云南腾冲热海三热泉细菌多样性的研究

应用变性梯度凝胶电泳(DGGE)对云南腾冲热海3个高温热泉中菌藻席和泉底沉积物的细菌多样性进行了初步研究。直接从环境样品中提取总DNA,用两套细菌通用引物进行PCR扩增,分别得到包含V8和V9高变区的16SrDNA片段,进行DGGE分析。结果表明：菌藻席和泉底沉积物中不仅物种组成差异较大,而且都存在丰富的细菌多样性;一些关键的生态因子,如氧气、温度等会对群落中微生物的物种组成有很大影响。     

腾冲热海四种不同颜色菌藻席的DGGE图谱比较

研究热泉水化学成分与菌藻席群落结构的关系。直接提取四种菌藻席(mat)总DNA,PER扩增获得16S rDNA的V8高变区片段,进行DGGE分析。结果：四种菌藻席的细菌组成差异很大,DGGE条带数目最低为20,最高为47;4种菌藻席共有的条带为1。结合文献报道的3个热泉的水化学性质分析,表明水化学成分是热泉生态系统中重要的生态因子,直接影响菌藻席的群落结构和物种组成。     

云南腾冲热海三热泉细菌多样性的研究*

应用变性梯度凝胶电泳 (DGGE)对云南腾冲热海 3个高温热泉中菌藻席和泉底沉积物的细菌多样性进行了初步研究。直接从环境样品中提取总DNA ,用两套细菌通用引物进行PCR扩增 ,分别得到包含V₈和V₉高变区的 1 6SrDNA片段 ,进行DGGE分析。结果表明 :菌藻席和泉底沉积物中不仅物种组成差异较大 ,而且都存在丰富的细菌多样性 ;一些关键的生态因子 ,如氧气、温度等会对群落中微生物的物种组成有很大影响。     

腾冲热海眼镜泉菌藻席细菌ARDRA指纹图谱分析

对腾冲热海眼镜泉中粉红色丝状菌藻席进行ARDRA指纹图谱分析,获得其细菌多样性的部分信息。直接提取菌藻席总DNA,以之为模板PCR扩增出细菌的16S rDNA,将其克隆、转化进入大肠杆菌,获得120个克隆子。用限制性内切酶(RsaⅠ、HhaⅠ、HapⅡ)筛选出了29个16S rDNA插入片段酶切带型差异明显的克隆,表明眼镜泉菌藻席细菌组成丰富。聚类分析显示这29个克隆聚为A、B两个大类,表明该菌藻席可能主要由两个遗传多样性丰富的分类群组成。     

腾冲热海眼镜泉粉红色菌藻席的细菌组成分析

应用免培养法系统研究眼镜泉粉红色菌藻席的细菌组成。经过克隆筛选,测定了23个克隆的16S rDNA插入片段的近全序列。与GenBank的序列进行比对和相似性分析,结果表明,组成该菌藻席的细菌分属于Proteobacteria、Firmicutes、Bacteroidetes、Actinobacter、Deinococcus-thermus、Aquificals 6个类群(phylum),表现出了高度的细菌多样性。结合分析温度相近的5个热泉：Octopus spring、Haegindi and Fluidir spring、Olkelduhals、Grendalur spring中的菌藻席细菌组成,表明生态位相近的不同环境中,其物种组成相近。Aquificales是中性或弱碱性高温热泉菌藻席群落的优势物种。     

舌苔细菌PCR-DGGE分析条件的建立与优化

目的针对口腔舌苔细菌16S rDNA序列进行变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)适用序列的筛选及电泳条件的优化。方法以DGGE图谱的高分辨率为指标,选择舌苔细菌DGGE分离最适的16S rDNA高变区、电泳电压和电泳时间,并采用优化的条件分析健康青年人舌苔细菌群落的分布。结果舌苔细菌16S rDNA V3高变区引物序列能获得更加丰富清晰的DGGE条带;基于该区,当变性剂浓度为30%～60%、电泳温度60℃、电压60 V和电泳时间14 h时能得到分辨率最佳的DGGE图谱。运用此优化条件对12个样本舌苔细菌群落的分析表明,舌苔微生物主要由厚壁菌门、梭杆菌门、拟杆菌门和变形菌门等组成。优化后的DGGE技术对舌苔细菌多样性的分析具有准确性、灵敏性和可重复性。结论 DGGE图谱显示,不同分析条件对图谱类型和细菌多样性指数均有所差异。利用优化的DGGE条件能有效分离舌苔细菌16S rDNA V3区序列,为口腔微生物群落结构分析提供可靠的技术支持,也为其他不同生态细菌的多样性分析提供参考。     

应用PCR-DGGE技术研究石莼、网地藻藻际微生物多样性

应用变性凝胶梯度电泳(PCR-DGGE)技术对石莼、网地藻藻际微生物多样性进行研究,并对其进行相似性、未加权聚类分析(UPGMA)及微生物多样性(Shannon)指数分析。结果表明,PCR-DGGE图谱显示,石莼、网地藻藻际微生物DGGE图谱有着明显的差异性,具体表现在条带数及密度值的不同。Quantity one图谱分析表明:石莼藻际微生物16S r RNA基因的V3区共分离得到25条DNA片段,网地藻为16条DNA片段。DGGE相似性及未加权聚类分析表明:网地藻藻际微生物间的相似性为77.78%,石莼藻际微生物细菌群落间的相似性为49.25%。Shannon指数分析表明,石莼藻际微生物多样性指数显著高于网地藻(P0.05)。石莼藻际微生物细菌群落较网地藻丰富。     

厦门海域2011年中肋骨条藻和血红哈卡藻赤潮期间细菌群落结构变化

【目的】研究2011年8月厦门海域爆发的由中肋骨条藻和血红哈卡藻共同引发的赤潮生消过程中细菌群落结构变化。【方法】应用变性梯度凝胶电泳技术(Denaturing gradient gel electrophoresis,DGGE)对两个赤潮站位和一个非赤潮站位的细菌群落结构进行研究。通过DGGE图谱分析确定赤潮生消过程中细菌群落中的关键菌群,借助Canoco软件分析细菌菌群与环境因子的相关性。【结果】在赤潮起始阶段细菌的群落结构与pH、N/P的相关性较大,在赤潮消亡阶段细菌的群落结构与盐度、温度呈明显的正相关。γ变形杆菌(Gammaproteobacteria)(47.7%)在赤潮期间处于主导位置,假交替单胞菌属(Pseudoalteromonas)、假单胞菌属(Pseudomonas)、交替单胞菌属(Alteromonas)、噬氢菌属(Hydrogenophaga)、Actibacter、Oleibacter等属均为优势菌群。香农-威列多样性指数表明,赤潮站位细菌多样性随着赤潮生消呈先升高后降低趋势,而非赤潮站位细菌多样性基本保持不变。通过Canoco对细菌菌群的主成分分析发现,在赤潮开始阶段Hydrogenophaga属为优势菌群,而在赤潮消退阶段则以Pseudomonas和Pseudoalteromonas属为主。赤潮站位藻际细菌和游离细菌群落多样性在藻密度较大时均达到最大值,然而两者与环境因子的相关性有较大差别。【结论】研究结果表明赤潮站位细菌群落多样性远高于非赤潮站位,细菌丰度随着赤潮藻密度的升高而增加,细菌与赤潮藻有着密切的关系。本文首次研究了多种优势藻引发的赤潮环境下细菌群落结构的变化,这对于多种藻引发的赤潮生消过程中细菌菌群结构有了深入的了解,为赤潮调控的研究提供了理论依据。     

香蕉轮作和连作土壤细菌主要类群

香蕉枯萎病是香蕉生产上最主要病害,而利用韭菜轮作能有效防控香蕉枯萎病的发生.本文直接对香蕉-韭菜轮作、香蕉连作的土壤样品抽提总DNA,并对总DNA的细菌16SrDNA V3高变区域序列进行PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)进行分离后,对两类土壤中主要差异条带进行回收测序,经NCBI比对分析来鉴定细菌类群.结果表明:韭菜轮作地的细菌多样性更为丰富,并且以拟杆菌门、变形菌门、放线菌门、绿湾菌门和酸杆菌门为主要类群;而香蕉连作地细菌多样性有所减少,但有明显的优势种群出现,以厚壁菌门、变形菌门、放线菌门和绿湾菌门为主要菌群.     

应用DGGE法对青海相邻两盐湖中细菌多样性的快速检测

分别对青海相邻两盐湖柯柯盐湖、茶卡盐湖土样、泥样进行富集培养后,从中提取的DNA用两套不同的细菌通用引物进行扩增,分别得到包含V8和V9高变区的16SrDNA片断。经变性梯度凝胶电泳(DGGE)分析,结果显示这两个盐湖富集样品中细菌多样性具有较大的差异,而且同一盐湖不同性质样品中的细菌多样性差异也较大,两湖的泥样富集样品中均表现出了稍丰富的多样性。     

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。     

PCR-DGGE法在慢性胃炎病人舌苔菌群结构研究中的应用

在早间未刷牙和进食的情况下,刮取胃炎病人与正常人舌苔,去除杂质,分离菌体,采用酚/氯仿法抽提细菌基因组DNA,并对其中16S rDNA V3可变区进行聚合酶链式反应(PCR)扩增和变性梯度凝胶电泳(DGGE)测定.用Bionumerics软件对DGGE分子指纹图谱进行舌苔菌群结构相似性分析.实验结果表明,采用该方法成功地扩增出16S rDNA V3区片段,为230 bp.DGGE分子指纹图谱结果表明,正常人的舌苔菌群最高相似性为0.74,胃炎病人的舌苔菌群的最高相似性为0.52,正常人的舌苔菌群与胃炎病人的舌苔菌群相似性最高为0.38,即胃炎病人舌苔菌群结构发生了变化.     

Biodiversity analysis of microbial community in the chem-bioflocculation treatment process

Total DNA was directly extracted from environmental samples and amplified with polymerase chain reaction (PCR) technique. The PCR products were fingerprinted via denaturing gradient gel electrophoresis (DGGE). Significant differences were observed in the microbial community structures between traditional treatment process and chem-bioflocculation process. The microbial community structure shift at different sampling locations in chem-bioflocculation process and on two typical operational conditions was studied. 16S rDNA V3 regions of some dominant species were sequenced and the species were identified. The microbial communities were stable in both the chem-bioflocculation process and the activated sludge process under various experimental conditions presented in this work. The attached growth treatment process was less stable when operational conditions changed.     

PCR指纹图谱技术分析人体口腔内微生物菌群结构

目的 应用PCR-DGGE指纹图谱技术对人体口腔微生物菌群结构进行系统性研究.方法 对1例健康人唾液周期性采集的样品和8例健康人个体的唾液与牙菌斑采集的样品,进行微生物群落总DNA的抽提.以此为模板扩增16S rRNA V3可变区,产物经DGGE指纹图谱分析其组成结构,并运用UVIBAND/MAP等软件比较所得群落指纹图谱的相似性指数.结果 同一健康人个体不同采样时间的唾液菌群结构相似性系数＞74%,通过对不同健康个体口腔样本的研究,发现同一个体的唾液与牙菌斑菌群结构存在差异（84%～95%）.结论 同一健康个体其唾液微生物菌群在一定时间内基本稳定,仅有微小的变化;唾液与同个体牙菌斑的微生物组成虽然存在差异,但这种差异要明显小于个体间的差异.     

Effect of monoculture soybean on soil microbial community in the Northeast China

Monoculture (MC) soybean, a common practice in the Northeast China, causes significant declines in soybean yield and quality. The objective of this study was to evaluate the responses of the soil microbial community and soybean yield to different soybean cropping systems. Three cropping systems were compared, (1) corn-soybean rotation (corn-corn-soybean, CS), (2) MC soybean for 3 years (S3), (3) MC soybean for 9 years (S9). Both bulk and rhizosphere soil samples were collected at three growth stages: two trifoliate (V2), full bloom (R2), and full seed (R6), respectively. Soil microbial DNA was analyzed using polymerase chain reaction (PCR)—denaturing gradient gel electrophoresis (DGGE) to assess changes in composition of bacterial and fungal communities. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant microbial populations. Some prominent differences were observed in bacterial DGGE patterns of amplified 16S rDNA (V3 region) among rhizosphere soils. These major differences included one DGGE band (showing 100% similarity to Arthrobacter sp.) that was enriched at R2 stages in CS and S9, and another band with 97% sequence similarity to an uncultured actinobacterium was detected at R6 stage in CS, and at R2 and R6 stages in S9. The bacterial community from bulk soil showed no significant band change in DGGE patterns among different cropping systems. In fungal DGGE patterns of the amplified 18S rDNA partial fragment, one specific band (showing 98% similarity to Trichoderma viride) occurred in rhizosphere soil of treatment CS at V2 and R6 stages and treatment S9 at R6 stage. None of the above bands were detected in treatment S3. The soybean yields and plant heights from CS and S9 were greater than those from S3. Moreover, catalase activities from CS and S9 at V2 and R2 stages were higher than those tested from S3 at the corresponding times in rhizosphere soil. The present results showed that DGGE patterns were not able to detect significant differences in diversity or evenness among microbial communities, but significant differences were found in the composition of bacterial and fungal community structures. Some distinguished bands from bacterial and fungal DGGE patterns were only enriched in CS and S9 soil, which could potentially play an important role in soybean growth development.     

茶园土壤微生物群落基因多样性

应用PCR技术,直接从土壤中抽提总DNA,扩增16S rDNA V3 片段,采用变性梯度凝胶电泳(DGGE)分析16S rDNA V3 片段的多态性,研究了杭州西湖梅家坞不同植茶年龄(8、50和90年)、不同利用方式(茶园、荒地和林地)的土壤微生物群落基因多样性.结果表明：不同植茶年龄和不同土地利用方式影响土壤微生物群落的基因多样性.荒地、茶园和林地土壤微生物群落基因多样性指数明显不同(P<0.05),其排列顺序为荒地>茶园>林地.不同植茶年龄的土壤中,50年茶园土壤的微生物群落基因多样性指数、微生物量碳和基础呼吸明显高于8年和90年茶园土壤(P<0.05).     

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技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Methanogenic profiles by denaturing gradient gel electrophoresis using order-specific primers in anaerobic sludge digestion

In the present study, the diversity of methanogenic populations was monitored for 25 days, together with the process data for an anaerobic batch reactor treating waste-activated sludge. To understand this microbial diversity and dynamics, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted at two different taxonomic levels: the domain and order levels. The DGGE profiles of the domain Archaea and the three orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales were comparatively analyzed after each DGGE band was sequenced to enable identification. The DGGE profiles of the three orders showed methanogens belonging to each order that were not detected in the DGGE profile of the Archaea. This discrepancy may have resulted from PCR bias or differences in the abundances of the three microbial orders in the anaerobic bioreactor. In conclusion, to fully understand the detailed methanogenic diversity and dynamics in an anaerobic bioreactor, it is necessary to conduct DGGE analysis with 16S rRNA gene primers that target lower taxonomic groups.