PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

新疆一号冰川土壤细菌多样性的研究

应用变性梯度凝胶电泳（DGGE）技术分离PCR扩增的16SrDNA来研究土壤微生物的多样性。直接从新疆一号冰川不同海拔高度的土壤样品中提取总DNA。用两套细菌通用引物分别扩增16SrDNA的V3和V6／V9高变区的特异性片段,PCR产物进行DGGE分析。PCR—DGGE图谱表明,PCR产物经DGGE检测到的条带清晰且分离效果好。结果表明,PCR—DGGE是一种快速研究微生物群落结构的有效方法。     

新疆一号冰川土壤细菌多样性的研究*

应用变性梯度凝胶电泳(DGGE)技术分离PCR扩增的16S rDNA来研究土壤微生物的多样性。直接从新疆一号冰川不同海拔高度的土壤样品中提取总DNA。用两套细菌通用引物分别扩增16S rDNA的V3和V6/V9高变区的特异性片段,PCR产物进行DGGE分析。PCR-DGGE图谱表明,PCR产物经DGGE检测到的条带清晰且分离效果好。结果表明,PCR-DGGE是一种快速研究微生物群落结构的有效方法。     

生物造粒流化床微生物群落结构及其动态变化

为了研究生物适粒流化床污水处理反应器颗粒污泥中微生物群落结构及其动态变化,分别从生物造粒流化床10、60、110 cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA.以细菌和古细菌16S rRNA基因通用引物530F/1490R,对活性污泥中提取的细菌基因组DNA进行PCR扩增,长约1 kb的PCR扩增产物纯化后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱.结果显示,生物造粒流化床反应器颗粒污泥中的微生物群落非常丰富,在10 cm处微生物的种属达到23种,60 cm处为21种,110 cm处为20种;生物造粒流化床不同高度都有一些各自的特有种属和共有种属,反应器不同高度的微生物群落演替不明显,微生物群落相似性为83.1%,群落结构较为稳定.     

生物造粒流化床微生物落结构及其动态变化

为了研究生物造粒流化床污水处理反应器颗粒污泥中微生物群落结构及其动态变化,分别从生物造粒流化床10、60、110cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA。以细菌和古细菌16S rRNA基因通用引物530F/1490R,对活性污泥中提取的细菌基因组DNA进行PCR扩增,长约1kb的PCR扩增产物纯化后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱。结果显示,生物造粒流化床反应器颗粒污泥中的微生物群落非常丰富,在10cm处微生物的种属达到23种,60cm处为21种,110cm处为20种;生物造粒流化床不同高度都有一些各自的特有种属和共有种属,反应器不同高度的微生物群落演替不明显,微生物群落相似性为83.1%,群落结构较为稳定。     

用于分析土壤微生物结构变化规律的变性梯度凝胶电泳条件研究

研究确定土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为进一步研究分析土壤中微生物结构变化规律提供理论依据。土壤微生物基因组DNA提取采用直接法和间接法进行比较; PCR扩增条件调整扩增体系、DGGE电泳条件调整变性剂范围,并对其结果进行比较分析。通过对DGGE电泳相关条件的研究,结果显示,土壤中粗基因组DNA采用直接法提取,然后进行纯化; PCR扩增体系中加入BSA,DGGE电泳系统组成中变性剂浓度范围为35%～55%。确定了土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为后续的相关研究提供理论依据。     

AM真菌DNA的提取与PCR-DGGE分析

分别从丛枝菌根(AM)真菌的单孢、宿主植物根系及土壤样品中提取DNA,对AM真菌的18SrDNA中NS31/Glol区进行Nested PCR特异性扩增,表明Nested PCR能很好地以微量DNA为模板扩增出目标产物;对扩增产物进行DGGE电泳,3种样品表现出不同的DGGE指纹图谱特征。本文认为,将Nested PCR与DGGE技术相结合,可以成为AM真菌分子生态学研究的有效途径。     

狮子头热泉菌席样品环境总DNA提取方法的比较研究

通过对狮子头热泉7个环境菌席样品所提取的总DNA进行纯度检测、提取得率计算和DGGE分析,比较了3种直接和1种间接DNA提取方法。结果表明：综合利用多种裂解方式比单一裂解方式更能充分释放环境DNA;其中3种方法获得的DNA片段能够进行后续16S rDNA扩增;针对同一样品,不同方法提取的环境DNA,可获得不同DGGE群落指纹图谱;间接提取法提取的总DNA,能更好地反映狮子头热泉菌席的微生物多样性。     

福建省稻田土壤细菌群落的16S rDNA-PCR-DGGE分析

用不依赖细菌培养的16S rDNA-PCR-DGGE方法对福建省6个不同地区12个取样点的稻田土壤进行细菌群落结构分析.对12份样品直接提取其总DNA,用F341GC/R534引物扩增16SrDNA基因的V3可变区,结合DGGE(denaturing gradient gel electrophoresis)技术分析样品细菌群落组成.结果表明,福建省不同地区的稻田土壤之间细菌群落结构存在较大差异.犬体上可分为闽东、闽南、闽北、闽西4个大类.同一地区的根际土和表土样品之间也存在差异,但差异相对较低,其中龙岩根际土和表土细菌群落结构相似性最大,永泰差异性最大.回收了DGGE图谱中11个条带,测序结果经过Blast比对表明其中10个条带代表的细菌是不可培养的,显示了DGGE技术的优越性.     

DNA提取对微生物多样性测序分析的影响

运用高通量测序技术分析复杂样品中微生物种群的变化情况,已经成为目前微生物研究领域的热点问题之一。而微生物的样品准备,如DNA提取和16S可变区的扩增等,对于测序完成后的数据分析以及微生物原始群落组成的影响是至关重要的。采用国产试剂盒(天根土壤微生物基因组提取试剂盒)和进口试剂盒(MOBIO土壤微生物基因组提取试剂盒)分别对土壤样品和羊瘤胃食糜样品进行DNA提取。然后选取总DNA起始量为25ng,对16S V3可变区进行PCR扩增和文库构建,最后通过数据分析比较不同试剂盒提取的DNA对微生物多样性变化的影响,包括OTU数目、稀释曲线、微生物数量及物种种类等。研究发现,在相同DNA模板量和PCR条件下,进口试剂盒提取的DNA能够获得更多的微生物种类。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.     

（鱼句）亚科花（鱼骨）型鱼类骨骼系统的比较

对我国花型Hemibarbuspattern鱼类作了骨骼系统比较,结果表明,此类型鱼类脑颅较长,副蝶骨平直或稍弯曲,眶蝶骨腹纵嵴发达(铜鱼Coreius septentrionalis例外),下颞窝和咽突中等大,基枕骨后突发达;脑颅中的上筛骨的后突、侧筛骨的外筛突,蝶耳骨的外突、上耳骨的后突、围眶骨和后颞窝等均有明显的差异;咽颅中的舌颌骨、尾舌骨、鳃盖骨和下咽齿的列数等又有显著的区别;附肢骨骼中的腰带骨、脊椎骨中的复合神经骨和第4椎骨腹侧的悬器等也有不同之处。据此,这些差异和区别可作为属间或种间的分类依据。