T. thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.     

两种类型栓皮栎软木细胞微观排列与形态特征

采用扫描电镜对秦岭北坡楼观台地区的厚皮、薄皮两种类型栓皮栎软木进行细胞微观构造观察分析,并与欧洲栓皮槠进行比较,以阐明厚皮、薄皮栓皮栎软木的相关特性,为中国栓皮栎软木的合理利用提供依据。结果表明:(1)两种类型栓皮栎软木细胞的排列结构较一致,均由内部中空的封闭型薄壁细胞紧密排列组成;在弦切面上呈蜂窝状排列,径切面和横切面上呈砖墙状排列;在径切面上,软木细胞侧高整齐地排列成行,且与树干轴向垂直;在横切面上,软木细胞侧高整齐地处于以树干轴为中心散发出来的射线上。(2)栓皮栎软木细胞大小、细胞壁和侧壁褶皱等受生长季节的影响;从软木细胞形态特征上看,厚皮类型软木细胞壁薄、细胞体积大,其软木质量优于薄皮类型。(3)与欧洲栓皮槠比较,发现厚皮类型栓皮栎早软木细胞棱柱高较小(20.6μm vs.(对比)30~40μm),软木细胞壁略厚(1.7μm vs.1~1.5μm),细胞实体积(细胞壁体积占细胞总体积比例)略大(18.75%vs.10%),厚皮类型栓皮栎软木比欧洲栓皮槠的软木质量差一些。(4)受树皮生长应力的影响,两种类型栓皮栎软木细胞侧高壁上多发生褶皱,早软木细胞褶皱严重,晚软木细胞没有褶皱,但在早晚软木交界或含有杂质处褶皱特别严重,表明厚皮类型软木细胞的侧壁褶皱程度高于薄皮类型。(5)对细胞形态特征及软木特性等的分析表明,薄皮类型栓皮栎软木质量比厚皮类型差,未来对软木资源的开发利用应更注重厚皮类型。     

Physiological, morphological and metabolic changes in Tetrahymena pyriformis for the in vivo cytotoxicity assessment of metallic pollution: Impact on d-β-hydroxybutyrate dehydrogenase

The individual cytotoxicity of cadmium chloride, iron sulphate and chromium nitrate has been investigated by using the freshwater ciliate Tetrahymena pyriformis. The metabolic enzymes and antioxidant defense biomarkers were assessed. The results obtained reveal that their metal salts have perturbed the physiology and morphology of T. pyriformis. Also, the biomarkers assessed were sensitive to the presence of metal salts and this sensitivity was metal salt and dose dependant. To estimate the impact of their metal salts on mitochondria, we studied their effects in vivo and in vitro on the d-β-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30) inner mitochondrial membrane enzyme. The results showed a high inhibition of BDH in terms of activity, protein expression and kinetic parameters.     

不结球白菜BcMPK4基因的分离及表达

为了进一步探讨植物MAPKs(mitogen-activated protein kinases)在植物防卫中的作用,该研究从不结球白菜抗病品种‘苏州青’中克隆到一个抗核盘菌(Sclerotinia sclerotiorum)相关基因,命名为BcMPK4(DDBJ登录号AB557751)。该基因核苷酸序列全长1 334bp,编码373个氨基酸,与已克隆的MPK4基因有不同程度的相似性。系统进化树分析表明,该基因在不同物种之间具有保守性。基因组DNA杂交表明,BcMPK4可能属于一个较小的多基因家族,属组成型表达。实时定量PCR检测表明,核盘菌能够诱导不结球白菜BcMPK4基因的转录表达;BcMPK4基因在不结球白菜叶片中的表达特征说明它可能参与寄主对核盘菌的抗性。     

Purification and properties of a membrane-bound L-asparaginase of Tetrahymena pyriformis

L-Asparaginase activity reaches maximal values at the stationary phase of growth of Tetrahymena pyriformis and fluctuates upon the growth conditions and the composition of the medium. Most of the L-asparaginase activity (80%) is associated with the endoplasmic reticulum, and the remaining with the pellicles. Detergents either alone or in combination with NaCl up to 0.5 M concentration failed to solubilize L-asparaginase. Solubilization can be accomplished by means of either the chaotropic agents KSCN and NaClO₄, or 0.1 M sodium phosphate buffer pH 8.0, following pretreatment of the particulates with 2% w/v Triton X₁₀₀. L-Asparaginase has been purified to near homogeneity by hydrophobic and gel filtration chromatography. The native enzyme has a relative molecular weight of 230000. It is a multiple subunit enzyme, with subunit size of 39000. Its isoelectric point is at pH 6.8. It acts optimally at pH 8.6 with a Km of 2.2 mM. It does not hydrolyse L-glutamine and its reaction is inhibited competitively by D-aspartic acid and D-asparagine as well as by Ir asparagine analogues with substituents at the 0 position.     

In vitro alterations of L-asparaginase activity of Tetrahymena pyriformis by lipids

A membrane-bound L-asparaginase (EC 3.5.1.1) of Tetrahymena pyriformis was purified to homogeneity. The purified enzyme is a lipoprotein, since it is inactivated by phospholipase C and its activity is restored by the addition of naturally occuring lipids, such as phosphatidylcholine, triolein and oleyl acetate. The relative effectiveness of a variety of phospholipids, free saturated and unsaturated fatty acids, or neutral lipids, such as esters of fatty, acids and glycerides, with respect to the activation of purified L-asparaginase is compared. Enzyme activity is reconstituted in the presence of lipids and evidence for the formation of an enzyme-phospholipid complex is presented. The data of this report suggest that L-asparaginase may have a requirement for lipids that reconstitute a physiological hydrophobic environment, similar to the one existing in vivo.Abbreviations DPPC Dipalmitoylphosphatidylcholine - DPPE Dipalmitoylphosphatidylethanolamine - DMPC Dimyristoylphosphatidylcholine - PS Phosphatidylserine - PI Phosphatidylinositol - IPC Lysophosphatidylcholine - PC Phosphatidylcholine - PE Phosphatidylethanolamine     

铜绿假单胞菌cntRLMN操纵子介导锌离子摄取的功能鉴定

【目的】本研究以铜绿假单胞菌PAO1 (Pseudomonas aeruginosa PAO1,菌种编号ATCC15692)为对象,研究cntRLMN在锌离子摄取中的功能。【方法】在ΔznuBC的基础上,以同源重组的方法构建了cntRLMN的各种突变菌株,通过质粒接合转移的方法构建其互补菌株及lacZ转录融合报告菌株,运用β-半乳糖苷酶酶活检测研究了Zur蛋白对cntRLMN的转录调控,凝胶阻滞实验(EMSA)检验Zur蛋白与cnt启动子及cnt启动子的突变片段的体外结合,并进一步通过生长曲线分析对cntRLMN中cntR、cntL、cntN等基因的锌离子摄取功能进行了分析和鉴定。最终,通过构建大蜡螟幼虫的侵染模型来研究cntRLMN对铜绿假单胞菌毒力发挥的影响。【结果】lacZ转录融合的酶活分析显示cntRLMN受Zur蛋白的负调控,其表达以Zur蛋白依赖的方式受锌离子饥饿的诱导;EMSA实验的结果显示cntRLMN的启动子可以与His-Zur结合形成DNA-蛋白质复合体,结合位点为GCGTTATAGTATATCAT;生长曲线和大蜡螟幼虫侵染实验的分析结果显示ZnuBC和CntRLMN的功能存在互补性,仅znuBC和cntRLMN双缺失突变时菌株在限锌培养条件下的生长和对大蜡螟幼虫的毒性才受到显著抑制,说明CntRLMN代表另一种独立的锌离子摄取系统。【结论】cntRLMN是受Zur直接负调控的另一种独立的铜绿假单胞菌锌离子摄取系统,对铜绿假单胞菌毒力的发挥起重要作用。     

皂荚提取物的杀螺活性

[目的]研究皂荚生物农药活性,开发利用皂荚资源,发展环境友好的绿色植物源农药。[方法]采用室内生测和田间试验研究皂荚壳乙醇提取物的杀螺活性。[结果]皂荚提取物对福寿螺有显著的毒杀活性,对幼螺和成螺72 h的LC₅₀分别为40.56、109.83 mg·L^-1。田间试验表明,皂荚提取物对福寿螺有较好的防效,施用40 g·m^-2的皂荚提取物处理7 d后卵块减少率为100.00%（成螺失去产卵的能力）,防效为（99.12±1.26）%。[结论]皂荚提取物对福寿螺较好的生物防治效果,是一种潜在的生物杀螺剂。     

致犊牛脑膜炎大肠杆菌新疆分离株ibeB基因的特性分析北大核心CSCD

【目的】分析致犊牛脑膜炎大肠杆菌分离株ibeB基因的分子生物学信息。【方法】以自脑炎死亡犊牛脑组织、肝组织分离鉴定的O161-K99-STa致病性大肠杆菌牛-EN株和牛-EG分离株为材料。根据GenBank中公布的脑膜炎大肠杆菌K1株RS218 ibeB基因序列设计1对引物,采用PCR方法,从分离株中成功克隆ibe B基因,比较分离株ibeB基因与不同来源大肠杆菌ibeB基因的部分生物信息学特性。【结果】分离株ibeB基因序列全长1500 bp,包含1371 bp开放阅读框,共编码457个氨基酸;生物信息学分析显示,牛-EN株与致人脑膜炎大肠杆菌K1 RS218的核苷酸和氨基酸同源性分别为90.5%和96.9%,牛-EG株与大肠杆菌K12的核苷酸和氨基酸同源性分别为99.4%和100.0%;ibeB蛋白为亲水性蛋白,分子质量为50.26 kDa,理论等电点为6.05;该蛋白无跨膜区,但具有信号肽序列;亚细胞定位显示,分泌信号通路位点(SP)占比例为0.939,说明该蛋白属于分泌型蛋白。【结论】从致脑膜炎大肠杆菌分离株中成功克隆ibeB基因,该基因与致人脑膜炎大肠杆菌K1 RS218 ibeB基因有较高的同源性,均有相似的生物学特性,属肠外致病性大肠杆菌。     

颗石藻Pleurochrysis carterae抗捕食特征

颗石藻Pleurochrysis carterae是沿海水域中常见钙化微藻,易形成高密度水华,也是养殖环境致害种之一。抗捕食防御能力可能是其种群增殖优势的一个重要原因。以卤虫作为捕食者,分析了颗石藻P.carterae抗捕食现象,以及在捕食压力下的重要生理生化响应特征,以期为颗石藻P.carterea抗捕食机制研究及其高密度增殖机理提供参考。研究结果显示:(1)当颗石藻P.carterae比例增加时,卤虫对微藻的摄食率显著降低,且存活率显著下降,显示该藻具抗捕食能力。(2)以卤虫饵料微藻球等鞭金藻(Isochrysis galbana)为对照,比较研究发现,相同的捕食压力下,饵料金藻的叶绿素荧光参数(电子传递速率ETR和最大量子产率Fv/Fm)显著降低,但颗石藻P.carterae的ETR和Fv/Fm没有显著变化,显示颗石藻P.carterae对卤虫抗捕食作用。(3)相对于没有捕食压力的对照组,捕食压力下,饵料金藻I.galbana的脂类组成没有显著差异。但是,颗石藻P.carterae的脂类组成则发生了显著变化,主要表现在对细胞叶绿体有重要作用的单半乳糖甘油二酯(MGDG),双半乳糖甘油二酯(DGDG),磷脂酰甘油二酯(PG)含量上升,与促细胞分裂相关的二酰甘油(DAG)和磷脂酰肌醇(PI)也上升。这些脂类代谢物的变化可能在其种群水平上抵抗捕食并实现种群增殖中发挥作用。(4)培养介质中磷的状态对颗石藻P.carterae细胞二甲基巯基丙酸(Dimethyl sulfonio propionate,DMSP)含量有显著影响,且影响颗石藻P.carterae对卤虫的致害效应:缺磷条件下生长的颗石藻P.carterae首先使卤虫受害。当培养液中仅以ATP为磷源时,颗石藻P.carterae的卤虫致害效应则降低。研究证明,颗石藻P.carterae具有抗捕食能力,细胞的脂类代谢物质以及DMSP可能在抗捕食防御中发挥作用。     

L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity

Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147–155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits. a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with -^32P ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg²⁺ and Ca²⁺ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.     

Molecular phylogeny of the Acre clade (Crassulaceae): Dealing with the lack of definitions for Echeveria and Sedum

The phylogenetic relationships within many clades of the Crassulaceae are still uncertain, therefore in this study attention was focused on the “Acre clade”, a group comprised of approximately 526 species in eight genera that include many Asian and Mediterranean species of Sedum and the majority of the American genera (Echeveria, Graptopetalum, Lenophyllum, Pachyphytum, Villadia, and Thompsonella). Parsimony and Bayesian analyses were conducted with 133 species based on nuclear (ETS, ITS) and chloroplast DNA regions (rpS16, matK). Our analyses retrieved four major clades within the Acre clade. Two of these were in a grade and corresponded to Asian species of Sedum, the rest corresponded to a European–Macaronesian group and to an American group. The American group included all taxa that were formerly placed in the Echeverioideae and the majority of the American Sedoideae. Our analyses support the monophyly of three genera – Lenophyllum, Thompsonella, and Pachyphytum; however, the relationships among Echeveria, Sedum and the various segregates of Sedum are largely unresolved. Our analyses represents the first broad phylogenetic framework for Acre clade, but further studies are necessary on the groups poorly represented here, such as the European and Asian species of Sedum and the Central and South American species of Echeveria.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Functional expression of Aquaspirillum magnetotacticum genes in Escherichia coli K12

Summary Gene libraries from the magnetotactic bacterium, Aquaspirillum magnetotacticum were constructed in Escherichia coli with cosmids pLAFR3 and c2RB as vectors. Recombinant cosmids able to complement the thr-1, leuB, and proA mutations of the host were identified. The Pro⁺ recombinant cosmid restored wild-type phenotype in proA and proB but not in the proC mutants of E. coli. The results of restriction endonuclease digestion and Southern hybridization analysis indicate that the relevent leu and pro biosynthetic genes of A. magnetotacticum are not closely linked on the chromosome.     

ConcanavalinA receptors and the chemosensory behaviour of Tetrahymena thermophila

The relationship between concanavalin A (ConA) receptors and the chemosensory behaviour of the ciliated protozoan Tetrahymena thermophila was studied using the peptide chemoattractants proteose peptone and fibroblast growth factor. Studies on the chemosensory behaviour in semisolid methylcellulose showed that 50 μg/ml ConA selectively inhibited the persistent element of swimming behaviour by reducing time runs of cells responding to proteose peptone from 12.2±4.5 min to 0.8±0.3 min. Methyl-alpha-D-mannoside, but not methyl-alpha-D-galactoside, abolished the inhibitory effect of ConA, suggesting that mannoside-containing ConA receptors are involved in maintaining a persistent swimming behaviour. Control experiments, carried out in liquids where persistent swimming is less important for cellular behaviour, showed that ConA did not affect proteose-peptone-induced chemoattraction under these conditions as measured by a two-phase assay for chemoattraction. Also, no inhibitory effect of ConA could be found on swimming rates when individual velocities of ConA-treated cells were determined. When tested in liquid chemoattraction assays, ConA was found to be a weak but significant chemoattractant. Studies of the cellular location of ConA receptors on the plasma membrane of starved cells showed an unequal distribution. A preferential clustering of receptors at the anterior end of the cell was observed when determined at high concentrations (100 μg/ml) of fluorescent ConA. Methyl-alpha-D-mannoside but not methyl-alpha-D-galactoside abolished the fluorescent ConA labelling, indicating a preferential clustering of these mannoside-containing receptors at the anterior part of the plasma membrane and cilia. At lower concentrations (25 μg/ml), FITC-ConA produced more general labelling of the entire cell membrane. The results suggest that ConA receptors are necessary for the persistent element of swimming and that binding of ConA to its receptors interferes with processes related to signal transduction rather than by limiting the free movement of cilia required for locomotion. The gradient of receptors seen at high FITC-ConA concentrations may be important for a putative spatial chemosensory mechanism, i.e. chemotaxis.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.     

毛白杨PtoGSTF4基因克隆及相关特性鉴定

该研究从毛白杨中克隆到1个Phi类谷胱苷肽S-转移酶（GST）基因（PtoGSTF4）,编码213个氨基酸。表达模式分析发现,PtoGSTF4在正常生长、H₂O₂和莠去津处理后的茎、叶以及茎的韧皮部均表达,属于组成型表达基因。在大肠杆菌中表达并纯化了PtoGSTF4重组蛋白,酶学性质分析表明PtoGSTF4对CDNB、NBD-Cl、NBC和Cum-OOH等4种底物均有活性。动力学分析发现,PtoGSTF4对GSH具有较高的亲和力,而对CDNB的亲和力相对较低。在不同pH及温度条件下对PtoGSTF4蛋白进行活性检测,发现PtoGSTF4在pH 7.5~10.5范围内或30 ℃~60 ℃温度范围内有较高的活性。研究推测,PtoGSTF4可能在毛白杨的抗逆生理中发挥重要作用。     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。