小麦随机扩增多态性DNA的反应体系探索

随机扩增多态性DNA(RAPD)是近年来发展起来的一种DNA多态性检测技术。通过以小麦基因组DNA为模板 ,对RAPD反应程序中的一些重要参数进行摸索和优化试验 ,建立了随机扩增多态性DNA分析应用反应体系。即反应体积为 2 5μ1,Mg2 浓度为 2 0mM ,dNTP浓度为 150 μM ,模板DNA量 5ng～2 5ng ,随机引物量 30ng～ 4 5ng ,Tap酶 1单位 ;反应过程为 90℃变性 30s ,38℃结合 1min ,72℃延伸 2min(最后一个循环 10min) ,共 4 0个循环。该体系可有效地应用在小麦遗传育种研究中 ,重复性好 ,可靠性高。     

番茄随机扩增DNA多态性体系的条件优化

利用改进的SDS法提取代号为03748的栽培番茄叶片基因组DNA。对影响番茄随机扩增DNA多态性（RAPD）扩增结果的因素进行了分析,确定了模板、Mg^2＋、dNTPs、引物和Tap DNA聚合酶的适宜浓度及反应的最佳循环次数。实验结果表明,在以下条件下,番茄的RAPD扩增效果较好：20μL反应体系中使用20-40ng的模板、1．5-2．0mmol／L的Mg^2＋、0．15＋0．20μmol／L的dNTPs、0．15-2．0μmol／L的引物、1．0U的Taq DNA聚合酶;94℃预变性5min,然后经94℃变性1min、360℃ 1min、720℃ 1．5min,进行35个循环,最后在72℃时再延伸10min。     

正交试验优化八角ISSR-PCR反应体系

利用正交试验设计研究Taq DNA聚合酶、DNA模板、引物(UBC 886)、dNTP、Mg2+浓度5个因素对云南八角ISSR-PCR反应的影响,建立其最佳反应体系。结果表明:25μL的反应体系中5个因子的最佳水平为:Mg2+3 mmol/L、Taq DNA聚合酶0.5 U、DNA模板0.016 ng、引物0.8μmol/L、dNTPs 0.2 mmol/L。PCR最佳反应程序为:94℃预变性5 min;94℃变性30 s,46℃退火45 s,72℃延伸1 min,40次循环;72℃最后延伸7 min,4℃保存。     

鲤TRAP分子标记反应体系的建立与优化

针对鲤(Cyprinus carpio)这一主要水产养殖品种设计了靶位区域扩增多态性(target region amplified polymorphism,TRAP)分子标记的反应体系,对影响TRAP反应体系的各参数,包括Mg2+、dNTPs、TaqDNA聚合酶、模板DNA和引物浓度进行了优化,建立了适合鲤的、稳定、可重复的TRAP-PCR反应体系。在15μlPCR反应体系中,Mg2+浓度为1.5mmol/L、dNTPs浓度为0.35mmol/L、两个随机引物浓度均为3pmol/L、固定引物浓度为10pmol/L、含DNA模板60ng、TaqDNA聚合酶1.0U。鲤的TRAP反应程序为:94℃4min,1个循环;94℃45s,35℃45s,72℃1min,5个循环;94℃45s,53℃45s,72℃1min,35个循环;72℃10min,1个循环。这一优化体系的建立为今后进行鲤群体遗传多样性、种质鉴定、遗传连锁图谱及亲缘关系分析等方面的研究提供了新的分子标记。     

均匀设计优化建兰ISSR-PCR体系

采用均匀设计,对影响建兰ISSR-PCR体系的引物、Mg^2＋、dNTP和Taq DNA聚合酶浓度等进行4因素5水平和4因素3水平两轮优化,建立了适合于建兰ISSR-PCR的反应体系：在20μL反应体系中,舍引物0．25μmol／L、Mg^2＋ 2．5mmol／L,dNTP0．2mmol／L、Taq DNA聚合酶1.5U和模板DNA40ng．在此基础上对扩增程序中的循环次数和退火温度,以及ISSR引物进行筛选．筛选获得的扩增程序为：94℃预变性5min;接着进行32个循环：94℃变性35S,52～56℃退火45S,72℃延伸90s;循环结束后,72℃延伸10min．同时筛选得到14个扩增稳定、多态性丰富的ISSR引物．     

苹果黑星病菌SSR反应体系的优化

通过对苹果黑星病菌基因组DNA的SSR反应中一些重要参数进行优化,结果表明,最适反应体系为:2 5μL体系中,10×Buffer Mg Cl2 2 0 mm ol/ L 2 .5μL ,d NTP 10 0μmol·L- 1 、引物0 .5μmol·L- 1 、Taq DNA聚合酶1.5 U ,DNA模板2 m g·L- 1 ,dd H2 O 19.5μL .PCR扩增程序为93℃,2 min,5 7℃,30 s,1个循环;72℃,1m in,93℃,30 s,5 7℃,30 s,4 0个循环;72℃,10 min.     

濒危药用植物厚朴ISSR引物筛选及反应条件的优化

以厚朴DNA为模板,利用正交试验分别对影响厚朴ISSR-PCR反应的Taq酶浓度、dNTP浓度、引物浓度、Mg~(2+)浓度、模板DNA浓度进行了优化,并通过梯度PCR确定不同引物的最佳退火温度和循环次数,最终确定厚朴最佳反应体系及扩增条件为:25μl 体系,其中包括1.5 mmol·L~(-1) MgCl_2,0.3 μmol·L~(-1)引物,0.04 U·μl~(-1)Taq 酶,0.2 mmol·L~(-1) dNTP,4 ng·μl~(-1)模板DNA,1 × Buffer;扩增程序:94℃预变性5 min,94℃变性30 s,50℃～60℃(退火温度随引物不同而定)退火45 s,72℃延伸90 s,共40个循环,然后72℃延伸8 min,4℃终止反应.此外,还利用优化的反应体系成功筛选出21条ISSR引物,并利用部分引物对厚朴个体进行了遗传多样性分析.     

西伯利亚蝗基因组DNA提取及RAPD分析条件的优化

以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、TaqDNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25μLPCR反应体系,10×buffer2·5μL;dNTP0·24mmol/L;MgCl22·0mmol/L;Taq DNA聚合酶1U;DNA模板45ng;引物30ng。扩增程序为:94℃预变性1min45s、94℃变性30s、35℃退火1min30s、72℃延伸2min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。     

葡萄ISSR-PCR反应体系的建立与优化

采用正交设计L9(34)对影响葡萄ISSR-PCR反应体系的4个因素(dNTP、TaqDNA聚合酶、引物、模板DNA)在3个浓度水平上进行试验,并通过直观分析初步确定其反应体系;在此基础上,通过单因素试验探讨了dNTP、TaqDNA聚合酶、引物、模板DNA、退火温度及循环次数等因素或条件对葡萄ISSR-PCR扩增结果的影响,确定最佳反应水平。最终建立了葡萄ISSR-PCR扩增的最佳反应体系:在25μL的反应体系中,dNTP浓度0.2 mmol/L,TaqDNA聚合酶的用量0.5 U,引物浓度0.4mmol/L,DNA模板用量40 ng。反应程序:94℃预变性5 min;94℃变性1 min,52℃退火1 min,72℃延伸1 min 30 s,40次循环;最后72℃延伸10 min,10℃保存。     

高质量人体粪便细菌16S rDNA V3区扩增方法

[目的]为获得应用于二代测序的高质量16S r DNA V3区PCR产物。[方法]以从人体粪便中提取微生物总DNA为模板,通过梯度PCR和touchdown PCR技术确定了循环条件,并通过调整反应体系中的相关浓度参数以使扩增结果得到优化。[结果]最终确定的循环条件为预变性95℃3 min,变性95℃30 s,退火69℃~62℃每个循环退火温度降0.5℃,退火时间30 s,延伸72℃60 s,共15个循环;变性95℃30 s,退火62℃30 s,延伸72℃60 s,共15个循环,最后72℃延伸5 min。反应体系为:在50μL体系中DNA模板量10~25 ng,pfu酶0.25~0.5 U,正反向引物均为0.06~0.1μmol/L,d NTPs浓度0.2~0.4 mmol/L,Mg2+浓度2~2.5 mmol/L。[结论]梯度PCR与touchdown PCR相结合可快速确定最佳的退火温度以及循环条件,通过调整反应体系中浓度参数可以解决扩增中一些问题。     

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.     

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.     

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.     

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.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

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.