新疆沙湾冷泉沉积物的细菌系统发育多样性

为了解新疆沙湾冷泉沉积物的细菌群落组成与类群多样性,利用免培养方法直接从沙湾冷泉沉积物中提取环境总DNA,构建细菌16S rRNA基因文库。对随机挑选的241个细菌阳性克隆子进行HaeIII酶切分型得到86个可操作分类单元(OTUs),系统发育分析将其归为11个门:放线菌门(Actinobacteria),酸杆菌门(Acidobacteria),拟杆菌门(Bacteroidetes),绿菌门(Chlorobi),蓝细菌门(Cyanobacteria),厚壁菌门(Firmicutes),芽单胞菌门(Gemmatimonadetes),硝化螺旋菌门(Nitrospirae),变形菌门(Proteobacteria),浮霉菌门(Planctomycetes),疣微菌门(Verrucomicrobia)。其中酸杆菌门和变型菌门为优势类群,分别占细菌克隆文库的48%和25%。超过1/3的OTUs序列与GenBank中已存序列具有较低相似性(相似性小于95%)。此外20%左右的克隆子与固氮细菌和硝酸盐氧化细菌相关。研究结果表明,新疆沙湾冷泉沉积物中细菌种类丰富,代谢类型多样而且存在大量未知类群。     

新疆顿巴斯他乌盐湖沉积物免培养古菌多样性

【目的】了解新疆顿巴斯他乌盐湖沉积物免培养古菌组成及多样性。【方法】利用免培养法直接从顿巴斯他乌盐湖沉积物样品中提取环境总DNA,采用古菌通用引物对16S rRNA基因进行扩增,构建基因克隆文库。对随机挑选的59个阳性克隆进行HaeⅢ限制性酶切分型并测序、BLAST比对及构建16S rRNA基因系统发育树。【结果】文库覆盖率为89%,Shannon-Wiener指数为2.69,共得到21个不同的可操作分类单元,分属于广古菌门(Euryarchaeota,92%)和泉古菌门(Crenarchaeota,8%),其中多数为盐杆菌科(Halobacteriaceae,88%)的盐杆菌属(Halobacterium,24%)、盐盒菌属(Haloarcula,18%)、盐碱红菌属(Natronorubrum,14%)、盐红菌属(Halorubrum,8%)等,与海盐环境(thalassohaline)获得的16S rRNA基因序列相似性最高(﹥95%);整个文库中约11%的克隆与可培养古菌多个属的相似性小于97%。【结论】顿巴斯他乌盐湖古菌多样性略低于同类高盐环境,组成较为一致,只是各类群所占百分比稍有不同,且可能存在一些潜在新物种或新类群。     

新疆两典型微咸水湖水体免培养古菌多样性

微咸水湖是湖泊演化过程中的一个重要中间状态,以新疆两典型微咸水湖-赛里木湖和柴窝堡湖水为研究对象,采用微孔滤膜收集菌体,SDS-酚-氯仿抽提法直接提取湖水总DNA,利用古菌16S rRNA基因通用引物进行PCR扩增,分别构建两湖古菌16S rRNA基因克隆文库。用限制性内切酶Hae Ⅲ对随机挑选的阳性克隆子进行酶切分型,分别得到7个和8个可操作分类单元(Operational Taxonomic Units, OTUs),两文库覆盖率均大于98%。BLAST比对和系统发育分析表明赛里木湖全部克隆子归属于泉古菌门(Crenarchaeota),97%的克隆子与不同环境免培养氨氧化泉古菌有较高的序列相似性(>97%)。柴窝堡湖水古菌归为3个门:Thaumarchaeota (81.2%)、广古菌门(Euryarchaeota)(13%)和泉古菌门(Crenarchaeota) (5.8%),81.2%的克隆子与具有氮代谢功能的氨氧化古菌纯培养物具有较高的序列相似性(97%-98%),13%的克隆子与已分离到的产甲烷古菌序列同源性大于97%。研究发现新疆微咸水湖可能存有大量新划分的古菌Thaumarchaeota门类群、可培养氨氧化及产甲烷古菌类群,两典型微咸水湖泊中古菌类群多样性较低且群落组成差异大。     

西藏米拉山土壤古菌16S rRNA及amoA基因多样性?分析

摘要：【目的】硝化作用在全球土壤氮循环中具有重要的作用,虽然细菌一度被认为单独负责催化这个过程的限速步骤,但是最近一些研究结果表明泉古菌具有氨氧化的能力。本文通过构建古菌16S rRNA 基因克隆文库和氨氧化古菌amoA基因文库,分析西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况,为揭示青藏高原高寒草甸土壤古菌的多样性提供理论基础。【方法】采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA 基因和氨氧化古菌amoA基因克隆文库。【结果】通过构建系统发育树,表明古菌16S rRNA 基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲。氨氧化古菌amoA基因克隆文库中序列均为泉古菌。通过DOTUR软件分析,古菌16S rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和 75个OTUs。【结论】西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用。所获得的一些序列与已知环境中土壤、淡水及海洋沉积物中获得的一些序列具有很高的相似性,其古菌及氨氧化古菌来自不同环境的可能性比较大,可能与青藏高原的地质历史变迁过程有关。米拉山古菌及氨氧化古菌与陆地设施土壤中相似性最高,说明与西藏米拉山高寒草甸土壤的退化有关。     

新疆断裂带含硫冷泉泉水细菌群落结构多样性

摘要：【目的】为了解新疆断裂带含硫冷泉泉水中细菌群落结构的组成和物种多样性。【方法】采用免培养法直接从冷泉水中提取环境总DNA,采用细菌通用引物对泉水中细菌的16S rRNA基因进行PCR扩增,构建16S rRNA基因克隆文库。使用限制性内切酶Hae Ⅲ对随机挑选的阳性克隆子进行限制性片段长度多态性分析(Restriction Fragment Length Polymorphism, RFLP),选出具有不同酶切图谱的序列进行测序、BLAST比对和构建16S rRNA基因系统发育树。【结果】共从细菌16S rRNA基因文库中筛选了228个阳性克隆,RFLP分型得到33个不同的操作分类单元 (Operational Taxonomic Unites, OTUs),覆盖度 (Coverage C) 为92%。BLAST比对、RDP归类及系统发育分析将这33个OTUs归为：变形菌门 (Proteobacteria)、拟杆菌门 (Bacteroidetes) 和厚壁菌门 (Firmicutes)。变形菌门为绝对优势类群,占整个细菌克隆文库的98%,,其中20%左右的类群与硫化物代谢相关的光合自养和化能自养类群纯培养菌具有高的相似性 (>97%)。此外,还发现大量类群 (总文库的64%,其中57%为军团菌属Legionella spp., 类群)与GenBank中已存细菌16S rRNA基因相似性小于96%。【结论】新疆断裂带含硫冷泉泉水中细菌类群的多样性较低,但可能存在大量潜在细菌新种和新分类。另外,该泉水可能是潜在的新军团菌病传播源,因而可能对下游人畜健康存在潜在威胁。     

俄罗斯堪察加地区热泉及其周边生境的泉古菌多样性

【目的】泉古菌为陆地热泉系统的主要古菌类群,可能在自然界生源元素的地球化学循环中发挥着重要作用。本研究旨在揭示俄罗斯堪察加地区热泉以及热泉周边区域的泉古菌多样性,同时基于之前已获得的我国云南地区热泉数据,比较两地区泉古菌群落差异。【方法】通过构建16S rRNA基因片段克隆文库获得序列信息和丰度,随后进行物种多样性、系统发育和群落结构差异分析。【结果】高温热泉Burlyashi Liza(BSL,89℃)中的泉古菌全部属于热变形菌纲(Thermoprotei)内的物种。中温热泉TF Vent 2(TFV,49℃)的群落结构主要由不确定的热变形菌纲类群、不确定的泉古菌类群、高温水环境泉古菌类群Ⅱ(HWCG-Ⅱ)和奇古菌下的Group1.1b类群组成。热泉周边常温环境的主要物种与热泉环境的代表性克隆pJP41一起聚成一个较大的遗传分枝。Jackknife聚类树和主坐标分析(Principal coordinates analysis,PCoA)的结果显示:温度相似的样点,其泉古菌群落结构相对来说更为相似。【结论】俄罗斯堪察加地区与我国云南地区热泉中的泉古菌存在着一定程度上的不同。陆地热泉系统影响着其周边环境的泉古菌类群。热泉中泉古菌群落结构受温度的明显影响。     

新疆乌鲁木齐10号冷泉古菌群落结构多样性

[目的]探究新疆地震断裂带含硫冷泉泉水古菌群落组成及多样性.[方法]利用酶解法直接从冷泉泉水样品中提取环境总DNA,采用古菌通用引物对16S rRNA基因进行扩增,构建16S rRNA基因克隆文库,通过Alu Ⅰ和AfaⅠ两种限制性内切酶对随机挑选的115个阳性克隆子进行酶切分型,将不同酶切带型对应的克隆子送样测序,测序结果与GenBank序列进行比对并构建16S rRNA基因系统发育树.[结果]古菌克隆文库中共得到44个不同的酶切带型,BLAST序列比对和系统发育分析将它们划分于广古菌门(Euryarchaeota,94.78％)和奇古菌门(Thaumarchaeota,4.35％).奇古菌门克隆与Nitrosopumilus.sp序列相似性达到了93％;而广古菌门类群较为多样,其中,42.61％的克隆子属于RC-V cluster,20.87％与13.91％的克隆子分别属于LDS cluster和Methanomicrobiales,4.35％的克隆子与甲烷厌氧氧化相关的类群(ANME-1a-FW)具有较高的的相似.另外,13.05％的克隆子属于广古菌门中的未知类群.[结论]乌鲁木齐10号泉水体中广古菌类群多样,可能蕴藏有大量潜在的古菌新类群.     

新疆红井子盐碱土壤非培养放线菌多样性

【目的】研究新疆红井子盐碱土壤中的放线菌物种多样性。【方法】应用基于16S rRNA基因序列系统发育分析的免培养方法进行放线菌物种多样性分析。利用放线菌特异性引物,以土壤样品总DNA为模板,扩增16S rRNA基因,构建16S rRNA基因克隆文库,并对文库中的插入序列进行RFLP分析。【结果】随机挑选的246个阳性克隆通过酶切筛选出61个不同图谱的重组克隆并测序。分析结果显示这61个克隆序列分属于42个OTUs,分布于放线菌纲(Actinobacteria)的放线菌亚纲(Actinobacteridae)、酸微菌亚纲(Acidimicrobidae)和红色杆菌亚纲(Rubrobacteridae);该环境中有71.4%的序列与已有效发表菌株的序列相似性小于97%,代表着放线菌新类群,其中部分序列形成了几个独立的进化分支,可能代表更高级的新分类单元。【结论】红井子土壤中的放线菌组成具有丰富的多样性,并有新放线菌分类单位的潜在资源,值得进一步进行开发研究。     

新疆艾比湖沉积物中免培养放线菌的多样性北大核心CSCD

【目的】采用免培养的方法研究新疆艾比湖湖底沉积物放线菌组成及多样性。【方法】采集并混合5份艾比湖湖底沉积物样本,并提取其总DNA,采用放线菌通用引物对其16S r RNA基因序列进行Touchdown PCR,构建放线菌16S r RNA基因文库。蓝白斑筛选后随机挑选白色克隆分析,利用限制性内切酶HhaⅠ进行酶切分型,挑选具有独特限制性片段差异的阳性克隆进行测序分析。序列经Chimera Check检测,BLAST同源比对及构建16S r RNA基因序列系统发育树。【结果】随机挑选192个白色克隆,其中166个为阳性克隆,选取51个具有独特限制性片段差异的克隆进行序列分析。测序结果进行比对以及Chimera Check检测后,共获得36个可操作单元(Operational Taxonomic Units,OTUs),Gen Bank注册号为KR182090-KR182131。文库覆盖度结果表明克隆文库涵盖了本环境中90.4%的放线菌类群。聚类结果显示,艾比湖湖底沉积物中放线菌分为2个类群,第1个类群属于放线菌门(Actinobacteria),放线菌纲(Actinobacteria)中的放线菌目(Actinomycetales)、丙酸杆菌目(Propionibacteriales)、微球菌目(Micrococcales)和棒杆菌目(Corynebacteriales)4个目,该类群占克隆文库18.1%;另外1个类群属于Unclassified Actinobacteria的类群,分为3个不同的group,占整个克隆文库的81.9%。【结论】新疆艾比湖湖底沉积物中存在多种未知的放线菌类群。     

引物应用策略：基于2对古菌16S rRNA基因通用引物对环境样品古菌多样性分析

【目的】找到适宜的16S rRNA基因通用引物应用策略,应对复杂环境微生物多样性调查,尤其目前高速发展的高通量测序技术带来的巨大挑战。【方法】用Oligocheck软件分别将两对应试的古菌16S rRNA基因通用引物与RDP(Ribosomal database project)数据库中古菌16S rRNA基因序列进行匹配比对。用两对应试引物分别构建海洋沉积物样品的古菌16S rRNA基因文库。【结果】软件匹配结果显示引物f109/r958与目的基因的匹配程度高于引物f21/r958。该结果与古菌16S rRNA基因文库RFLP分析、古菌多样性指数分析结果相吻合。数据还表明,2对引物的综合文库能更好满足该沉积物样品的古菌多样性分析。【结论】选用与数据库中目的基因匹配性高的通用引物和多个引物的联合使用,可以有效提高环境样品微生物多样性调查的分辨率。     

Microbial diversity of cold-seep sediments in Sagami Bay, Japan, as determined by 16S rRNA gene and lipid analyses

Microbial communities in Calyptogena sediment and microbial mats of Sagami Bay, Japan, were characterized using 16S rRNA gene sequencing and lipid biomarker analysis. Characterization of 16S rRNA gene isolated from these samples suggested a predominance of bacterial phylotypes related to Gammaproteobacteria (57-64%) and Deltaproteobacteria (27-29%). The Epsilonproteobacteria commonly found in cold seeps and hydrothermal vents were only detected in the microbial mat sample. Significantly different archaeal phylotypes were found in Calyptogena sediment and microbial mats; the former contained only Crenarchaeota clones (100% of the total archaeal clones) and the latter exclusively Euryarchaeota clones, including the anaerobic oxidation of methane archaeal groups ANME-2a and ANME-2c. Many of these lineages are as yet uncultured and undescribed groups of bacteria and archaea. Phospholipid fatty acid analysis suggested the presence of sulphate-reducing and sulphur-oxidizing bacteria. Results of intact glyceryl dialkyl glyceryl tetraether lipid analysis indicated the presence of nonthermophilic marine planktonic archaea. These results suggest that the microbial community in the Sagami Bay seep site is distinct from previously characterized cold-seep environments.     

云南热带户用沼气池的原核生物群落结构研究

【目的】揭示云南热带农村户用沼气池中的原核生物(细菌和古菌)的群落结构特征。【方法】采用16S r RNA基因克隆文库技术对云南(北)热带代表性气候区的户用沼气池中的原核生物(细菌和古菌)多样性进行研究。【结果】得到细菌330条有效序列,划分为108个OTUs,文库覆盖度为81.5%;古菌有效序列185条,划分为17个OTUs,文库覆盖度为97.8%。通过Gen Bank数据库进行相似性比对与系统发育分析,结果表明:大部分细菌为未知细菌(Unclassified bacteria,占24.19%),优势细菌类群归属拟杆菌门(Bacteroidetes,占23.58%)、绿弯菌门(Chloroflexi,占21.46%)、厚壁菌门(Firmicutes,占13.91%)和变形菌门(Proteobacteria,占8.74%);古菌主要的优势类群为乙酸盐营养型的甲烷八叠球菌目(Methanosarcinales)的鬃毛甲烷菌属(Methanosaeta,占76.75%);此外还检测到少量未培养的泉古菌门细菌(Crenarchaeota,占9.19%)。【结论】云南(北)热带代表性气候区的农村户用沼气池中的微生物种类十分丰富,不同微生物种类的丰度存在明显差异,并存在明显优势种群,且细菌比古菌具有更丰富的多样性。     

Communities of archaea and bacteria in a subsurface radioactive thermal spring in the Austrian Central Alps, and evidence of ammonia-oxidizing Crenarchaeota

Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (alpha, beta, gamma, and delta), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (delta-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.     

Phylogenetic Analyses of Archaeal Ribosomal DNA Sequences from Salt Pan Sediment of Mumbai,India

The archaeal diversity in salt pan sediment from Mumbai, India, was investigated by 16S rDNA-dependent molecular phylogeny. Small-subunit rRNA (16S rDNA) from salt pan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. Thirty-two unique phylotypes were obtained by PCR-based RFLP of 16S rRNA genes using endonucleases Hae111 and Msp1, which were most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain Archaea including both Crenarchaeota and Euryarcheaota. However, none of the retrieved Crenarchaeota sequences could be grouped with previously cultured Crenarchaeota. Of all the Euryarcheaota sequences, three sequences were related to Haloarchaea, two were related to cultured Methanosarcina sp., and the remaining sequences were affiliated with uncultured Methanosarcina sp., Methanogenium sp., and Methanolobus sp. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of a highly diverse archaeal population and may represent novel sequences and organisms specially adapted to the salt pan sediment of Mumbai. These findings are of fundamental value for understanding the complexity of salt pan ecosystems.     

Phylogenetic diversity of planktonic archaea in the estuarine region of East China Sea

To examine the diversity and structure of archaeal communities in the Yangtze River estuarine region of East China Sea (ECS), the 16S rRNA gene clone libraries of two typical sites were constructed with the archaea-specific primers. In total, 71 clones randomly selected were screened by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and 21 clones with unique RFLP pattern were sequenced. All the sequences are clustered into the two groups of Marine Group I (MGI) and Marine Group II (MGII) which are affiliated with the phyla Crenarchaeota and Euryarchaeota, respectively. MGI clones dominate both libraries with 20 MGI sequences obtained. The majority of sequences are closely related to uncultured marine archaea except for two sequences of which the nearest neighbor is a newly identified isolate of nitrifying marine archaeon Nitrosopumilus maritimus (98% identity). The results indicate that ECS coastal waters are inhabited by archaeal community with low dominance and high diversity corresponding to the complex estuarine environments, suggesting that archaea may perform an important role in the estuarine ecosystem.     

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。