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。     

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。     

菌群16S rRNA基因测序的聚类分析方法研究进展

16S核糖体RNA（16S rRNA）基因测序是微生物分析的重要手段。16S rRNA基因测序的原始数据复杂,存在许多误差,分析前一般需要先进行序列质量控制,即对数据进行去噪、去冗余和去嵌合,最终将质量控制后的数据分为可操作分类单元（OTU）或ASV,在OTU或ASV基础上再进行菌群的各种分析。经过多年改进,聚类方法逐渐以UPARSE、DADA2、Deblur和UNOISE3等为主流,OTU聚类已经不能满足研究的需求,而ASV聚类使得菌群分析更加准确。本文除了综述聚类方法的研究进展外,还介绍了USEARCH、MOTHUR和QIIME等多种16S基因测序分析工具软件的相关研究进展。

     

基于16S rRNA基因测序分析微生物群落多样性

微生物群落多样性的研究对于挖掘微生物资源,探索微生物群落功能,阐明微生物群落与生境间的关系具有重要意义。随着宏基因组概念的提出以及测序技术的快速发展,16S rRNA基因测序在微生物群落多样性的研究中已被广泛应用。文中系统地介绍了16S rRNA基因测序分析流程中的四个重要环节,包括测序平台与扩增区的选择、测序数据预处理以及多样性分析方法,就其面临的问题与挑战进行了探讨并对未来的研究方向进行了展望,以期为微生物群落多样性相关研究提供参考。     

16S rRNA基因高通量测序分析水性涂料微生物群落结构及多样性

微生物对水性涂料产品的稳定性起着重要作用,本实验以广东省某涂料厂的水性涂料为研究对象,利用16S rRNA基因高通量测序技术检测其微生物群落结构及多样性。结果表明5个水性涂料样本在97%的相似水平下共获得有效序列总数224 801,涵盖了10门16纲30目46科59属的细菌;物种组成与相对多样性由高到低依次分别为A4、A5、A1、A2和A3;水性涂料样本A1、A2和A3微生物群落结构与聚集规律较为相似,优势菌群均为类芽孢杆菌门、芽胞乳杆菌属和固氮菌;水性涂料样本A5中相对丰度为57.3%的产己酸细菌属于特异菌群。本研究解析了水性涂料中微生物群落结构、相对丰度及多样性,为建立和完善水性涂料中微生物防控体系提供理论支撑。     

基于16S rRNA基因序列分析1例健康人龈上菌群

目的构建细菌16S rRNA基因文库分析健康人龈上菌群的组成。方法取1例健康成年女性龈上菌斑并构建细菌16S rRNA基因文库,分析其龈上菌群组成。结果 1例健康人龈上菌斑细菌的种水平分类有62种,其中可以培养的细菌有34种而尚无法培养的细菌有28种(45.1%);新发现的细菌物种有17种(27.4%);缓症链球菌是克隆子数最多的优势菌种;链球菌属、奈氏菌属和嗜血杆菌属占文库的60%,为主要菌群;构建的细菌16S rRNA基因文库的覆盖率为95%,文库的均匀度值为0.016。结论 1例健康人龈上菌群中45.1%的细菌尚无法分离培养、27.4%的物种尚不清楚,未培养菌及尚不清楚的菌种中可能藏匿着与口腔疾病密切相关的致病菌,全面地了解健康人龈上菌群的组成有助于研究龋病的发病机制。     

基于16S rRNA基因检测双歧杆菌的方法

本文综述了基于16S rRNA序列(16S rDNA)检测、鉴定双歧杆菌的方法。包括基因探针法、荧光原位杂交法、PCR扩特异性片段法、多重PCR法、荧光定量PCR法和PCR-DGGE/TGGE法等。     

地衣芽孢杆菌16S rRNA基因的TD-PCR扩增及系统发育分析

运用16SrRNA基因序列分析了中国工业微生物菌种保藏管理中心(CICC)保存的30株地衣芽孢杆菌的系统发育关系,结果显示:24株菌株位于地衣芽孢杆菌系统发育分支;3株菌株位于蜡状芽孢杆菌-苏云金芽孢杆菌系统发育分支;1株菌株位于枯草芽孢杆菌系统发育分支;2株菌株与其它地衣芽孢杆菌菌株间序列同源性为96.4%~97.4%,明显低于其它地衣芽孢杆菌菌株间同源性,分类地位不明确,有待进一步讨论。通过比较分析16SrRNA基因5′端500bp、3′端500bp以及其全基因的系统发育树,表明16SrRNA基因5′端500bp可以很好的代表全基因序列进行系统发育研究,可用于区分地衣芽孢杆菌、枯草芽孢杆菌以及蜡状芽孢杆菌分支。     

基于16S rRNA基因高通量测序研究狞猫肠道微生物多样性

[目的]研究狞猫肠道微生物多样性特征。[方法]对7只健康成年野生狞猫（2只雄性,5只雌性;2只来自济南野生动物园,5只来自威海野生动物园）粪便微生物16S rRNA基因V3-V4区进行高通量测序,对狞猫肠道微生物多样性进行研究。[结果] 7只狞猫共获得肠道微生物16S rRNA基因V3-V4区有效序列1458741条,平均208392条,序列平均长度433 bp。通过以97%的序列相似性进行分类,获得操作分类单元（OTU）平均 233个。经序列比对和分类鉴定,这些OTU都属于细菌域,包括13个门,26个纲,43个目,75个科,119个属。其中,丰度最高的细菌门是厚壁菌门（Firmicutes,平均占61.7%）、放线菌门（Actinobacteria,12.42%）、拟杆菌门（Bacteroidetes,7.79%）、梭杆菌门（Fusobacteroidetes,7.79%）和变形菌门（Proteobacteria,7.53%）。丰度最高的科依次是消化链球菌科（Peptostreptococcaceae,平均占16.15%）,梭菌科I（Clostridiaceae_I,14.78%）,毛螺菌科（Lachnospiraceae,13.13%）,红蝽杆菌科（Coriobacteriaceae,12.31%）等。丰度最高的属是柯林斯氏菌属（Collinsella,11.44%）,艰难梭菌属（Peptoclostridium,10.91%）,狭窄梭菌属1（Clostridium_sensu_stricto_1,10.3%）,拟杆菌属（Bacteroides,7.41%）,消化链球菌属（Peptostreptococcus,5.21%）等。7只狞猫的肠道微生物中有平均15.35%的OTU没有归类到属。样本间聚类分析结果表明,来自同一动物园的样本聚为一支。[结论]本文通过高通量测序技术研究了狞猫肠道微生物的组成和多样性特征,为狞猫的救护饲养和消化生理学研究提供基础数据。     

16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

结合宏基因组末端随机测序和16S rDNA技术分析温室黄瓜根围土壤细菌多样性

土壤细菌在温室土壤环境中具有十分重要的生态功能,与温室作物以及微生物内部存在互作关系。研究土壤细菌的群落结构组成,有助于了解土地利用变化与生态环境效应之间的关系。结合16S rRNA基因克隆文库和宏基因组末端测序对温室黄瓜根围土壤细菌的多样性进行了分析。在16S文库中,根据97%的序列相似性水平划分OTU,共有35个OTU,其中优势菌群是γ-Proteobacteria,其次为Firmicutes,Bacillus为优势细菌。在纲分类水平上,16S文库和宏基因组末端测序结果均包含γ-Proteobacteria、α-Proteobacteria、δ-Proteobacteria、β-Proteobacteria、Actinomycetales和Firmicutes,各纲比例有差别;在优势种群属水平上,末端测序的结果包含的属多于16S文库(4035);在优势细菌种类上,两者反映的结果一致,均为Bacillus。但是,宏基因组末端测序包含了大多数的弱势种群,更能反映细菌多样性的真实水平。与露地土壤细菌16S文库相比较,土壤细菌多样性降低,这可能与温室多年连作,种植蔬菜种类单一直接相关。     

PCR-amplification of mixed 16S rRNA genes from an anaerobic, cyanide-degrading consortium

Abstract The microorganisms participating in the anaerobic biodegradation of cyanide were characterized using 16S rRNA genes as genetic markers of diversity. Segments of mixed population 16S rRNA genes were amplified using the polymerase chain reaction (PCR) and prokaryote-specific amplification primers. Restriction fragment length polymorphism (RFLPs) and screening with the 926f universal sequencing primer were used to categorized the cloned PCR products. Six unique prokaryote sequence were obtained, including four similar to methanogens and two similar to Gram-positive eubacteria.     

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis

Abstract: Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.     

嗜盐菌HBCC-2的16S rRNA基因测序分析及其培养特性

从连云港台南盐场海盐生产区中分离纯化到一株嗜盐古菌HBCC-2,该菌株经PCR扩增后,测定其16S rRNA基因序列,采用BLAST软件对基因库中基因序列进行同源性比较,选取其相似性序列,采用Clustalx1.8和MEGA3.1软件对其16S rDNA序列进行了系统发育分析研究,结果表明HBCC-2菌株与菌株Halorubrum sp.GSL5.48的相似性达99%,结合其形态观察及生理生化反应特性,初步确定该菌株属于嗜盐红菌属(Halorubrum),菌株HBCC-2的16S rDNA序列已登陆到GenBank,其序列号为EF687739.通过比较不同NaCl浓度、pH和培养温度对该菌株生长的影响情况,研究了该菌株的生长特性,结果表明NaCl浓度为4mol/L、温度为35℃和pH为7.0的培养条件下其生长最佳.     

Characterization of the depth-related changes in the microbial communities in Lake Hovsgol sediment by 16S rRNA gene-based approaches

The undisturbed sediment of Lake Hovsgol (Mongolia) is scientifically important because it represents a record of the environmental changes that took place between the Holocene (the present age) and Pleistocene (the last ice age; 12,000 14C years before present day). Here, we investigated how the current microbial communities change as the depth increases by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes of the microbial communities. The microbial diversity, as estimated by the Shannon index, decreased as the depth increased. In particular, significant changes in archaeal diversity were observed in the middle depth (at 39-42 cm depth of total 60 cm depth) that marks the border between the Holocene and Pleistocene. Phylotype belonging to Beta-and Gamma-Proteobacteria were the predominant bacteria and most of these persisted throughout the depth examined. However, as the depth increased, some bacteria (some genera belonging to Beta-Proteobacteria, Nitrospira, and OP8-9) were not detectable while others (some genera belonging to Alpha-, Beta-, Gamma-Proteobacteria) newly detected by DGGE. Crenarchaea were the predominant archaea and only one phylotype belonging to Euryarchaea was found. Both the archaeal and bacterial profiles revealed by the DGGE band patterns could be grouped into four and three subsets, respectively, subsets that were largely divided by the border between the Holocene and Pleistocene. Thus, the diversity of the current microbial communities in Lake Hovsgol sediments decreases with increasing depth. These changes probably relate to the environmental conditions in the sediments, which were shaped by the paleoclimatic events taking place between the Holocene and Pleistocene.     

16S rRNA analysis of Listeria monocytogenes and Brochothrix thermosphacta

Abstract Listeria monocytogenes and Brochothrix thermosphacta were investigated by the 16S rRNA cataloguing approach in order to determine their phylogenetic relationship. Both species are specifically, although moderately related, forming one of several sublines within the Bacillus-Lactobacillus-Streptococcus cluster of the ' Clostridium ' subbranch of Gram-positive eubacteria.     

Turkey fecal microbial community structure and functional gene diversity revealed by 16S rRNA gene and metagenomic sequences

The primary goal of this study was to better understand the microbial composition and functional genetic diversity associated with turkey fecal communities. To achieve this, 16S rRNA gene and metagenomic clone libraries were sequenced from turkey fecal samples. The analysis of 382 16S rRNA gene sequences showed that the most abundant bacteria were closely related to Lactobacillales (47%), Bacillales (31%), and Clostridiales (11%). Actinomycetales, Enterobacteriales, and Bacteroidales sequences were also identified, but represented a smaller part of the community. The analysis of 379 metagenomic sequences showed that most clones were similar to bacterial protein sequences (58%). Bacteriophage (10%) and avian viruses (3%) sequences were also represented. Of all metagenomic clones potentially encoding for bacterial proteins, most were similar to low G+C Gram-positive bacterial proteins, particularly from Lactobacillales (50%), Bacillales (11%), and Clostridiales (8%). Bioinformatic analyses suggested the presence of genes encoding for membrane proteins, lipoproteins, hydrolases, and functional genes associated with the metabolism of nitrogen and sulfur containing compounds. The results from this study further confirmed the predominance of Firmicutes in the avian gut and highlight the value of coupling 16S rRNA gene and metagenomic sequencing data analysis to study the microbial composition of avian fecal microbial communities.     

16S rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca

Mucosa-associated microbiota from different regions of the gastrointestinal (GI) tract of adult broilers was studied by analysis of 16S rRNA gene sequences. The microbiota mainly comprised Gram-positive bacteria along the GI tract. Fifty-one operational taxonomic units (OTUs) (from 98 clones) were detected in the ceca, as compared with 13 OTUs (from 49 clones) in the crops, 11 OTUs (from 51 clones) in the gizzard, 14 OTUs (from 52 clones) in the duodenum, 12 OTUs (from 50 clones) in the jejunum and nine OTUs (from 50 clones) in the ileum. Ceca were dominantly occupied by clostridia-related sequences (40%) with other abundant sequences being related to Faecalibacterium prausnitzii (14%), Escherichia coli (11%), lactobacilli (7%) and Ruminococcus (6%). Lactobacilli were predominant in the upper GI tract and had the highest diversity in the crop. Both Lactobacillus aviarius and Lactobacillus salivarius were the predominant species among lactobacilli. Candidatus division Arthromitus was also abundant in the jejunum and ileum.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。