一种高效可直接用于PCR分析的土壤总微生物DNA抽提方法

以CTAB-溶菌酶-蛋白酶K-冻融裂解法直接抽提土壤总微生物的基因组DNA,利用G8000沉淀和纯化DNA.结果表明,该方法是一种简便、有效可直接应用于PCR分析的土壤总微生物基因组DNA的抽提方法.采用含聚乙烯吡咯烷酮(PVP)的缓冲液预洗,添加CaCl₂和BSA,可以去除腐殖酸;用PEG8000沉淀DNA,可以提高DNA质量;采用冻融法破碎细胞,CTAB、溶菌酶和蛋白质酶K共同作用以裂解细胞,可以保证获得大片段的DNA,提高DNA产率.用该方法抽提的七子花林下土壤总微生物DNA产率为9.22 μg·g^-1,A260/A280为1.65,可适用于 PCR扩增及扩增rDNA限制酶切分析(ARDRA)技术,适宜的模板DNA浓度为0.67 ng·μl^-1.快速、有效、可直接用于PCR分析的土壤总微生物DNA提取方法的建立,为大规模的土壤微生物分子生态学研究提供了可能.     

环氧丙烷皂化废水活性污泥DNA提取方法的比较与优化

探索适宜的环氧丙烷废水中活性污泥微生物总DNA提取的方法并进行优化。采用11种方法提取环氧丙烷废水中活性污泥微生物总DNA,即Na Cl溶液-提取缓冲液-溶菌酶-十二烷基磺酸钠(SDS)法、蛋白酶K-SDS法、SDS-反复冻融法、Tris-EDTA-Na Cl-聚乙烯吡咯烷酮(PVP)(TENP)法和反复冻融-SDS-蛋白酶K法等。通过对这11种方法进行比较,以确定最佳试验方案并结合单因素法对最佳方案进行优化。琼脂糖凝胶电泳结果显示:Na Cl溶液-提取缓冲液-溶菌酶-SDS法提取DNA亮度高,有大片断DNA的存在且拖带较少,条带集中;酶法所提取的DNA效果仅次于提取缓冲液-溶菌酶-SDS法,其他方案没有提取出DNA。单因子试验结果表明:10 mg/m L溶菌酶,100g/LSDS,反应时间为30 min,反应温度为45℃时效果最佳。Na Cl溶液-提取缓冲液-溶菌酶-SDS提取法为环氧丙烷废水中微生物群落在分子水平上的研究提供了一种简便、可靠的DNA提取方法。     

四种土壤微生物总DNA的纯化方法的比较

比较了4种从土壤中直接抽提的微生物总DNA的纯化方法,实验结果表明1 %的琼脂糖凝胶电泳纯化方法及葡聚糖凝胶G 2 0 0离心层析纯化方法均不能完全纯化从土壤中抽提的微生物总DNA。若将直接抽提的总DNA先经葡聚糖凝胶G 2 0 0离心层析纯化,再用1 %的琼脂糖凝胶电泳纯化,则能取得较好的纯化效果。含2 %PVP的1 %琼脂糖凝胶电泳纯化,用DNA凝胶回收试剂盒回收后没有得到纯化后的土壤微生物总DNA。     

油气田土壤DNA提取方法及油气指示菌基因定量结果的比较

采用4种提取方法对油田上方6个土壤样品微生物总基因组DNA进行提取,并比较了其纯度和浓度。利用定量PCR技术定量分析各土壤中甲烷单加氧酶基因（pmoA）和丁烷单加氧酶基因（brnoX）。与DNA试剂盒法相比,玻璃珠击打法、液氮研磨法、反复冻融法得到DNA纯度较低,需纯化才能进行后续的PCR扩增。液氮研磨法得到的DNA完整性、纯度和得率较其他提取方法均较好,尤其对于生物量较少的深层油田土壤DNA提取较为实用,成本较低。甲烷单加氧酶基因（pmoA）和丁烷单加氧酶基因（bmoX）的定量PCR结果表明,液氮研磨法提取DNA样品的定量结果在气区和油区均出现一定的高值。液氮研磨法较其他方法更适合于油田土壤DNA的提取。下一步研究可以把丁烷氧化茵作为油气指示茵研究中的重点检测对象。     

茶园土壤微生物总DNA不同提取方法的比较

传统的微生物分离培养方法,在反映茶园土壤微生物基因信息上有很大的局限性,因此,目前逐步被分子生态学的方法替代,而获得高质量、大片段、无偏好的土壤微生物总DNA则是茶园土壤微生物分子生态学研究的基础.本文采用SDS高盐法、变性剂加SDS高盐法、脱腐SDS高盐法、CTAB法和Krsek改进法5种土壤微生物DNA提取方法分别从茶园土壤微生物中提取总DNA,并对5种方法提取的DNA的片段大小、质量和产量进行了综合评价.结果表明,Krsek改进法提取到的DNA片段最大(>23 kb)、纯度最高(OD260/OD280>1.70;OD260/OD230>1.35)、产量较高(>34.50μg/g dry soil)且不需纯化就可以用于PCR扩增和RFLP分析.因此,Krsek改进法是一种高效、可靠且适合于茶园土壤微生物分子生态学研究的DNA提取方法.     

改进的SDS-CTAB法提取濒危植物连香树总DNA

对珍稀濒危植物连香树(Cercidiphyllum japonicum)的6种总DNA提取方法进行了对比试验,结果表明改进的SDS-CTAB法更适合于连香树总DNA提取。该方法提取的DNA经紫外消光值检测,其A260/A280为1.8532,优于CTAB法(1.4872)、SDS法(1.3552)、PVP法(1.5079)、尿素法(1.1858)和高盐低pH法(1.4534)。琼脂糖凝胶电泳和PCR扩增结果也得出同样的结论。     

新生隐球菌总RNA抽提方法的实验研究

目的探讨快速获取高质量的新生隐球菌总RNA的实验方法。方法选取新生隐球菌的荚膜株、荚膜缺陷株,分别设计采用4种方法提取总RNA:酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法。用紫外线分光光度计测量其OD260、OD280的值,并且进行琼脂糖凝胶电泳,同时应用定量PCR法鉴定RNA质量。结果酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法的RNA产量分别为0.2μg/105细胞、0.4μg/105细胞、0.1μg/105细胞、0.6μg/105细胞。结论冷酸洗玻璃珠联合Yeast RNA kit法提取的RNA均一性和完整性最好,是简便、快捷地提取具有荚膜和细胞壁双重屏障的新生隐球菌RNA的理想方法。     

东北设施黑土土壤微生物总DNA提取方法探讨

目的:找到一种适合东北设施高腐殖含量黑土土壤微生物总DNA简单易行的提取方法。方法:采用5种不同的DNA提取方法进行土壤总DNA提取。结果:5种提取方法的DNA产量在6.88～29.71μg/g,OD260/OD280值为1.03～1.27,OD260/OD230值为0.65～0.88,经纯化后均可进行PCR扩增。但经改进的方法 E提取DNA产量最高,纯度最好。结论:方法 E—加入TENP和PBS缓冲液预处理的提取方法是一种适宜东北设施黑土土壤微生物总DNA批量提取简单易行的方法。     

三种提取苹果绵蚜基因组DNA方法的比较

参考国内外昆虫基因组DNA提取的常用方法,选取KAc法、氯仿-异戊醇法和盐析法3种方法对苹果绵蚜(Eriosoma lanigerum)基因组DNA进行提取.通过基因组DNA直接琼脂糖凝胶电泳、SSR引物扩增产物的琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳检测,对3种方法所提基因组DNA的质量进行了比较,并综合分析了提取所需时间及所用试剂的毒性大小等.结果表明,盐析法操作程序较简便快捷,节时省工,可得较好质量的DNA样本,且对试验操作人员无伤害,是一种值得推广应用的基因组DNA提取方法.     

组培枣苗基因组DNA提取方法及其含量的比较

本文以组培二倍体木枣、四倍体变异苗和二倍体京枣苗为供试材料,采用琼脂糖凝胶电泳和紫外分光光度检测技术,研究了不同提取方法对其叶片基因组DNA的提取效果.结果表明,CTAB法提取枣叶片DNA的效果优于SDS法,表现为蛋白质杂质干扰少,DNA结构完整、纯度与得率高;用不同方法提取的样液中DNA片段的大小和分布是不同的,以改进的CTAB法提取的样液中大片段的DNA分子较多,所以在其底部分布较多,而用SDS法提取的样液中小片段的DNA分子较多,故在其上部分布较多;电泳时不同的加样量获得的DNA带也有较大差异,试验选出CTAB法提取样液的适宜的加样量为5 μL (7.485 μg ),SDS法提取样液的适宜的加样量为4 μL (5.988 μg);枣品种间DNA的含量明显不同,木枣四倍体的DNA含量(3.074 μg/μL)相当于组培木枣二倍体(1.497 μg/μL)的2倍,也明显地高于京枣二倍体(2.182 μg/μL).这些结果为枣树遗传种质资源的研究奠定了一定的科学与技术基础.     

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.