一种直接用于PCR扩增的山羊瘤胃微生物DNA提取方法的建立

目的建立提取高质量的瘤胃微生物DNA的方法,为采用免培养技术研究山羊瘤胃微生物奠定基础。方法采集山羊瘤胃内容物,用SDS高盐法提取微生物总DNA,以通用引物扩增细菌和古细菌的16SrDNA。结果提取到的瘤胃微生物总DNA片段大于23kb,PCR能够扩增出细菌和古细菌的16SrDNA片段。结论用该提取方法得到的山羊瘤胃微生物总DNA能够满足后续实验的需要。     

昆明盐矿古老岩盐沉积中可培养细菌多样性研究

为了了解昆明盐矿古老岩盐沉积中可培养细菌的多样性,用MBA和ISP2分离和培养了昆明盐矿卤水和盐晶中的细菌44株。发现盐晶中的可培养好氧细胞数量(3·1×103~3·7×106CFU/g)远远高于卤水中的数量(1·3~6·3×103CFU/L)。分离所得纯培养物的16SrDNA序列系统发育分析结果表明,44株菌可分为4大类群34个不同的分类单元(16SrDNA序列相似性大于97%为同一分类单元)。24株属于厚壁菌门(Firmicutes,54·6%),2株属于变形菌门α亚群(α-Proterbacteria,4·6%),4株属于变形菌门γ亚群(γ-Proterbacteria,9·1%),14株属于放线细菌门(Actinobacteria,31·7%)。卤水和盐晶中的优势菌都是Bacillus属菌(26·1%和59·9%)。据16SrDNA序列相似性分析发现7株菌为可能的新种或属。此外,还筛选到7株抗菌活性菌株。研究表明,昆明盐矿古老岩盐沉积中,不仅含有较为丰富的微生物物种多样性,并且存在许多未被认识的新物种和生物活性菌株,为古老岩盐沉积中微生物的深入研究奠定了基础。     

太平洋帕里西维拉海盆细菌多样性的非培养的初步分析

从环境中直接提取总DNA ,构建了含 32个有效转化子的太平洋帕里西维拉 (PareceVela)海盆 5 0 10米深处底泥的细菌 16SrRNA基因文库。测序结果表明 ,可以将 32条有效序列分为 17个不同的分类单元。大部分序列与已知细菌类群的 16SrDNA序列相似性较高 ,归属于Proteobacteria的gamma亚群、alpha亚群和海洋非培养细菌类群 ,主要分布在Pseudoalteromonas属、Halomonas属、Alcanivorax属、Photobacterium属、Acinetobacter属 ;部分序列与已知细菌类群的 16SrDNA序列同源性较低 ,可能代表新的分类单位。研究结果表明 ,帕里西维拉海盆不仅含有丰富的微生物物种 ,并且存在尚未被认识的新物种     

东北虎粪细菌区系的16S rRNA基因序列分析

为研究东北虎粪微生物区系建立了东北虎粪细菌的16SrDNA文库。通过EcoRⅠ和HindⅢ分别对阳性克隆进行酶切分析,从东北虎的16SrDNA文库中分别获得了15个具有酶切差异的克隆。BLAST分析结果显示,在15个克隆中,10个克隆与梭菌属成员有97%以上的同源性,其中有6个序列与诺维梭菌A型(Clostridiumnovyitype A)有99%的同源性,为诺维梭菌A型;4个序列与猪粪细菌RT-18B(Swine manure bacteriumRT-18B)有97%的同源性,为消化链球菌属(Peptostreptococcus)成员。其它序列与GenBank中登录的序列同源性低于97%,为5种未培养细菌,其中4种16SrRNA基因序列分别与Clostridiumpascui、破伤风梭菌E88(ClostridiumtetaniE88)、梭菌(Clostridiumsp.)14505及产气荚膜梭菌(Clostridiumperfringens)有94%~95%的相似性。第5种与肉杆菌(Carnobacteriumsp.)R-7279株有94%的同源性。     

细菌群体感应抑制活性微生物Zou 03的筛选与鉴定

从近海区生态环境中分离纯化98株海洋菌株,以根癌农杆菌WCF47为敏感检测菌株,筛选出1株具细菌群体感应抑制活性的菌株Zou03,对其进行形态、生理生化特征鉴定和16S rDNA分子鉴定。结果显示,Zou03具枯草芽胞杆菌(Bacillus subtilis)的典型特征,其16S rDNA序列通过对比分析,与GenBank中枯草芽胞杆菌16SrDNA的部分序列同源性为100%。综合形态、生化特征及16S rDNA序列对比分分析,鉴定菌株Zou03为枯草芽胞杆菌。表明近海区生态环境中存在具有抑制细菌群体感应活性的微生物,有利于海洋微生物资源开发,为以致病菌群体感应系统为靶点的新型疗法提供新技术。     

沙坡头地区根瘤菌DNA同源性及16SrDNA全序列

数值分类和多位点酶电泳分析表明,分离自宁夏沙坡头 地区的12株根瘤菌构成一个独立的表观群。对这一菌群进行了DNA同源性和群内中心菌株1 6SrDNA全序列分析。12个菌株的G+C mol%在56.4～62.2范围内;群内DNA同源性为72.3% ～9.5%,大于70%,属种内水平;中心株N220的16SrDNA全序列与参比菌株的序列比较,从 模拟系统发育树看出,它与三株土壤杆菌、三株根瘤菌的16SrDNA序列同源性在94.8%～99 .2%的相似性水平上构成一个分支,看来沙坡头地区这群根瘤菌是一个独立的新种群。     

一株拮抗姜瘟青枯假单胞杆菌的链霉菌的分离及鉴定

从生姜田土中分离到一株对姜瘟青枯假单胞杆菌(PseudomonassolanacarumSmith)有强拮抗作用的链霉菌菌株SR 11,研究拮抗性表明,对革兰氏阳性细菌、革兰氏阴性细菌以及多种病原真菌均有很强的抑制作用。对该菌株进行形态特征、培养特征、生理生化、细胞壁组分分析及16SrDNA序列分析。基内菌丝无横隔、不断裂,气生菌丝多分枝;孢子丝波曲至螺旋形,孢子椭圆形,表面光滑。细胞壁化学组分Ⅰ型,糖型C。在培养成熟后,气丝变为灰色,可闻到浓烈的土味。以16SrDNA序列为基础构建了包括13株相关种属细菌在内的系统发育树,其中,与12个模式链霉菌株的16SrDNA序列的同源性为96 5 %～98 3%。     

生物造粒流化床污水处理反应器的微生物多样性

为了研究生物造粒流化床污水处理反应器颗粒污泥的微生物种群多样性,分别从生物造粒流化床10、60和110cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA,PCR扩增后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱,对特征条带进行序列测定及序列同源性分析。16S rRNA序列分析表明,获得的18个OTUs均属于细菌域,其中61%属于变形菌,17%属于放线菌,11%属于低G C革兰氏阳性菌,11%属于其它未知细菌。     

海南温泉嗜热菌的16S rDNA分析

目的:确定24株海南温泉嗜热菌菌株的分类地位。方法:Blastn分析菌株16S rDNA序列同源性;邻接法构建菌株16SrDNA序列系统发育进化树并分析菌株的进化位置;Clustax比对分析菌株的相似度和进化距离。结果:菌株LY5和LY4的16SrDNA序列与Geobacillus pallidus strain B1,partial sequence(GenBank:HM030740.1)的16S rDNA序列同源性分别为98%和97%,其他菌株的16S rDNA序列与Geobacillus subterraneus,strain R-35641(GenBank:FN428689.1)的16S rDNA序列的同源性均大于96%。Clustax比对分析表明26株菌16S rDNA序列前段(1～70bp)、中段(70bp～1 420bp)、后段(1 420～1 484bp)的相似度分别为40%、100%和60%,进化距离分析表明菌株GT7、LY4和LY5与其他菌株进化距离较远,其余菌株之间进化距离差异不明显。综上所述,初步将24株温泉嗜热菌鉴定为土芽孢杆菌属(Geobacillus sp.)。结论:16S rDNA序列分析可用于温泉嗜热菌的鉴定。     

山东肥城寒武纪竹叶状石灰岩菌群分析

为了探讨微生物活动与地层形成的关系,对山东肥城万灵山寒武纪竹叶状石灰岩中的微生物进行了培养分离,用PCR方法获得了它们的16S DNA序列,通过序列比对确定了这些微生物的种属,并对这些细菌进行了系统发育进化分析.结果 表明这些细菌属于芽胞杆菌属(Bacillus)、节杆菌属(Arthrobacter)及未培养细菌(Uncultured bacterium),万灵山竹叶状石灰岩的微生物菌群分布以革兰阳性芽胞杆菌占优势.     

日粮中添加鱼油后肉牛瘤胃细菌区系的变化

目的：研究肉牛日粮中添加2%鱼油后瘤胃细菌区系的变化。方法：分别于添加鱼油前后取瘤胃液提取总DNA,根据细菌通用引物27F/1492R扩增16S rDNA基因序列,分别构建2个16S rDNA基因文库;从每个文库中各随机挑取384个克隆,对阳性克隆用HhaⅠ进行限制性片段长度多态性（RFLP）分析、聚类,取各RFLP类群中的代表克隆进行16S rDNA序列测定,构建系统发育树,研究细菌区系多样性的变化。结果：添加鱼油前、后,文库的RFLP图谱分别可以分成74和41个RFLP类群;添加鱼油前后的优势菌群均为噬纤维菌-屈挠杆菌-拟杆菌（CFB）和低（G＋C）含量的革兰阳性菌（LGCGPB）,但添加鱼油后文库中LGCGPB比例降低,而且未检测到纤维菌门细菌;有50%～60%的测定序列与GenBank数据库中的序列相似性高于97%,90%以上为未培养的细菌;添加鱼油后瘤胃细菌的多样性减少。结论：日粮添加鱼油后CFB比例增高但多样性下降,LGCGPB、纤维杆菌比例下降,这一结果为调控瘤胃发酵提供了依据。     

Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes

Comparative DNA sequence analysis of 16S rRNA genes (rDNA) was undertaken to further our understanding of the make-up of bacterial communities in the rumen fluid of dairy cattle. Total DNA was extracted from the rumen fluid of 10 cattle fed haylage/corn silage/concentrate rations at two different times. Rumen samples were collected on two separate occasions from five cows each. In experiment 1, 31 cloned rDNA sequences were analysed. In experiment 2, DNA extractions were amplified using either 12 or 30 cycles of PCR in order to examine biases introduced during the reactions. A set of 53 sequences were analysed in experiment 2 from DNA amplified using 12 cycles and 49 sequences from PCR using 30 cycles. Sequences from the 5' end of 16S rRNA gene were compared with existing sequences in the Ribosomal Database Project. Clones from experiment 1 produced a data set in which 55% of the sequences were similar to low G+C Gram-positive bacteria related to the genus Clostridia, the majority of which were closely related to bacteria in Cluster XIV. Approximately 30% of the cloned sequences were related to bacteria in the Prevotella-Bacteroides group. Clones from experiment 2 produced a data set in which the majority of sequences were related to the Prevotella-Bacteroides group, regardless of the number of cycles of PCR. The remaining sequences clustered with members of the genus Clostridia. The majority of rDNA sequences analysed in this study represent novel rumen bacteria which have not yet been isolated.     

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) was estimated by 16S rDNA homology analysis. Two rumen 16S rDNA libraries were constructed. Of the 194 clones in the library of yak rumen, the sequences were mainly clustered to two phyla, low G+C Gram-positive bacteria (LGCGPB, 54.12% total clones) and Bacteroidetes (30.93%), respectively. While in the 197 clone-library of the cattle rumen, the sequences were mainly related to three phyla, Bacteroidetes (39.59%), gamma-Proteobacteria (26.9%) and LGCGPB (22.34%), respectively. The sequence analysis indicated that more than half of the species harbored in yak rumen belonged to the not-yet-cultured groups at <90% 16S rDNA similarity levels with cultured species, while 36% 16S rDNA sequences amplified from the rumen of Jinnan cattle fell in these catalogues. By comparing the uncultured sequences in yak rumen with those in Jinnan cattle and cow, the former formed distinct clusters loosely related to the later, implying that yak rumen could harbor some special prokaryote phyla. 10.8% sequences retrieved in yak rumen were related to the known rumen fibrolytic bacterial species; however none was related to the known amylolysis species. While 4% and 17.8% sequences retrieved from Jinnan cattle rumen were related to cultured fibrolytic and amylolysis species, respectively. The bacterial structures seemed to be in accordance with the feed of the two kinds of animals. In both rumens, retrieved methanogenic Archaea-related 16S rDNA sequences were at an unreasonable low level; in addition, none sequence was related to Ruminococcus albus, a classical rumen fibrolytic species. The reason can be due to the experimental biases.     

共存于厌氧真菌分离培养液中瘤胃甲烷菌的检测及其多样性分析

对分离自山羊瘤胃的真菌分离培养液中甲烷菌进行16SrDNA扩增、DGGE分析、RFLP及测序分析,研究共存于真菌分离培养液中甲烷菌的种类及其多样性。DGGE结果显示:从厌氧真菌分离至第45代,甲烷菌多样性指数由1·32降至0·99,相似性最低为34·7%;第45代至62代,多样性指数由0·99升至1·15,相似性最低为89·2%。RFLP多态性分析69个克隆共得到5个操作分类单元,选择其中6个具有代表性的序列进行测序。序列及系统进化分析表明,属于其中3个操作分类单元的克隆最相似菌都是UnculturedarchaealsymbiontPA202,相似性均为95%,没有与这些克隆相似性较高的已培养甲烷菌;属于另外2个操作分类单元的克隆最相似菌都是Unculturedrumenmethanogen956,相似性均为97%,最相似已知菌为Methanobrevibactersp.NT7,相似性为97%。结果表明,真菌培养液中存在目前尚未分离培养的瘤胃甲烷菌。     

应用rpoB和16S rDNA基因的变性梯度凝胶电泳技术对山羊瘤胃细菌多样性的研究

采用免培养的rpoB和16S rDNA基因的变性梯度凝胶电泳技术(DGGE)对3种山羊(波尔山羊,内蒙古绒山羊,四川南江黄羊)瘤胃细菌优势菌群结构进行了比较分析。研究结果显示rpoBDGGE图谱中条带数目少于16S rDNA图谱,并且条带分离效果明显,更有利于分析瘤胃细菌群落组成。从两种DGGE图谱中均可以发现3种山羊瘤胃细菌具有一定的相似性,种内个体间相似性明显高于种间相似性,这说明寄主品种是影响瘤胃细菌种群构成的一个重要因素。同时进行了部分优势细菌16S rDNA基因V6-V8区序列的系统发育分析。基因序列分析表明,DGGE图谱中优势条带的16S rDNA基因序列中有4条克隆的序列与基因库最相似菌的相似性大于97%,余下的克隆序列相似性在89%～96%之间,其中13条序列的与之相似性最高的序列均来自于未被鉴定的瘤胃细菌。     

Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs

Aims:  To screen a pair of primers suitable for denaturing gradient gel electrophoretic (DGGE) analysis of ruminal methanogenic Archaea and to detect the archaeal communities in the rumen of goat.
Methods and Results:  Nine primer pairs for 16S rDNA of methanogenic Archaea , including six for directed polymerase chain reaction (PCR) and three for nested PCR were first evaluated by PCR amplification of the total DNA from rumen fluids and bacteria. The DGGE analysis of rumen fluids was then conducted with three primer sets (344fGC/915r, 1106fGC/1378r and 519f/915rGC) of the nine pairs tested. Good separation and quality of patterns were obtained in DGGE analysis with primer pairs 1106fGC/1378r and 519f/915rGC. A total of 40 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic Archaea while primer pair 519f/915rGC had better amplification ranges than the other two primer pairs.
Conclusions:  The procedure of DGGE analysis with primer pair 519f/915rGC was more suitable for investigating methanogenic archaeal community in the rumen. The dominant methanogenic Archaea in the rumen of goat was Methanobrevibacter sp. and an unidentified methanogenic Archaea .
Significance and Impact of the Study:  One pair of primers suitable for DGGE analysis of ruminal methanogenic Archaea was obtained and the molecular diversity of ruminal methanogenic Archaea in goat was investigated by PCR-DGGE.     

Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA.

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and WEISSELLA: Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant's life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.     

16S rDNA library-based analysis of ruminal bacterial diversity

Bacterial 16S rDNA sequence data, incorporating sequences > 1 kb, were retrieved from published rumen library studies and public databases, then were combined and analysed to assess the diversity of the rumen microbial ecosystem as indicated by the pooled data. Low G+C Gram positive bacteria (54%) and the Cytophaga-Flexibacter-Bacteroides (40%) phyla were most abundantly represented. The diversity inferred by combining the datasets was much wider than inferred by individual studies, most likely due to different diets enriching for bacteria with different fermentative activities. A total of 341 operational taxonomic units (OTU) was predicted by the Chao1 non-parametric estimator approach. Phylogenetic and database analysis demonstrated that 89% of the diversity had greatest similarity to organisms which had not been cultivated, and that several sequences are likely to represent novel taxonomic groupings. Furthermore, of the 11% of the diversity represented by cultured isolates (> 95% 16S rDNA identity), not all of the bacteria were of ruminal origin. This study therefore reinforces the need to reconcile classical culture-based rumen microbiology with molecular ecological studies to determine the metabolic role of uncultivated species.     

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.