微生物分子生态学技术在湖泊微生物多样性研究中的应用

微生物是湖泊生物圈物质循环和能量流动的主要参与者,在湖泊的生态系统中起着重要的作用。但是,湖泊中存在着大量不可培养的细菌,利用传统的培养技术,无法对湖泊微生物的多样性进行深入而全面的研究,而不依赖培养的分子生物学技术的发展为此方面研究开辟了新的路径。微生物分子生态学作为分子生物学与微生物生态学交叉产生的学科,在研究湖泊微生物多样性方面已经得到了广泛的应用。主要综述了变性梯度凝胶电泳(PCR-DGGE)技术,末端限制性酶切片段长度多态性技术(T-RFLP),16SrDNA克隆文库技术等微生物分子生态学技术在研究湖泊微生物多样性方面的应用情况。     

生物技术在分子微生物生态学上的应用

分子生物学及其技术的发展和变革开辟了生态学研究的新途径。分子生物学与生态学交叉的研究,越来越引起人们的重视,于是分子生态学便应运而生了。而分子微生物生态学则为其中的重要分支,它是运用分子的方法和技术,在基因水平上估计种的个体丰度,查明种的变异情况以及探究群落中微生物间相互关系的科学。生物技术的应用,使我们不必培养微生物,而直接通过对环境中的遗传物质的研究来达到目的,它为微生物生态学的研究开辟了新的途径。１用于分子微生物生态学的主要生物技术１．１核酸探针检测技术探针是能与特定核苷酸序列发生特异性互…     

PCR-DGGE技术在微生物生态学中的应用

PCR-DGGE技术是随着现代分子生物学发展起来的一种很重要的分析手段,与传统的种群鉴定方法相比,PCR-DGGE技术具有快速和操作简便等优点,对于不可培养的微生物也能达到分离的效果,因而在微生物生态学中受到普遍关注与重视。介绍了该技术的基本原理、主要影响因素等研究动态以及在微生物生态学中的应用现状,并对其应用前景作了综述。     

FISH技术在微生物生态学中的研究及进展*

分子生物学技术在微生物生态学研究中具有灵敏、精确和快速的优势,但不能提供微生物的形态学、数量性状、空间分布等信息。荧光原位杂交技术结合了分子生物学的精确性和显微镜的可视性信息,可以在自然生境中监测和鉴定不同的微生物个体,尤其是对难培养和未被培养的微生物进行检测。荧光原位杂交技术被广泛用于微生物群落结构诊断和评价,现已成为微生物分子生态学研究中的热点技术。对荧光原位杂交技术的发展和在微生物分子生态学中的应用进行了综述,探讨了该技术应用中存在的问题和发展前景。     

分子生物学技术在生物修复中的应用研究进展

随着分子生物学技术如分子杂交、PCR、电泳技术等的发展,微生物学研究领域发生了深刻的变革,灵敏的检测和精确的细菌鉴定成为可能.微生物分子生态学作为分子生物学与微生物生态学交叉而形成的学科,在生物修复方面得到广泛应用.从分子生物学实验技术角度综述了各种微生物分子生物学技术在生物修复中的应用研究情况.     

FISH技术在微生物生态学中的研究及进展

分子生物学技术在微生物生态学研究中具有灵敏、精确和快速的优势,但不能提供微生物的形态学、数量性状、空间分布等信息。荧光原位杂交技术结合了分子生物学的精确性和显微镜的可视性信息,可以在自然生境中监测和鉴定不同的微生物个体,尤其是对难培养和未被培养的微生物进行检测。荧光原位杂交技术被广泛用于微生物群落结构诊断和评价,现已成为微生物分子生态学研究中的热点技术。对荧光原位杂交技术的发展和在微生物分子生态学中的应用进行了综述,探讨了该技术应用中存在的问题和发展前景。     

分子微生物生态学及其研究进展

分子微生物生态学是分子生物学实验技术应用于微生物生态学研究领域而发展形成的一门交叉学科,在研究微生物生态系统组成结构、功能的分子机理以及微生物与生物和非生物环境之间相互关系等方面显示了巨大的潜力．十几年来分子微生物生态学研究所取得的成就证明：分子生物学研究技术向微生物生态学领域的不断渗透,为微生物生态学研究领域注入了新的活力,尤其在微生物多样性、微生物区系分子组成及变化规律以及微生物系统进化研究方面取得了重大突破．本文根据近年分子微生物生态学的研究进展,就分子微生物生态学概念的提出、发展历程、主要研究领域、主要研究方法以及未来研究热点领域作以简要综述。     

环境微生物转录组学研究进展

环境微生物转录组学是一门针对复杂生境中RNA转录本中基因转录情况及基因表达活性进行研究的分子生物学手段。本研究概述了环境微生物转录组学的基本概念及其涉及的关键技术,并总结了其在各生境中(土壤,淡水湖泊,海洋以及一些特殊的生态环境)的应用进展。环境微生物转录组学作为一种新颖且有效的技术方法,极大地丰富了微生物生态学的研究手段,并且随着该技术在不同复杂生境中的广泛运用,将对环境科学研究的进步产生巨大的促进作用。     

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。     

微生物分子生态学技术在污水处理系统中的应用

微生物分子生态学作为分子生物学与微生物生态学交叉而形成的学科,在污水处理方面广泛应用。本文从分子生态学实验技术角度,综述了目前污水处理系统中微生物群体结构、多样性及其与功能相关性的研究进展,探讨了分子生态学技术的发展与应用前景,并指出研究该体系微生物对于认识微生物系统发育地位具有重要意义。     

现代分子生物学技术在冰川微生物生态研究中的应用

研究冰川中微生物的多样性对于揭示环境气候变迁,研究生物进化,开发微生物资源具有重要意义,现代分子生物学的发展为研究冰川微生物的多样性提供了行之有效的方法.简要综述了16S rDNA文库构建、变性梯度凝胶电泳、限制性片段长度多态性和荧光原位杂交等技术的原理及在冰川微生物生态研究中的应用现状.     

Increase in bacterial community diversity in subsurface aquifers receiving livestock wastewater input

Despite intensive studies of microbial-community diversity, the questions of which kinds of microbial populations are associated with changes in community diversity have not yet been fully solved by molecular approaches. In this study, to investigate the impact of livestock wastewater on changes in the bacterial communities in groundwater, bacterial communities in subsurface aquifers were analyzed by characterizing their 16S rDNA sequences. The similarity coefficients of restriction fragment length polymorphism (RFLP) patterns of the cloned 16S ribosomal DNAs showed that the bacterial communities in livestock wastewater samples were more closely related to those in contaminated aquifer samples. In addition, calculations of community diversity clearly showed that bacterial communities in the livestock wastewater and the contaminated aquifer were much more diverse than those in the uncontaminated aquifer. Thus, the increase in bacterial-community diversity in the contaminated aquifer was assumed to be due to the infiltration of livestock wastewater, containing high concentrations of diverse microbial flora, into the aquifer. Phylogenetic analysis of the sequences from a subset of the RFLP patterns showed that the Cytophaga-Flexibacter-Bacteroides and low-G+C gram-positive groups originating from livestock wastewater were responsible for the change in the bacterial community in groundwater. This was evidenced by the occurrence of rumen-related sequences not only in the livestock wastewater samples but also in the contaminated-groundwater samples. Rumen-related sequences, therefore, can be used as indicator sequences for fecal contamination of groundwater, particularly from livestock.     

Increase in Bacterial Community Diversity in Subsurface Aquifers Receiving Livestock Wastewater Input

Despite intensive studies of microbial-community diversity, the questions of which kinds of microbial populations are associated with changes in community diversity have not yet been fully solved by molecular approaches. In this study, to investigate the impact of livestock wastewater on changes in the bacterial communities in groundwater, bacterial communities in subsurface aquifers were analyzed by characterizing their 16S rDNA sequences. The similarity coefficients of restriction fragment length polymorphism (RFLP) patterns of the cloned 16S ribosomal DNAs showed that the bacterial communities in livestock wastewater samples were more closely related to those in contaminated aquifer samples. In addition, calculations of community diversity clearly showed that bacterial communities in the livestock wastewater and the contaminated aquifer were much more diverse than those in the uncontaminated aquifer. Thus, the increase in bacterial-community diversity in the contaminated aquifer was assumed to be due to the infiltration of livestock wastewater, containing high concentrations of diverse microbial flora, into the aquifer. Phylogenetic analysis of the sequences from a subset of the RFLP patterns showed that the Cytophaga-Flexibacter-Bacteroides and low-G+C gram-positive groups originating from livestock wastewater were responsible for the change in the bacterial community in groundwater. This was evidenced by the occurrence of rumen-related sequences not only in the livestock wastewater samples but also in the contaminated-groundwater samples. Rumen-related sequences, therefore, can be used as indicator sequences for fecal contamination of groundwater, particularly from livestock.     

Molecular and functional diversity in soil micro-organisms

Traditional approaches to the study of microbial diversity have relied on laboratory cultivation of isolates from natural environments and identification by classical techniques, including analysis of morphology, physiological characteristics and biochemical properties. These approaches provide information on fine-scale diversity but suffer from bias, resulting from the media and cultivation conditions employed, and from the inability to grow and isolate significant proportions of natural communities in the laboratory. An alternative approach is the amplification of ribosomal RNA and functional genes from nucleic acids extracted directly from environmental samples, with subsequent analysis by `fingerprinting' methods or by sequencing and phylogenetic analysis. This approach avoids the need for laboratory cultivation and has provided major insights into species and functional diversity of bacterial and archaeal populations. This article reviews molecular approaches to the characterisation of prokaryote diversity in natural environments, their more recent application to fungal diversity and the advantages and limitations of molecular analyses.     

分子生物学方法在环境微生物生态学中的应用研究进展

随着分子生物学方法的不断发展和改进,微生物在生态系统中的作用被更好的挖掘出来。目前快速发展的先进的分子生物学技术,已经开始应用于分析环境微生物的多样性、微生物的生物地理学及微生物对气候变化的响应等。一般环境微生物的研究目标主要有3个,即确定微生物的种类和多样性、微生物的功能或潜在作用及在特定时间点活跃的微生物等。然而,现有微生物的研究方法复杂多样,容易给研究者在方法的选择上带来困惑。将从微生物的多样性和功能研究两个方面介绍和分析相应的分子生物学方法,尤其是近年来快速发展的高通量测序、宏组学和单细胞水平研究方法(如纳米二次离子质谱与荧光原位杂交相结合的方法)等新技术及其应用情况,以期为研究者选择合适的研究方法进行环境微生物的研究提供依据。     

分子生物学方法在水体微生物生态研究中的应用

微生物是生态系统的重要组成部分,研究水体中微生物的多样性和群落结构对于开发微生物资源、进行水体生物修复具有重要意义。现代分子生物学的发展为研究水体微生物提供了行之有效的方法。综述了16S rDNA文库构建、变性梯度凝胶电泳、限制性片段长度多态性、末端标记限制性片段长度多态性等技术的原理以及在水体微生物研究中的主要应用。     

Ecosystem processes and interactions in a morass of diversity

High diversity in natural communities is indicated by both traditional, cultivation-based methods and molecular techniques, but the latter have significantly increased richness estimates. The increased ease and reduced cost associated with molecular analysis of microbial communities have fuelled interest in the links between richness, community composition and ecosystem function, and raise questions about our ability to understand mechanisms controlling interactions in highly complex communities. High-throughput sequencing is increasing the depth of sequencing but the relevance of such studies to important ecological questions is often unclear. This article discusses, and challenges, some of the often implicit assumptions made in community studies. It suggests greater focus on ecological questions, more critical analysis of accepted concepts and consideration of the fundamental mechanisms controlling microbial processes and interactions in situ. These considerations indicate that many questions do not require deeper sequence analysis and increased phylogenetic resolution but, rather, require analysis at smaller spatial scale, determination of phenotypic diversity and temporal, rather than snapshot, studies. Increasing realisation of the high richness of microbial communities, and potentially high physiological diversity, also require new conceptual approaches.     

真菌多样性研究方法进展

真菌广泛存在于自然界,在生态系统中发挥着重要的作用.随着分子生物学技术在微生物多样性研究中的广泛应用以及宏基因组学技术的出现,打破了传统微生物培养法的局限性,人们对于真菌多样性的认识日渐提高.该文主要综述了研究真菌多样性方法的主要发展历程,介绍几种有关真菌多样性研究常用的分子生物学方法,包括基于PCR的克隆文库方法、变性梯度凝胶电泳、末端限制性长度多态性、实时荧光定量PCR、荧光原位杂交、序列标签标记的高通量测序以及宏基因组技术,并且阐述宏基因组学技术在此领域蕴含的巨大发展潜力.     

Natural Populations of Chickpea Rhizobia Evaluated by Antibiotic Resistance Profiles and Molecular Methods

The aims of this study were to investigate the hypothesis that intrinsic antibiotic resistance (IAR) profiles of chickpea rhizobia are correlated with the isolates site of origin, and to compare the discriminating power of IAR profiles with molecular approaches in rhizobial strain identification and differentiation. Rhizobial diversity from five Portuguese soils was assessed by IAR profiles and molecular methods [16S rDNA restriction fragment length polymorphism (RFLP) analysis, direct amplified polymorphic DNA (DAPD) fingerprinting, and SDS–PAGE analysis of protein profiles]. For each analysis, a dendrogram was generated using the software BioNumerics. All three molecular methods generated analogous clustering of the isolates, supporting previous results on 16S rDNA sequence-based phylogeny. Clusters obtained with IAR profile are similar to the species groups generated with the molecular methods used. IAR groups do not correlate significantly with the geographic origin of the isolates. These results may indicate a chromosomal location of antibiotic resistance genes, and suggest that IAR is species related. DAPD and IAR profiles proved to be the most discriminating approaches in strain differentiation and can be used as fast methods to screen diversity in new isolates.     

Comparative microbial diversity in the gastrointestinal tracts of food animal species

Molecular tools based on small subunit (SSU) rDNA gene sequencesoffer a powerful and rapid tool for the analysis of complexmicrobial communities found in the gastrointestinal tracts (GIT)of food animal species. Extensive comparative sequence analysisof SSU rRNA molecules representing a wide diversity of organismsshows that different regions of the molecule vary in sequenceconservation. Oligonucleotides complementing regions of universallyconversed SSU rRNA sequences are used as universal probes, whilethose complementing more variable regions of sequence are usefulas selective probes targeting species, genus, or phylogeneticgroups. Different approaches derive different information andthis is highly dependent on the type of target nucleic acidemployed and the conceptual and technical basis used for nucleicacid probe design. Generally these approaches can be dividedinto DNA-based methods employing empirically characterized probesand rRNA-based methods based on comparative sequence analysisfor design and interpretation of "rational" probes. Polymerasechain reaction (PCR) based techniques can also be applied tothe analysis of microbial communities in the GIT. Direct cloningof SSU rDNA genes amplified from these complex communities canbe used to determine the extent of diversity in these GIT communities.Denaturing gradient gel electrophoresis (DGGE) is another powerfultool for profiling microbial diversity of microbial communitiesin GI tracts. Sequence analysis of the excised DGGE ampliconscan then be used to presumptively identify predominant bacterialspecies. Examples of how these molecular approaches are beingused to study the microbial diversity of communities from steersfed different diets, swine fed probiotics, and Atlantic salmonfed aquaculture diets are presented.