直接从土壤中提取DNA的方法

研究微生物的多样性 ,即微生物的种类和数量的多少是评价土壤质量的重要指标。由于土壤微生物种类繁多 ,数量巨大 ,加上土壤中 99%的种类难以通过传统的平板分离技术来进行培养[1],人们必须借助其他技术来解决。近年发展的非培养技术 ,如ＢＩＯＬＯＧ微量板分析技术[2 ]、细胞壁磷脂酸分析技术[3]和分子生物学方法[4 - 6 ],克服了培养的环节 ,对微生物生态学研究产生极大的推动作用。其中分子生物学是应用最广、最有发展潜力的技术。它的主要步骤是通过直接提取土壤中的ＤＮＡ ,经纯化处理后 ,利用合适的引物扩增 1 6ＳｒＲＮＡ基因 ,通过分…     

土壤微生物生态学研究中的非培养方法

土壤微生物种类和数量是评价土壤健康质量的重要指标之一。然而环境中90%以上的微生物不能够通过传统的培养基培养方法获得。最近发展起来的分析方法如磷脂脂肪酸(PLFA)、BIOLOG微孔板和分子生物学的方法可从不同方面对土壤微生物群落进行更为详尽的分析。论文就土壤微生物生态学研究中使用的主要方法进行了综述。     

转基因作物对土壤微生物多样性影响的研究方法

土壤微生物是土壤生态系统的一个重要组成部分,对土壤中的生物化学循环起着不可替代的驱动作用。转基因作物在生长过程中会不可避免地与土壤微生物发生交流,开展转基因作物对土壤微生物群落影响的研究对于科学评价转基因作物的潜在风险具有重要意义。随着现代生物技术的不断发展,土壤微生物多样性及其分析方法已经从传统的分离培养发展到从种群角度去研究整个土壤微生态系统内的微生物。但是由于土壤微生物的各种特性(如大部分不可培养、体积微小及群体效应等)和仪器设备检测性能的局限性,单一的研究方法会存在一些弊端,还需要结合其他的手段共同研究土壤生态系统中的微生物多样性。目前,人们对土壤微生物多样性的研究主要包括物种多样性、功能多样性、结构多样性及遗传多样性等4个方面,国内外关于转基因作物对土壤微生物多样性及其群落结构影响的研究方法很多,对常见的研究方法进行了总结,并提出了今后转基因作物对土壤微生物多样性及群落结构影响的研究策略。     

DNA指纹图谱技术在土壤微生物多样性研究中的应用

土壤中的微生物多样性是十分丰富的,传统培养方法对土壤微生物多样性的研究有很大局限性。近年来,各种基于16S rDNA基因的指纹图谱分析技术取得了长足的进步,并广泛应用于土壤微生物多样性的研究。这些技术主要有变性梯度凝胶电泳(DGGE)/温度梯度凝胶电泳(TGGE)、单链构象多态性(SSCP)、随机引物扩增多态性DNA(RAPD)、限制性片段长度多态性(RFLP)和扩增核糖体DNA限制性分析(ARDRA)等。对这些技术近年来在土壤微生物多样性研究领域的应用予以简短综述,并初步探讨未来几年土壤微生物分子生态学发展的方向。     

污染土壤微生物群落结构多样性及功能多样性测定方法

土壤微生物在促进土壤质量和植物健康方面发挥着重要的作用,土壤微生物群落结构和组成的多样性及其变化在一定程度上反映了土壤质量.为了更好地了解土壤健康状况,非常有必要发展有效的方法来研究污染土壤微生物的多样性、分布以及行为等.回顾了近年来国内外污染土壤微生物群落结构多样性及功能多样性的测定方法,包括生物化学技术和分子生物学技术,现将它们的原理、优缺点、实用性及其发展动态作一阐述,同时指出结合这两种技术可为微生物群落分析提供一个更全面的、精确的方法.     

极端干旱区防护林地土壤微生物多样性

土壤微生物在森林土壤中有重要功能.采用传统培养、脂肪酸鉴定和PCR-DGGE 3种方法分别研究了塔里木沙漠公路防护林地土壤微生物数量、脂肪酸种类和细菌DNA片段的多样性,从微生物学角度为塔里木沙漠公路防护林的管理提供理论依据.结果表明,塔里木沙漠公路防护林的建设促进了风沙土土壤微生物的发育,随着防护林定植年限的增加,土壤微生物数量、脂肪酸和细菌DNA片段的多样性指数明显增大;土壤微生物区系组成中,细菌是优势类群,占微生物总数80%以上,而真菌很少,不到微生物总数1%,但在不同土层间有所差异;传统培养法与现代生物标记和分子生物学方法对塔里木沙漠公路防护林地土壤微生物多样性的研究结果基本一致,说明塔里木沙漠公路防护林的建设使林地土壤生物活性有所增强,有利于林地土壤养分循环与利用.     

土壤动物肠道微生物多样性研究进展

随着分子生物学技术方法的快速发展,动物肠道微生物已成为医学、动物生理学与微生物生态学等研究领域热点。土壤动物种类繁多,分布广泛,其作为陆地生态系统重要组分,是驱动生态系统功能的关键因子。土壤动物体内的微生物由于与宿主长期共存,在与宿主协同进化中形成了丰富多样的群落结构,能够影响土壤动物本身的健康,进而介导土壤动物生态功能的实现。近些年,土壤动物肠道微生物工作方兴未艾,日渐得到重视。总结了四个部分内容：1)首先总结了土壤动物肠道微生物多样性领域的研究现状,该领域年发文量逐年增长,且近十年增长快速。土壤模式生物肠道微生物多样性研究较多且更为深入。土壤动物肠道微生物多样性组成与驱动机制、共存机制及群落构建的理论研究是该领域前沿;2)进而展示了土壤动物肠道微生物多样性组成和研究方法,土壤动物肠道菌群组成以变形菌门、厚壁菌门、放线菌门和拟杆菌门为主。早期工作基于传统分离培养,近年来新一代测序技术推动了该领域发展;3)接着关注了土壤动物肠道微生物的生态学功能,总体上体现在肠道微生物能帮助宿主分解食物基质、参与营养利用、影响寿命和繁殖及提高宿主免疫能力,且其能够影响土壤动物的气体排放及介导其对生态系...     

植被对土壤微生物群落结构的影响

研究了不同土壤及覆盖其上的植被与土壤微生物群落结构和多样性的关系．植被使土壤中的微生物种类更丰富,群落多样性更高．表层土壤微生物群落中没有明显的优势种群,种间竞争作用较弱．并介绍了研究土壤微生物群落的分子生物学方法．     

用SRAP标记研究根际土壤微生物的遗传多样性

将近年来新建立的分子标记技术——相关序列扩增多态性(sequence-related amplified polymorphism,SRAP)应用于土壤微生物遗传多样性研究.采用22对引物组合对20种植物根际土壤微生物进行了分析,共获得237个扩增位点,其中多态性位点221个,占93.2％.平均每对引物组合的多态位点比...     

污染土壤微生物群落结构分子鉴定技术研究进展

土壤微生物群落结构多样性检测是土壤修复、监测、评估时的一个重要参数。由于绝大多数微生物在实验室条件下是不可培养,因而早期依赖于微生物培养的检测结果代表性不强。20世纪90年代以来,不依赖于微生物培养的分子生物学方法是研究微生物群落结构的重要手段。该文对近年来土壤污染微生物群落结构研究所采用的主要分子生物学方法按照其原理进行了比较、分析、总结。根据不同技术的灵敏度、优缺点分析了其适用范围。指出了目前技术中存在的一些共性问题和缺陷并展望了土壤修复领域分子生物学技术的发展趋势。     

Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices

Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.     

Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.     

Characterization of microbial communities found in the human vagina by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.     

Community structure analyses are more sensitive to differences in soil bacterial communities than anonymous diversity indices

Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.     

Development of a fluorophore-ribosomal DNA restriction typing method for monitoring structural shifts of microbial communities

DNA restriction fragment polymorphism technologies such as amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) have been widely used in investigating microbial community structures. However, these methods are limited due to either the low resolution or sensitivity. In this study, a fluorophore-ribosomal DNA restriction typing (f-DRT) approach is developed for structural profiling of microbial communities. 16S rRNA genes are amplified from the community DNA and digested by a single restriction enzyme Msp I. All restriction fragments are end-labeled with a fluorescent nucleotide Cy5-dCTP via a one-step extension reaction and detected with an automated DNA sequencer. All 50 predicted restriction fragments between 100 and 600 bp were detected when twelve single 16S rRNA gene sequences were analyzed using f-DRT approach; 92% of these fragments were determined with accuracy of ±2 bp. In the defined model communities containing five components with different ratios, relative abundance of each component was correctly revealed by this method. The f-DRT analysis also showed structural shifts of intestinal microbiota in carcinogen-treated rats during the formation of precancerous lesions in the colon, as sensitive as multiple digestion-based T-RFLP analysis. This study provides a labor and cost-saving new method for monitoring structural shifts of microbial communities.     

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Soil microbial community analysis using two-dimensional polyacrylamide gel electrophoresis of the bacterial ribosomal internal transcribed spacer regions

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of digested genomic DNA has been previously used in comparative genomics studies of closely related bacteria species. However, a two-dimensional gel electrophoresis approach for examining microbial community structures in environmental samples has not yet been developed. We determined that it is theoretically possible to separate internal transcribed spacer regions (ITS) of bacterial communities into hundreds of operational taxonomic units (OTUs) using 2D-PAGE. Application of 2D-PAGE for separating Bacterial ITS sequences that have been PCR-amplified from replicate soil samples taken from along a Zn gradient resulted in reproducible gels containing hundreds of spots. Clear differences in spot patterns were observed between soil samples that differed in both sampling location and Zn content. The number of OTUs detected using 2D-PAGE of ITS regions was much greater than that observed using Automated Ribosomal Internal Transcribed Spacer Analysis (ARISA), Terminal Restriction Fragment Length Polymorphism (T-RFLP), or Denaturing Gradient Gel Electrophoresis (DGGE). Principal Component Analysis (PCA) of community spot patterns resulted in similar groupings of samples as those obtained using other molecular methods, however, excised spots were found to contain a far lower diversity of different sequences than excised ITS bands of the same length, as determined by RFLP analysis of excision clone libraries and subsequent sequencing of DNA eluted from excised spots. This increase in resolution makes 2D-PAGE of Bacteria ITS fragments from complex microbial communities a viable method for detecting differences between highly similar communities, as well as in streamlining follow-on sequencing efforts by reducing the level of homoplasy (co-migration of heterogeneous sequences) often seen in band-based community fingerprinting methods.     

内蒙古草原典型植物对土壤微生物群落的影响

为了分析内蒙古草原不同植物物种对土壤微生物群落的影响, 采用实时荧光定量PCR (real-time PCR)以及末端限制性片段长度多态性分析(terminal restriction fragment length polymorphism, T-RFLP)等分子生物学技术, 测定了退化-恢复样地上几种典型植物的根际土壤和非根际土壤中细菌和真菌的数量及群落结构。结果表明, 不同植物物种对根际和非根际细菌及根际真菌数量均有显著影响。根际土壤中的细菌和真菌数量普遍高于非根际土壤, 尤其以真菌更为明显。对T-RFLP数据进行多响应置换过程(multi-response permutation procedures, MRPP)分析和主成分分析(principal component analysis, PCA), 结果表明, 大多数物种的根际细菌及真菌的群落结构与非根际有明显差异, 并且所有物种的真菌群落可以按根际和非根际明显分为两大类群。此外, 细菌和真菌群落结构在一定程度上存在按物种聚类的现象, 以细菌较为明显。这些结果揭示了不同植物对土壤微生物群落的影响特征, 对理解内蒙古草原地区退化及恢复过程中植被演替引起的土壤性质和功能的变化有一定的帮助。     

Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Analysis of Soil Whole- and Inner-Microaggregate Bacterial Communities

Although soil structure largely determines energy flows and the distribution and composition of soil microhabitats, little is known about how microbial community composition is influenced by soil structural characteristics and organic matter compartmentalization dynamics. A UV irradiation-based procedure was developed to specifically isolate inner-microaggregate microbial communities, thus providing the means to analyze these communities in relation to their environment. Whole- and inner-microaggregate fractions of undisturbed soil and soils reclaimed after disturbance by surface coal mining were analyzed using 16S rDNA terminal restriction fragment polymorphism (T-RFLP) and sequence analyses to determine salient bacterial community structural characteristics. We hypothesized that inner-microaggregate environments select for definable microbial communities and that, due to their sequestered environment, inner-microaggregate communities would not be significantly impacted by disturbance. However, T-RFLP analysis indicated distinct differences between bacterial populations of inner-microaggregates of undisturbed and reclaimed soils. While both undisturbed and reclaimed inner-microaggregate bacterial communities were found dominated by Actinobacteria, undisturbed soils contained only Actinobacteridae, while in inner-microaggregates of reclaimed soils Rubrobacteridae predominate. Spatial stratification of division-level lineages within microaggregates was also evidenced, with Proteobacteria clones being prevalent in libraries derived from whole microaggregates. The fractionation methods employed in this study therefore represent a valuable tool for defining relationships between biodiversity and soil structure.