荧光原位杂交技术及其在环境微生物生态学中的应用研究

荧光原位杂交技术是一种能够同时对微生物进行定性、定量和研究微生物群落空间分布情况的有力工具。简要介绍了荧光原位杂交技术的方法,并对其在人为创制环境和自然环境中特征性微生物种群及群落生态学中的应用研究进行了讨论,指出了该种技术在应用中存在的问题与缺陷,最后对荧光原位杂交技术在堆肥微生物生态中的应用及与其他方法的组合应用进行了展望。     

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。     

二次离子显微镜及其在生物医学中的应用

二次离子显微镜是由离子源激发样品表面原子产生二次离子,通过质谱仪将不同原子产生的离子分离并在显示系统上成像,以确定不同元素在样品中的分布图。较高的图像分辨率(0.5—1μm)和极高的灵敏度(浓度低于10^-19g/μm³)使得生物样品含有极低浓度甚至是痕迹量的元素分析成为可能。目前,二次离子显微镜已广泛应用于细胞生物学、核医学、植物生理学和人类病理学等领域。     

荧光原位杂交技术及其在微生物生态学中的应用

综述了荧光原位杂交技术 (fluorescence in situ hybridization FISH)在微生物生态学领域的各种应用 ,同时就其发展过程、原理及种类做了介绍     

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。     

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。     

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。     

DGGE技术在微生物生态学研究中的应用

简要介绍了DGGE（denaturing gradient gel electrophorests）的基本原理,及其在研究微生物类群多样性,环境中微生物变化的动态监测,微生物新物种的发现,不同DNA提取方法效果的比较和功能基因多样性研究等微生物生态学领域中的应用,并对该技术自身存在的缺陷进行了评价。     

PCR-DGGE技术及其在微生物生态学中的应用

现代分子生物学技术PCR-DGGE是一种分析微生物群落的有效工具,可以用于研究生态系统中微生物多样性和群落动态性。本文简要介绍了PCR-DGGE技术原理及其在微生物生态学领域的应用,并对该技术的局限性进行了评价。     

变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

由于从环境样品中分离和培养细菌的困难,分子生物学方法已发展用来描述和鉴定微生物群落。近年来基于DNA方法的群落分析得到了迅速的发展,如PCR扩增技术,克隆文库法,荧光原位杂交法,限制性酶切片段长度多态性法,变性和温度梯度凝胶电泳法。DGGE已广泛用于分析自然环境中细菌、蓝细菌,古菌、微微型真核生物、真核生物和病毒群落的生物多样性。这一技术能够提供群落中优势种类信息和同时分析多个样品。具有可重复和容易操作等特点,适合于调查种群的时空变化,并且可通过对切下的带进行序列分析或与特异性探针杂交分析鉴定群落成员。DGGE分析微生物群落的一般步骤如下：一是核酸的提取,二是16S rRNA,18S rRNA或功能基因如可容性甲烷加单氧酶羟化酶基因(mmoX)和氨加单氧酶a一亚单位基因(amoA)片段的扩增,三是通过DGGE分析PCR产物。DGGE使用具有化学变性剂梯度的聚丙烯酰胺凝胶,该凝胶能够有区别的解链PCR扩增产物。由PCR产生的不同的DNA片段长度相同但核苷酸序列不同。因此不同的双链DNA片段由于沿着化学梯度的不同解链行为将在凝胶的不同位置上停止迁移。DNA解链行为的不同导致一个凝胶带图案,该图案是微生物群落中主要种类的一个轮廓。DGGE使用所有生物中保守的基因片段如细菌中的16S rRNA基因片段和真菌中的18S rRNA基因片段。然而同其他分子生物学方法一样,DGGE也有缺陷,其中之一是只能分离较小的片段,使用于系统发育分析比较和探针设计的序列信息量受到了限制。在某些情况下,由于所用基因的多拷贝导致一个种类多于一条带,因此不易鉴定群落结构到种的水平。此外,该技术具有内在的如单一细菌种类16S rDNA拷贝之间的异质性问题,可导致自然群落中微生物数量的过多估计。DGGE是分析微生物群落的一种有力的工具。不过为了减少DGGE和其它技术的缺陷,建议研究者结合DGGE和其它分子及微生物学方法以便更详细的观察微生物的群落结构和功能。     

Geochemistry and Microbial Populations in Sediments of the Northern Baffin Bay,Arctic

The Northern Baffin Bay between Greenland and Canada is a remote Arctic area restricted in primary production by seasonal ice cover, with presumably low sedimentation rates, carbon content and microbial activities in its sediments. Our aim was to study the so far unknown subseafloor geochemistry and microbial populations driving seafloor ecosystems. Shelf sediments had the highest organic carbon content, numbers of Bacteria and Archaea, and microcosms inoculated from Shelf sediments showed highest sulfate reduction and methane production rates. Sediments in the central deep area and on the southern slope contained less organic carbon and overall lower microbial numbers. Similar 16S rRNA gene copy numbers of Archaea and Bacteria were found for the majority of the sites investigated. Sulfate in pore water correlated with dsrA copy numbers of sulfate-reducing prokaryotes and differed between sites. No methane was found as free gas in the sediments, and mcrA copy numbers of methanogenic Archaea were low. Methanogenic and sulfate-reducing cultures were enriched on a variety of substrates including hydrocarbons. In summary, the Greenlandic shelf sediments contain vital microbial communities adapted to their specific environmental conditions.     

Microbial community composition and function in wastewater treatment plants

Biological wastewater treatment has been applied for more than a century to ameliorate anthropogenic damage to the environment. But only during the last decade the use of molecular tools allowed to accurately determine the composition, and dynamics of activated sludge and biofilm microbial communities. Novel, in many cases yet not cultured bacteria were identified to be responsible for filamentous bulking and foaming as well as phosphorus and nitrogen removal in these systems. Now, methods are developed to infer the in situ physiology of these bacteria. Here we provide an overview of what is currently known about the identity and physiology of some of the microbial key players in activated sludge and biofilm systems. This revised version was published online in August 2006 with corrections to the Cover Date.     

微生物生态学研究方法进展

微生物培养及显微技术作为鉴定微生物种群的手段有很大的局限性,因为环境中大多数微生物处于“存活但不能培养”的状态。因此．不依赖于微生物培养的生物化学以及分子生物学方法正被广泛地用于微生物生态学研究。主要介绍了荧光技术。基于PCR的分析技术和PLFA等技术在表征微生物多样性研究中的某些进展。     

The effect of FISH and CARD-FISH on the isotopic composition of C- and N-labeled Pseudomonas putida cells measured by nanoSIMS

The use of nanoSIMS for the exploration of microbial activities in natural habitats often implies that stable isotope tracer experiments are combined with in situ hybridization techniques (i.e. fluorescence in situ hybridization (FISH) or catalyzed reporter deposition (CARD)-FISH). In this study, Pseudomonas putida grown on ¹³C- and ¹⁵N-labeled carbon and nitrogen, collected in exponential growth and stationary phases, was hybridized and analyzed by nanoSIMS. It was shown that ¹³C and ¹⁵N fractions decreased after FISH and CARD-FISH in comparison to chemically untreated cells. However, the fractions were influenced differently by various treatments. After paraformaldehyde fixation of exponentially growing cells, a reduction of the ¹³C and ¹⁵N fractions was measured from 94 ± 1.2% and 89.5 ± 3.8% to 90.2 ± 0.8% and 64 ± 4.6%, respectively, indicating that nitrogen isotopic composition was most influenced. A further decrease of the ¹³C and ¹⁵N fractions to 80.7 ± 6.5 and 59.5 ± 4.1%, respectively, was measured after FISH, while CARD-FISH decreased the fractions to 57.4 ± 3.0% and 47.1 ± 4.1%, respectively. The analysis of cells collected in different growth phases revealed that the effect of various treatments seemed to be dependent on the cell's physiological state. In addition, a mathematical model that can be used in further studies was developed in order to calculate the amount of carbon introduced into the cells by chemical treatments. These results can be valuable for environmental FISH-nanoSIMS studies where the isotopic composition of single cells will be used to quantitatively assess the importance of specific populations to certain biochemical processes and determine budget estimations.     

A simple computer-based video image analysis system and potential applications to microbiology

An inexpensive microcomputer-based image analysis system is described in which an Apple microcomputer acquires data from a video camera or video cassette recorder and measures the brightness of the image received at specified points or areas. Suggested uses for this apparatus include measurements of chlorophyll fluorescence in algal cells, determination of the effects of ultraviolet illumination on chlorophyll fluorescence, estimation of total amounts of chlorophyll in a microscope field, and microspectrophotometic and microdensitometic measurements. A similar ssytem using the IBM personal computer with a different interface is also described.     

元基因组文库分析技术研究进展

随着新的分析技术的不断出现和成熟,促进了微生物分子生态学及相关学科的诞生和迅速发展。其中,元基因组文库分析技术即是近年来微生物分子生态学研究领域兴起的一种新的分析技术。就元基因组分析技术诞生的背景及该技术的原理进行了讨论,着重阐述了元基因组文库分析技术在寻找新基因、开发新的生物活性物质、研究群落中微生物多样性、人类元基因组测序等方面的应用。另外,归纳总结了目前国际上常用的诸如PCR为基础的筛选、荧光原位杂交（fluorescent in situ hybridization,FISH）、底物诱导的基因表达筛选（substrate induced gene expression screening,SIGEX）、基因芯片等元基因组文库筛选方法,并就不同方法的优缺点进行了分析和讨论,指出了目前元基因组文库分析技术存在的主要问题并对今后该技术的发展进行了展望。     

High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use

Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation—and therefore substrate use—by secondary ion mass spectrometer imaging (NanoSIMS). Laboratory experiments demonstrated that Chip-SIP can detect isotopic enrichment of 0.5 atom % ¹³C and 0.1 atom % ¹⁵N, thus permitting experiments with short incubation times and low substrate concentrations. We applied Chip-SIP analysis to a natural estuarine community and quantified amino acid, nucleic acid or fatty acid incorporation by 81 distinct microbial taxa, thus demonstrating that resource partitioning occurs with relatively simple organic substrates. The Chip-SIP approach expands the repertoire of stable isotope-enabled methods available to microbial ecologists and provides a means to test genomics-generated hypotheses about biogeochemical function in any natural environment.     

实时荧光定量PCR及其在微生物生态学中的应用

定量描述微生物群落的组成,在微生物生态学的许多研究领域都是非常重要的。然而由于可培养技术的局限性,定量描述微生物群落成为比较困难的事情。最近包括PCR技术在内的分子生物学技术为人们提供了有力的工具,使对微生物群落的分布、丰度等有了进一步的了解。实时荧光定量PCR技术作为核酸定量检测技术,自从发明以来在微生物生态学研究中逐渐得到了广泛的应用。从微生物生态学角度,综述了实时荧光定量PCR技术的原理、发展、优缺点及其在微生物生态学研究中的应用与研究进展,并探讨了实时荧光定量PCR技术的发展和应用前景。