变性梯度凝胶电泳在微生物多样性分析中的应用及其技术发展

变性梯度凝胶电泳技术是目前研究微生物群落遗传多样性及种群动态性有效手段,已被广泛应用于土壤、活性污泥、生物膜、动物肠道、热泉、湖泊等环境微生物生态学研究。阐述了变性梯度凝胶电泳的原理,分析其在微生物多样性分析方面的优越性及局限性;并着重介绍了PCR-DGGE应用过程中的技术发展,包括Cloning/DGGE,GC-DGGE,Nested PCR-DGGE和DG-DGGE。     

川纹笛鲷消化道优势菌群PCR-DGGE指纹图谱比较分析

采用免培养的16S rDNA梯度凝胶电泳技术（DGGE）对集约化海水网箱养殖川纹笛鲷（Lutjanus sebae）的消化道胃壁、胃内容物、肠壁、肠内容物优势菌群结构进行了比较分析。研究结果显示，川纹笛鲷消化道存在着丰富多样的细菌群落，对DGGE指纹图谱聚类分析表明菌群组成相似度高于55％，其中胃内容物及胃壁细菌组成相似度最高（90％），这可能与摄食饵料在消化道推移有关；而胃壁与肠壁相似度相对最差，可能反应了由于生理环境不同引起的宿主差异性。通过建立川纹笛鲷消化道16S rDNA-DGGE指纹图谱及比较分析，为澄清川纹笛鲷消化道微生物区系奠定了基础。     

南极菲尔德斯半岛表层土壤样品中细菌多样性的系统发育分析

通过PCR结合变性梯度凝胶电泳(DGGE)技术对从南极长城站附近表层土壤样品中获得16SrDNA序列特征片段V3区序列进行分离。对其中的主要12条DGGE条带进行胶回收,获得的DNA片段经测序以及计算机比对分析发现,它们分别属于β、γ、δ-变形细菌(Proteobac-teria)、噬纤维菌-屈挠杆菌-拟杆菌(Cytophaga-Flexibacter-Bacteroides,CFB)群细菌、放线细菌(Actinobacteria)、蓝细菌属(Cyanobacteria)、酸杆菌属(Acidobacteria)和绿屈挠菌属(Chlo-roflexi)等系统分类群。南极表层土壤样品中的大部分16S rDNA序列与从其他土壤或沉积物样品中直接获得的序列相似性较高(93%-100%)。     

Molecular identification of Prorocentrum (Dinophyceae) species

A fragment of a large sub-unit ribosomal DNA (LrDNA) of 12 strains ofProrocentrum species was amplified by polymerase chain reaction (PCR). The PCR products were digested by 3 restriction endonucleases (Cfo I, Hae III, and RSA I) and then resolved in agarose gels. Results show that different species had different RFLP patterns, except forP. arcuatum (ME 131), which had the same pattern toP. micans (ME160 and 04). The same fragment of 19 strains of the genus was also amplified and subjected to denaturing gradient gel electrophoresis (DGGE). 11 different patterns were resolved. Different cultures of a same species had the same pattern. The results of RFLP and DGGE analyses showed that eight newly isolated epibenthicProrocentrum species were different from each other, and also from other cultured ones examined in this study.P arcuatum (ME132) could not be differentiated fromP. micans (ME160 and 04), it was probably mis-identified, since they are quite different morphologically.P. redfieldii (ME138) could also not be distinguished formP. triestinium (ME132), it should be regarded as a synonym ofP. triestinium. Unexpectedly, a restriction site was found inP. micans, compared with previous sequence data. Project supported by National Basic Research Priorities Program (2001CB409701, 2001CB409710) and supported by NSFC (40376040, 40025614)     

Combined effects of heavy metal and polycyclic aromatic hydrocarbon on soil microorganism communities

Soil contaminated sites contain a variety of pollutants, especially heavy metals and polycyclic aromatic hydrocarbons (PAHs). Interactions between heavy metals have been relatively well studied, but little is known about interactions between heavy metals and PAHs. The combined effect of heavy metals and PAHs on soil microorganism was studied in laboratory conditions and evaluated by random denaturing gradient gel electrophoresis. We extracted DNA directly from contaminated soils and then amplified the V3 sequences of the 16S rDNA. The results showed that with different culture time, the gene diversity of the single and combined contaminated soil differed as well. After 15 days of culture, the microorganisms were stimulated and accommodated. After 45 days of cultivation, the quantities of the soil microorganisms were affected. It is concluded that some of the microorganisms utilize phenanthrene as important carbon resources. Microorganisms directly isolated from soil could reflect the diversity of soil microorganism and population distribution conditions.     

Spatial heterogeneity in a deep artificial lake plankton community revealed by PCR-DGGE fingerprinting

To explore the spatial heterogeneity of plankton communities in a deep artificial lake(Songhua Lake,China),samples were collected at seven sites. Samples were investigated by denaturing gradient gel electrophoresis(DGGE) analysis of the PCR-amplified 16 S and 18 S r RNA genes and specif ic bands were sequenced. Cluster analysis of the DGGE profiles revealed that all of the samples grouped into two distinct clusters,in accordance with sampling site; while in each cluster,the divergence of sub-clusters correlated with sampling depth. Sequence analysis of selected dominant DGGE bands revealed that most sequenced phylotypes(84%) exhibited ≥97% similarity to the closest sequences in Gen Bank,and were affiliated with ten common freshwater plankton phyla(Proteobacteria,Actinobacteria,Bacteroidetes,Cyanobacteria,Bacillariophyta,Pyrrophyta,Cryptophyta,Ciliophora,Stramenopiles,and R otifera). Several of these groups are also found worldwide,indicating the cosmopolitan distribution of the phylotypes. The relationships between DGGE patterns and environmental factors were analyzed by redundancy analysis(RDA). The results suggested that,total nitrogen,nitrate,nitrite,ammonia,and CODM n concentrations,and water temperature were strongly correlated with the variation in plankton composition.     

东南极企鹅和海豹粪土层细菌群落的变化特征

海洋动物粪土是南极陆地生态系统养分的重要来源,粪土层细菌群落对南极苔原碳、氮循环过程起重要作用,但目前对南极海洋动物粪土层中细菌群落的变化特征却鲜有报道。本文采集了东南极四个企鹅、海豹粪土剖面样品,通过酶活性、微生物量碳、微生物熵、土壤呼吸和DNA浓度测定以及16 S r DNA-DGGE分析,探讨了粪土层中细菌群落变化特征及其与环境因子的关系。土壤转化酶、脲酶和磷酸酶活性、微生物量碳、土壤呼吸和微生物熵随深度增加而递减,而代谢熵增加,表明随深度增加,微生物生存环境恶劣,造成细菌丰度和酶分泌量减小,需更多有机碳进行能量代谢。土壤DNA浓度与pH、含水率、TOC、TN、微生物量碳、土壤呼吸以及酶活性呈高度正相关,表明这些因子对粪土层中细菌种群的丰度有重要影响。DGGE图谱分析证实：东南极海洋动物粪土中含丰富的细菌群落结构,随深度增加泳道带型数量和亮度递减,表明细菌多样性和丰度递减。聚类分析表明：四个剖面随深度泳道带型差异明显,细菌遗传相似性为46%,企鹅、海豹粪土细菌群落差异性尤为明显;发现四个粪土剖面中,细菌丰度与企鹅、海豹粪的相对含量呈协调一致性变化,且表层5cm粪土层细菌丰度最高,表明东南极企鹅和海豹排泄物对土壤中细菌群落多样性变化起重要作用。