PCR-DGGE和FAMEs技术在土壤微生物多态性研究中的应用

将PCR-DGGE和FAMEs等生物技术新方法用于土壤微生物分析可有效的克服传统培养方法的局限性,为土壤微生物多样性、群落结构及动态变化的研究提供了更全面、可靠的方法,受到了国内外研究者的重视.本文综述了PCR-DGGE和FAMEs技术的原理及其在土壤微生物多样性、群落结构及动态变化方面的应用效果,指出了实际应用存在的问题.     

变性梯度凝胶电泳在环境微生物生态学中的应用

PCR-变性梯度凝胶电泳(PCR-DGGE)具有可靠性强、重复性好、方便快捷等优点,已被广泛应用于环境生态学中微生物群落多样性、动态性分析和功能细菌的跟踪。本文综述了PCR-DGGE技术的基本原理,不同DNA提取方法的比较,不同PCR方式的比较及其在环境生态学中研究微生物群落多样性、环境中微生物群落变化的动态监测、硝化菌-反硝化菌和硫酸还原菌(SRB)的动态分析和监测等领域中的应用,并对该技术自身存在的局限性和应用前景进行了评价。     

基于PCR-DGGE技术的红树林区微生物群落结构

【目的】为了解红树林沉积物中细菌的群落结构特征。【方法】应用PCR-DGGE技术对福建浮宫红树林的16个采样站位样品细菌的群落结构进行了研究。根据DGGE指纹图谱,对它们的遗传多样性进行了分析。【结果】各站位样品细菌多样性指数(H)、丰度(S)和均匀度(EH)均有所不同,这些差异与它们所处站位的不同有关,红树林区细菌多样性高于非红树林区细菌多样性。对不同站位细菌群落相似性分析,它们的相似性系数也存在一定的规律,同一断面的细菌群落结构相近性较高。对DGGE的优势条带序列分析,同源性最高的微生物分别属于变形菌门(Proteobacteria)、酸菌门(Acidobacteria)和绿菌门(Chlorobi),它们均为未培养微生物,分别来自于河口海岸沉积物。【结论】应用PCR-DGGE技术更能客观地反映红树林沉积物中真实的细菌群落结构信息。另外,研究也表明红树林区微生物多样性丰富,在红树林区研究开发未知微生物资源具有巨大的潜力。     

两株芽孢杆菌对黄瓜和番茄根际土壤微生物群落结构影响

采用聚合酶链式反应/变性梯度凝胶电泳技术(PCR-DGGE),研究了盆栽条件下,接种多粘类芽孢杆菌(Paenibacillus polymyxa)和枯草芽孢杆菌(Bacillus subtilis)对不同时期黄瓜和番茄根际土壤微生物群落结构的影响.通过DGGE图谱的主成分分析表明,这2株生防菌对根际微生物的影响因作物的种类不同而不同:对黄瓜根际细菌和真菌群落结构均无显著影响,真菌群落随着取样时期而发生明显变化;对番茄根际细菌群落结构无明显影响,但不同采样时期细菌群落结构发生明显变化,而2株生防菌对其真菌群落结构的变化有显著影响.蔬菜种类是决定根际微生物群落结构的主要因素,接种生防细菌对根际细菌群落结构影响不显著,而对根际真菌群落结构的影响因蔬菜种类的不同而有差异.     

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。     

不同施肥制度土壤微生物量碳氮变化及细菌群落16S rDNA V3 片段PCR产物的DGGE分析

应用化学分析和变性梯度凝胶电泳（DGGE）技术分离PCR扩增的16S rDNA的方法,研究了不同施肥制度对土壤微生物量碳、氮变化及微生物多样性的影响。结果表明,连续15a长期试验下,土壤微生物量碳（SMB-C）和微生物量氮（SMB-N）的含量大小均为长期撂荒（CK0）土壤高于农田土壤,而在农田土壤中,长期施肥的处理（NPK、NPKM、NPKSt和NPKF）高于长期不施肥处理（CK）,不同的种植制度中,长期复种轮作（NPKF）高于长期复种连作（NPK）;各处理的SMB-C/SOC（土壤有机碳）和SMB-N/TN（全氮）的比值的变化趋势与SMB-C和SMB-N变化一致;从PCR-DGGE分析,长期氮磷钾化肥配施有机肥（NPKM）处理的微生物量碳、氮的含量最高,微生物丰度最高,细菌物种最多,其次为长期撂荒（CK0）,CK处理细菌物种最少。UPGMC聚类分析表明NPK和NPKF处理细菌的群落结构相似,CK和CK0处理细菌的群落结构相似,而NPKM和NPKSt处理细菌的群落结构相似。     

用PCR-DGGE研究长期施用无机肥对种稻红壤微生物群落多样性的影响

以中国科学院红壤生态试验站的发育于第四纪红粘土的种稻红壤为研究对象,采用PCR-DGGE方法研究了长期施用无机肥对土壤微生物群落多样性的影响。在种植双季稻、连续13a施用不同无机肥后,土壤中细菌、古菌、放线菌和真菌的群落结构发生了较大的变化。未种植水稻的土壤与种稻土壤间四类微生物SSUrDNADGGE带谱相似性只有33%~66%。施磷肥的处理NP、PK、NPK之间微生物群落结构相似性较高,4类微生物的SSUrDNADGGE带谱相似性高达75%~81%。施氮钾肥(NK)、不施肥(CK)处理与施磷肥处理间土壤微生物群落结构的差异较大,其四类微生物的SSUrDNADGGE带谱相似性分别为69%~77%、55%~77%。研究的目的是深入地了解土壤中微生物群落的多样性,为科学施肥、合理利用土壤、保护微生物多样性和实现农业生态系统的可持续发展提供科学依据。     

油藏微生物群落研究的方法学

油藏微生物群落的解析和认知是开发和应用微生物采油技术的基础。利用各种提高油藏微生物可培养性的方法和非培养技术解析不同油藏微生物的群落结构、功能和多样性,对定向调控油藏微生物群落、开发和应用有效微生物驱油技术具有重要的指导意义。通过调查新近发展的提高微生物可培养性的方法和措施以及不依赖于培养的分子微生物生态学技术,总结了油藏微生物群落研究方法学的最新进展。提高微生物可培养性的方法和措施主要通过模拟微生物的生存环境,减少富营养的毒害作用、添加信号分子维持微生物细胞间的作用和提供新型电子供体和受体等手段采用稀释法、高通量培养法等方法得以实现;不依赖于培养的分子微生物生态学技术主要包括荧光原位杂交、末端限制性片断长度多态性分析、变性梯度凝胶电泳和构建克隆文库等技术。这些方法学的进展为更有效的获得各种油藏微生物资源、调控油藏微生物群落以提高石油采收率提供理论指导。     

磷脂脂肪酸谱图分析方法及其在微生物生态学领域的应用

应用磷脂脂肪酸谱图分析技术对微生物群落进行定量分布,克服了传统的微生物培养方法和显微技术的局限性。介绍了磷脂脂肪酸谱图分析方法及其在微生物生态学领域中的应用,包括对微生物群落的生物量、群落结构、营养状况和新陈代谢活动等方面的研究。     

含苯酚废水生物处理的细菌群落结构初探

本文利用PCR-DGGE技术对苯酚生产污水处理中常用的连续缺氧-好氧工艺、连续好氧工艺和缺氧SBR工艺的微生物的群落变化进行了对比研究。研究结果表明,针对苯酚生产废水的生物处理,与A/O工艺和缺氧SBR相比较,连续好氧工艺处理效果最佳,同时还具有较好的耐冲击力;生物的种群特性与所选择的工艺有密切关系,连续好氧体系中微生物的多样性最高,缺氧SBR的微生物多样性最低;在同一种运行工艺中,随水质或负荷的改变,微生物的种群多样性发生变化,而体系中的优势种群并没发生明显改变。     

应用DGGE研究微生物群落时的常见问题分析

变性梯度凝胶电泳(DGGE)是通过核酸片段对微生物群落进行研究,可以监测未培养细菌及其功能基因,被广泛地应用于微生物群落多样性和动态分析,并成为微生物分子生态学研究中的重要手段之一。文中论述了DGGE操作过程中遇到的常见问题,并提出了相应的解决方法。全面分析了样品预处理过程和PCR扩增效果对DGGE分析的影响,探讨了DGGE图谱的优化过程和图谱分析方法,并对DGGE的应用前景进行了综述。     

PCR-DGGE技术在细菌多样性研究中的条件优化

基于聚合酶链式反应的变性梯度凝胶电泳（Polymerase chain reaction denaturing gradient gel electrophoresis,PCR-DGGE）技术作为研究微生物物种多样性和动态变化的分子检测工具之一,具有可靠性强、重复性好、方便快捷等优点。该技术无需传统的微生物分离培养,便能快速、准确地获取复杂样品中微生物的菌群多样性、动态性及其功能菌的遗传信息,被广泛用于环境生态学的研究中。该文概述了PCR-DGGE技术的基本原理,并对该技术的各个环节如DNA提取、PCR扩增、DGGE条件以及图谱染色等条件的优化进行探讨,提出可能出现的影响因素,并对该技术自身存在的局限性和应用前景进行了评价。     

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。     

新疆一号冰川土壤细菌多样性的研究

应用变性梯度凝胶电泳（DGGE）技术分离PCR扩增的16SrDNA来研究土壤微生物的多样性。直接从新疆一号冰川不同海拔高度的土壤样品中提取总DNA。用两套细菌通用引物分别扩增16SrDNA的V3和V6／V9高变区的特异性片段,PCR产物进行DGGE分析。PCR—DGGE图谱表明,PCR产物经DGGE检测到的条带清晰且分离效果好。结果表明,PCR—DGGE是一种快速研究微生物群落结构的有效方法。     

Observation of bias associated with re-amplification of DNA isolated from denaturing gradient gels

DNA from environmental PCR products separated by denaturing gradient gel electrophoresis (DGGE) was isolated from the background smear rather than from discrete bands of the DGGE gel. The "interband" region was considered as a potential source of less dominant members of natural microbial communities. Surprisingly, instead of detecting new bands from the re-amplified PCR products, patterns very similar to the original ones were obtained regardless of the position of the "interband" region. The results suggest that the separation of amplicons by DGGE may not be perfect and band re-amplification based sequence analyses need careful interpretation.     

Variable selection in high-dimensional multivariate binary data with application to the analysis of microbial community DNA fingerprints

In order to understand the relevance of microbial communities on crop productivity, the identification and characterization of the rhizosphere soil microbial community is necessary. Characteristic profiles of the microbial communities are obtained by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rDNA from soil extracted DNA. These characteristic profiles, commonly called community DNA fingerprints, can be represented in the form of high-dimensional binary vectors. We address the problem of modeling and variable selection in high-dimensional multivariate binary data and present an application of our methodology in the context of a controlled agricultural experiment.     

Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences

The phylogenetic affiliation of 91 operational taxonomic units, randomly sampled from three aquatic microcosm experiments, was investigated by two PCR based and one culture dependent method. The occurrence of multiple melting domains and poor coupling between Tm and DGGE retardation was demonstrated to cause poor resolution at the species level in PCR-DGGE analysis of microbial communities. We also showed that the problem of multiple melting domains was particularly prone for brackish water bacterioplankton in the Flavobacterium genus, providing characteristic band morphology for this genus. Banding patterns from DGGE analysis may therefore be misinterpreted in terms of the species richness in natural bacterial communities, when using commonly applied universal primers.     

Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study

Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.     

Assessing the impact of the biological control agent Bacillus thuringiensis on the indigenous microbial community within the pepper plant phyllosphere

Although biological control agents (BCAs) have been used extensively for controlling insects and pathogens of plants, little is known regarding the effects of such agents on the indigenous microbial communities within the plant phyllosphere. We assessed the effect of the BCA Bacillus thuringiensis (Bt) on the microbial communities within the pepper plant phyllosphere using culture-independent methodologies. Phospholipid fatty acid (PLFA) analysis suggested that the bacterial and fungal biomass were not significantly affected following Bt application. However, principal component analysis of PLFA data indicated that Bt did change the phyllosphere microbial community structure significantly. 16S rRNA gene-directed PCR with denaturing gradient gel electrophoresis (DGGE) also suggested a significant change in the phyllosphere bacterial community structure following Bt inoculation. Phylogenetic analysis of excised DGGE bands suggested a change in bacterial phyla; bands from untreated samples predominantly belonged to the Firmicutes, while Gammaproteobacteria abounded in the treated samples.     

Salecan Enhances the Activities of β-1,3-Glucanase and Decreases the Biomass of Soil-Borne Fungi

Salecan, a linear extracellular polysaccharide consisting of β-1,3-D-glucan, has potential applications in the food, pharmaceutical and cosmetic industries. The objective of this study was to evaluate the effects of salecan on soil microbial communities in a vegetable patch. Compositional shifts in the genetic structure of indigenous soil bacterial and fungal communities were monitored using culture-dependent dilution plating, culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR. After 60 days, soil microorganism counts showed no significant variation in bacterial density and a marked decrease in the numbers of fungi. The DGGE profiles revealed that salecan changed the composition of the microbial community in soil by increasing the amount of Bacillus strains and decreasing the amount of Fusarium strains. Quantitative PCR confirmed that the populations of the soil-borne fungi Fusarium oxysporum and Trichoderma spp. were decreased approximately 6- and 2-fold, respectively, in soil containing salecan. This decrease in the amount of fungi can be explained by salecan inducing an increase in the activities of β-1,3-glucanase in the soil. These results suggest the promising application of salecan for biological control of pathogens of soil-borne fungi.