油藏水样细菌群落16S rRNA基因的RFLP分析

通过16S rRNA基因的限制性酶切片段长度多态性分析(RFLP)方法,考察了油藏水样中细菌群落及多样性。从水样中分离纯化微生物总DNA,选择性扩增细菌16S rRNA基因,并构建16S rDNA克隆文库。337个16S rDNA克隆片段分别用限制性内切酶HinfⅠ和HaeⅢ酶切分析,得到74个操作分类单元(OTUs),其中数量最多的4个OTUs共占克隆子总数的73.6%,另外70个OTUs的丰度均处于较低水平,有57个OTUs仅含有1个克隆子。结果表明,运用RFLP方法分析16S rDNA克隆片段能够有效评估油藏水样中的细菌群落和多样性。     

应用16S rDNA克隆文库解析人工快速渗滤系统细菌种群多样性

本文出不穷通过构建16S rDNA克隆文库开展了人工快速渗滤(CRI)系统表层(10cm深)细菌种群多样性研究,结果表明,在CRI系统表层填料中细菌多样性十分丰富,存在7个主要类群,其中不可培养的酸杆菌纲(uncultured Acidobacteria)和其它不可培养细菌(uncultured bacteria)在整个克隆文库中比例最大,比例为53.72%,其次是浮霉菌纲(uncultured planctomycete)和β-变形菌纲(β-proteobacterium),分别占文库比例的13.89%和8.33%;克隆文库中反硝化菌的比例高于亚硝化单胞菌属细菌(O.925%),没有出现Nitrospira属的克隆子.本文通过16S rDNA克隆文库揭示了在生物膜上存在的优势茵为不可培养菌,而假单胞菌(Pseudomonas aeruginosa)和紫色色杆菌(Chromobacterium sp.)等在平板分离培养中出现的频率较高,两种方法之间的结果存在差异.本研究采用16S rDNA克隆文库方法揭示的结果,将为CRI系统生物降解的进一步提高提供依据.     

应用16S rDNA克隆文库解析人工快速渗滤系统细菌种群多样性

本文通过构建16S rDNA克隆文库开展了人工快速渗滤(CRI)系统表层(10 cm深)细菌种群多样性研究, 结果表明, 在CRI系统表层填料中细菌多样性十分丰富, 存在7个主要类群, 其中不可培养的酸杆菌纲(uncultured Acidobacteria)和其它不可培养细菌(uncultured bacteria)在整个克隆文库中比例最大, 比例为53.72%, 其次是浮霉菌纲(uncultured planctomycete)和β-变形菌纲(β-proteobacterium), 分别占文库比例的13.89%和8.33%; 克隆文库中反硝化菌的比例高于亚硝化单胞菌属细菌(0.925%), 没有出现Nitrospira属的克隆子。本文通过16S rDNA克隆文库揭示了在生物膜上存在的优势菌为不可培养菌, 而假单胞菌(Pseudomonas aeruginosa)和紫色色杆菌(Chromobacterium sp.)等在平板分离培养中出现的频率较高, 两种方法之间的结果存在差异。本研究采用16S rDNA克隆文库方法揭示的结果, 将为CRI系统生物降解的进一步提高提供依据。     

大庆油田油藏采出水的细菌群落结构

采用ARDRA (扩增性rDNA限制性酶切片段多态性分析)技术对大庆油田聚驱、水驱和过渡带3种油藏采出水中的细菌群落的基因组总DNA的16S rDNA克隆文库进行分析,研究了细菌群落结构.结果表明：随机挑取的596个阳性克隆可分为85个操作分类单元 (OTUs),其中聚驱、水驱和过渡带文库分别含有28、41和33个.通过对优势OTUs测序,并与GenBank进行序列比对,发现油藏采出水中的优势菌群为不动杆菌属、弓形杆菌属、厚壁菌门、假单胞菌属和硫磺单胞菌属.聚驱样品中细菌群落组成最简单,优势菌群为不动杆菌属,占库容的85%,假单胞菌属占7%;水驱样品中的优势菌群也是不动杆菌属,占库容的62%,假单胞菌属和硫磺单胞菌属各占20%和6%;过渡带文库的细菌群落的优势菌群为弓形杆菌属,占库容的50%,不动杆菌属和厚壁菌门各占19%和18%.     

四川冬菜中细菌群落组成及多样性

【目的】了解腌制4年的四川南充冬菜中细菌群落组成及多样性。【方法】通过16S rDNA多样性分析样品细菌落组成;采用16S rDNA-RFLP方法分析从样品中分离出的纯培养细菌。【结果】16S rDNA多样性分析结果表明,样品中细菌主要属于变形杆菌门(Proteobacteria)和厚壁菌门(Firmicutes),分别占克隆文库的87.9%、7.1%,其中包括Virgibacillus kekensis,Marinococcus albus,Salinicoccus sp.,Lactobacillus halophilus和Halomonas等中度嗜盐菌,仅有5%属于放线菌门(Actinobacteria)。通过纯培养方法从冬菜中分离到35株菌,16S rDNA-RFLP分析结果表明,34株属于厚壁菌门(Firmicutes),包括Virgibacillus,Bacillus megaterium和Gracilibacillus saliphilus等中度嗜盐菌,1株属于放线菌门(Actinobacteria)。【结论】冬菜中细菌群落多样性较低,以中度嗜盐菌为主。     

垃圾填埋场渗滤液中古细菌群落16S rRNA基因的ARDRA分析

利用特异性的引物对,选择性扩增垃圾填埋场渗滤液中古细菌群落的18S rRNA基因片断,在此基础上建立16S rDNA克隆文库,经古细菌通用寡核苷酸探针的原位杂交筛选后,克隆文库内古细菌16S rDNA扩增片断的多样性通过ARDRA分析（amplified rDNA restriction analysis)而获得,利用PCR将各组重克隆子内的16S rDNA外源片断再扩增出来后,两种限制性内切酶－Hha I和HaeⅢ－被分别用于16S rDNA克隆片断的限制酶切分析,结果表明,随机选出的70个古细菌16S rDNA克隆片断被妥为21个不同的ARDRA型（组）,其中的两个优势型总共占了所有被分析克隆子的60％,而其余19个型的相对丰度均处于较低的水平,当中的14个型更仅含有1个克隆子,通过对16S rRNA基因的PCR扩增,克隆及其ARDRA分析,能快速地获得有关填埋场渗滤液中古细菌群落的结构及其多样性的初步信息。     

PCR-RFLP分析桉树人工林土壤微生物的群落结构

本研究利用限制性内切酶片段长度多态性(RFLP)和16S rDNA序列分析相结合的方法研究了广西南宁高峰林场人工桉树林中土壤微生物的群落结构.通过采用直接法提取桉树人工林土壤中微生物总DNA,构建细菌的16S rDNA克隆文库,从中随机挑取了192个阳性克隆进行检测,分析显示188个阳性克隆子确定含有16S rDNA并分归为52个不同的类群OTUs,随机挑取其中35个克隆进行测序,并构建了系统发育进化树.研究结果表明:桉树人工林土壤中细菌种类较为丰富,含有大量的未培养微生物,进一步的序列分析表明桉树人工林土壤中占优势的类群分别为Acidobacteria(40%)和Proteobacteria(27%).本研究对桉树人工林土壤微生物群落结构进行了研究,为进一步研究桉树人工林环境质量变化提供基础资料.     

昆明盐矿古老岩盐沉积中的原核生物多样性

应用PCR-DGGE和rRNA分析法研究了昆明盐矿古老岩盐沉积中的原核生物多样性。样品的细菌DGGE分析得到27条带,古菌得到18条带。样品与纯培养得到的19个属菌株的DGGE图谱对比分析发现,细菌18个属菌株,只有1个属菌株与样品中的1条带迁移位置都不一致;古菌1个属的菌株不与样品中任何条带迁移位置一致。表明纯培养所得菌株并非该环境中的优势类群。同时,建立了样品细菌和古菌的16S rDNA克隆文库,从中分别挑取36个细菌克隆和20个古菌克隆进行ARDRA分析。细菌可分为10个OTUs,其中3个OTUs是优势类群,分别占38.9%,25.0%,16.7%,其余7个OTUs各含有1个克隆。古菌分为8个OTUs,没有明显的优势类群。每个OTU的代表克隆16S rDNA序列分析表明,细菌分属3大类群:α-Proteobacteria,γ-Proteobacteria和Actinobacteria,以Pseudomonas属菌为优势,含有其它岩盐沉积中没有发现的Actinobacteria。古菌主要是Halorubrum属、Haloterrigena属菌和未培养古菌。本研究表明,昆明盐矿古老岩盐沉积具有较丰富的原核生物多样性,含有大量未知的、未培养或不可培养的原核生物,但在原核生物物种组成和丰度上,免培养与此前的纯培养研究结果存在一定差异。因此,结合使用两类方法才能较全面地认识高盐极端环境微生物的多样性。     

接种微生物菌剂对猪粪堆肥过程中细菌群落多样性的影响

利用PCR-DGGE方法研究了接种外源微生物菌剂对鲜猪粪高温好氧堆肥过程中细菌群落多样性的影响.结果表明：接种外源微生物菌剂可以促进堆肥的顺利进行,比不接种处理的高温期提前2 d.DGGE图谱分析表明,堆肥中优势细菌群落组成发生了明显的更迭现象,不同堆肥时期细菌群落的Shannon-Wiener指数呈显著差异.目的条带克隆测序结果表明,整个堆肥过程Clostridium stercorarium subsp. thermolacticum sp.一直是优势菌属,不经培养细菌、Bacillus coagulans sp.、Clostridium thermocellum sp.在接种外源微生物菌剂处理的第10、16天成为优势菌属,不经培养的Firmicutes sp.和不经培养的 delta proteobacterium分别在未接种外源微生物菌剂处理堆肥发酵的第5天和第16天成为优势菌属.非优势菌属Ureibacillus thermosphaericus、不经培养的Silvimonas sp.出现在堆肥腐熟后期,不经培养的土壤细菌主要出现在堆肥初期和高温初期.UPGMC聚类分析表明,接种外源微生物菌剂明显影响了堆肥不同时期的细菌群落结构组成.堆肥化过程中细菌DGGE图谱主成分分析表明,细菌群落变化主要受外源接种微生物菌剂的影响.     

培养和非培养法分析冷藏鸡肉胴体中的细菌多样性

运用纯培养和非培养法对冷藏鸡肉胴体上细菌多样性进行了对比研究。采用培养法从鸡肉胴体中初步分离到45株细菌菌株,16S rDNA-ARDRA分析得到9株代表性细菌,其16S rDNA序列系统发育分析表明,这些菌株隶属于Bacillus sp.、Shigella sp.、Pseudomonas sp.、Citrobacter sp.、Klebsiella sp.和Escherichia sp.6个属。16S rDNA-ARDRA联合PAGE和16S rDNA全序列分析的非培养法结果表明,冷藏鸡肉中细菌主要属于Acinetobacter sp.、Bacillus sp.、Acidovorax sp.、Brochothrix thermosphacta、Lactococcus garvieae和Leuconostoc lactis等16个属。非培养法揭示的细菌多样性比培养法丰富,但二者结合使用能让肉品中微生物多样性得到更全面的展示。     

应用16S rDNA克隆文库法分析有机物料腐熟菌剂细菌组成

应用16SrDNA克隆文库法对有机物料腐熟菌剂A和B样品中的细菌组成进行分析研究。结果表明,样品A有14个OTU,主要是融合乳杆菌(Weissella confusa)、枯草芽孢杆菌(Bacillus subtilis)和短小芽孢杆菌(Bacillus pumilus),其比例分别占总克隆文库的28.6%、30.4%和23.2%;样品B有43个OTU,主要是布氏乳杆菌(Lactobacillus buchneri)、香肠乳杆菌(Lactobacillus farciminis)和耐酸乳杆菌(Lactobacillus acetotolerans),占总克隆文库的比例分别为18.03%、18.86%和13.12%;所得出的结果均与产品标注存在差异,样品A未提及细菌的种类,而样品B只标注短小芽孢杆菌。研究表明这一方法在微生物菌剂细菌组成分析及其质量检测中具有良好的应用前景。     

河西走廊酒泉地区盐碱土未培养放线菌多样性

采用免培养技术对河西走廊酒泉地区的原生盐碱土、次生盐碱土和农田土中的放线菌群落结构及其多样性进行分析.结果表明: 河西走廊酒泉地区原生盐碱土克隆文库分归于19个OTUs,分属于微球菌亚目、丙酸杆菌亚目、棒状杆菌亚目、弗兰克氏菌亚目、假诺卡氏菌亚目和放线菌目未知类群;次生盐碱土克隆文库分归于14个OTUs,分属于微球菌亚目、丙酸杆菌亚目和放线菌目未知类群;农田土克隆文库分归于7个OTUs,分属于微球菌亚目和棒状杆菌亚目;微球菌亚目是3种不同类型土壤中的共有种群,是原生盐碱土和农田土中的优势种群.多样性指数和稀释性曲线分析结果显示,3种不同类型土壤中放线菌种群丰富度为原生盐碱土>次生盐碱土>农田土;原生盐碱土、次生盐碱土的稀释性曲线均未趋于平稳,说明盐碱土中放线菌多样性比实际更加丰富.     

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.     

Assessment of fungal diversity in deep-sea sediments by multiple primer approach

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.     

Developing SSU rDNA metagenomic profiles of aquatic microbial communities for environmental assessments

Five water samples from three sources, two municipal reservoirs in central North Carolina and Toolik Lake in Alaska, were processed to conduct a comparative survey of microbial small subunit rDNA sequences. Genomic DNA was extracted and amplified by PCR using universal SSU rDNA primers to generate 16S and 18S rDNA clone libraries and 50 clones from each library were sequenced and placed in operational taxonomic units (OTUs). Through this recovery and analysis of SSU rRNA genes, a metagenomic profile of the microbial community emerged for each environmental sample. Analyses of these profiles, including species diversity estimates and rank-abundance curves, revealed that approximately 64% of prokaryotic OTUs and 80% of eukaryotic OTUs were novel. Diversity estimates were consistent with predicted ecosystem characteristics: they were greater for the mesotrophic to eutrophic temperate lakes, than for the oligotrophic arctic lake. Sample comparisons showed that community similarity declined as geographic distance between sites increased. Real-time quantitative PCR results showed that OTUs which had been recovered from only one library were actually present in other samples, but at much lower frequencies, suggesting that many, if not most, microorganisms are cosmopolitan. Together, these results support the potential value of using the microbial community as an indicator of local environmental conditions. In other words, it may be realistic to monitor water quality using a single, comprehensive suite of microorganisms by analyzing patterns of relative abundance.     

兰坪铅锌尾矿区豆科根瘤菌遗传多样性ARDRA分析

通过16S rDNA扩增产物限制性片段长度多态性分析(ARDRA),对兰坪铅锌尾矿区豆科植物根瘤菌的遗传多样性进行了研究。采用限制性内切酶Hae Ⅲ、Hind Ⅲ、Hinf Ⅰ和Taq Ⅰ对16S rDNA扩增产物进行了酶切分型,根据ARDRA酶切图谱的不同,进行树状聚类。结果表明:49株根瘤菌在40%的相似水平上按氮含量不同及铅锌含量的采集地不同分别聚为OTU1、OTU2和OTU33个群,说明根瘤菌的遗传多样性及分布与土壤中的氮含量和铅锌含量有关。代表菌株的16S rDNA测序结果分析表明,它们在系统发育树上属于Rhizobium sp.、Sinorhizobium sp.和Bradyrhizobium sp.3个系统发育分支,进一步说明兰坪铅锌尾矿区豆科植物根瘤菌多样性较丰富。     

Statistical approaches for estimating actinobacterial diversity in marine sediments

Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with >/= 99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpson's index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries. F(ST) and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.     

利用小亚基核糖体RNA 技术分析温室黄瓜近根土壤古菌和真菌多样性

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97％的标准分成若干个可操作分类单元 (OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌 (甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80％以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。     

南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。