大港孔店油田水驱油藏微生物群落的分子分析

通过多聚酶链式反应温度梯度凝胶电泳（PCRTGGE）和构建16S rRNA基因克隆文库两种方法对比研究了大港油田孔二北断块注水井和采油井的微生物群落结构。16S rDNA V3区PCR扩增产物的TGGE图谱分析表明,这两个油井的微生物群落结构差异很大。注水井样品的TGGE图谱中有6条主要条带,而采油井样品中只有一个条带占绝对优势。同时,建立了两个样品的16S rRNA基因克隆文库,从中分别挑选了108和50个克隆进行限制性酶切片段长度多样性分析（ARDRA）。注水井样品有33个操作分类单元（OUT）,其中6个OUT是优势类型;而采油井样品只有8个OUT,有1个OUT在文库中占绝对优势。克隆文库和TGGE的研究结果一致,均表明注水井样品的微生物多样性比采油井丰富很多。每个OUT的代表克隆序列分析结果表明,注水井样品中的细菌主要属于α、β、γ变形菌纲和放线菌纲,尤其是红细菌亚纲（47%）。采油井样品的细菌主要属于α、β、γ变形菌纲,尤其是假单胞菌属（62%）。油藏微生物多样性的分子分析可为开展微生物采油技术研究奠定基础。     

Analyses of soil fungal communities in adjacent natural forest and hoop pine plantation ecosystems of subtropical Australia using molecular approaches based on 18S rRNA genes

Soil fungal communities were studied using 18S rDNA-based molecular techniques. Soil DNA was analyzed using temperature gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP), cloning and sequencing methods, following community DNA extraction and polymerase chain reaction (PCR). The extracted community DNA was successfully amplified using the primer pair of EF4f-Fung5r which produced ca. 550bp 18S rDNA fragments. TGGE screening of the PCR products showed some differences in band position and intensity between two soil samples in adjacent natural forest (YNF) and hoop pine plantation (YHP) ecosystems at Yarraman in subtropical Australia. TGGE and SSCP could be used for screening PCR products. However, care must be exercised when interpreting the TGGE and SSCP results with respect to microbial diversity, because one band may not necessarily represent one species. It is recommended that the PCR products should be purified before TGGE or SSCP screening. SSCP screening of the clone sequences revealed differences among the clones. Sequence and phylogenetic analyses revealed that all obtained clones were affiliated to the kingdom Fungi, including three phyla, i.e., Zygomycota, Ascomycota and Basidiomycota. Our results suggested that community DNA extraction, PCR, cloning, SSCP screening of clones, sequencing of selected clones and phylogentic analyses could be a good strategy in investigation of soil fungal community and diversity.     

Molecular profiling of Bacteroides spp. in human feces by PCR-temperature gradient gel electrophoresis

A group-specific PCR-based temperature gradient gel electrophoresis (TGGE) method was developed to study the population composition of genus Bacteroides in human gut. Highly reproducible and well-separated bands in TGGE fingerprints of ten unrelated human fecal samples showed complex and host-specific Bacteroides species composition. Dynamic monitoring over 22 months of samples from one healthy 10-year-old boy indicated a relatively stable population profile of Bacteroides. The species identity of each band in TGGE gel of this boy was also resolved via comigration analysis of sequenced inserts in a Bacteroides group-specific clone library. This work provides a rapid and effective technique for analyzing the species composition of Bacteroides in human gut.     

Temperature Gradient Gel Electrophoresis Analysis of 16S rRNA from Human Fecal Samples Reveals Stable and Host-Specific Communities of Active Bacteria

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.     

Diversity and seasonal variability of beta-Proteobacteria in biofilms of polluted rivers: analysis by temperature gradient gel electrophoresis and cloning

The beta-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for beta-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described betaI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga: RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the beta-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters.     

Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries

Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.     

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.     

Isolation of a single messenger RNA and of the corresponding gene in Plasmodium berghei

A genomic library of Plasmodium berghei DNA was constructed using lambda 47.I as a vector. It represents 90% of Plasmodium genome. Genes expressed during the intraerythrocytic stage of P. berghei were isolated among the recombinant clones of the library using labelled cDNA complementary to the polyA + Plasmodium mRNA extracted during this stage. The purified coding strand of an expressed clone was utilized to catch the corresponding mRNA(s). The hybridized mRNA fraction was eluted and in vitro translated. Translation products were analyzed by gel electrophoresis; the gel fluorography revealed a single protein band of 32.500 daltons of molecular weight, corresponding to a 900bp coding region in the examined clone.     

Comparison of pmoA PCR primer sets as tools for investigating methanotroph diversity in three Danish soils

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.     

Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns

Technical developments in molecular biology have found extensive applications in the field of microbial ecology. Among these techniques, fingerprinting methods such as denaturing gel electrophoresis (DGE, including the three options: DGGE, TGGE and TTGE) has been applied to environmental samples over this last decade. Microbial ecologists took advantage of this technique, originally developed for the detection of single mutations, for the analysis of whole bacterial communities. However, until recently, the results of these high quality fingerprinting patterns were restricted to a visual interpretation, neglecting the analytical potential of the method in terms of statistical significance and ecological interpretation. A brief recall is presented here about the principles and limitations of DGE fingerprinting analysis, with an emphasis on the need of standardization of the whole analytical process. The main content focuses on statistical strategies for analysing the gel patterns, from single band examination to the analysis of whole fingerprinting profiles. Applying statistical method make the DGE fingerprinting technique a promising tool. Numerous samples can be analysed simultaneously, permitting the monitoring of microbial communities or simply bacterial groups for which occurrence and relative frequency are affected by any environmental parameter. As previously applied in the fields of plant and animal ecology, the use of statistics provides a significant advantage for the non-ambiguous interpretation of the spatial and temporal functioning of microbial communities.     

焦化废水处理厂接触氧化池中降酚菌群的苯酚羟化酶大亚基基因多样性

研究了某焦化废水处理厂接触氧化池中降酚菌群的苯酚羟化酶大亚基基因(thelargestsubunitofthemulti-componentphenolhydroxylase,LmPH)的多样性。通过温度梯度凝胶电泳(temperaturegradientgelelectrophoresis,TGGE)对比分析了氧化池4个区段(O1—O4)中降酚菌群LmPH的组成。它们的TGGE图谱完全一样,相似性为100%,表明该处理池中不同区段的降酚菌群的功能基因组成是高度相似的。以O4段的菌群为代表建立LmPH基因克隆文库,从中挑选了49个克隆测序。依据LmPH基因的DNA序列所推测的氨基酸序列完全相同的归为一类的原则,49个克隆被分为16种类型,其中优势LmPH基因主要有5种类型(多于4个克隆),而另外11种类型都只有1个克隆。与已知基因同源性超过90%的有7种类型,低于80%的有2种类型。基于氨基酸序列的系统进化树分析表明,LmPH文库中绝大部分的类型都属于低亲和常数(low-Ks)的LmPH,占所有克隆的92%。只有一个类型属于高亲和常数(high-Ks)的。因此,处理焦化废水的工业装置中不仅具有丰富多样的苯酚羟化酶基因类型,而且以编码低亲和常数的占优势地位,而过去报道的通过富集培养分离得到的降酚菌则多带有高亲和常数的酶。这提示我们传统的富集培养方法并不能筛选到生态环境中的真正优势功能菌。     

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。     

Comparison of pmoA PCR Primer Sets as Tools for Investigating Methanotroph Diversity in Three Danish Soils

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.     

TGGE分析焦化废水处理系统活性污泥细菌种群动态变化及多样性

应用TGGE指纹图谱技术对两个曝气池细菌种群的动态变化及多样性进行了研究。每3d进行1次,共8个监测时期中同一曝气池活性污泥的16SrDNAV3-PCRTGGE指纹图谱基本一致,图谱间的相似性系数(Cs)为100％。同一曝气池不同位点活性污泥的TGGE指纹图谱也完全一致。功能不同曝气池活性污泥TGGE指纹图谱存在差异,Cs为83．3％。对TJ1活性污泥TGGE图谱中9条主要条带回收、扩增、克隆建库,每个条带选4个转化子进行序列分析,结果显示TGGE条带是由序列不同的片段组成。32个序列在97％的相似性下分成16个分类操作单元(OTU),14个OTU与GenBank中已登录的细菌种群的同源性≥97％,2个OTU的同源性为95％和94％。与10个OTU同源性较高的细菌类群是在活性污泥或污染环境分离或发现的,与8个OTU相似的细菌类群目前尚无法分离培养。     

Thermal Gradient Gel Electrophoresis Analysis of Bioprotection from Pollutant Shocks in the Activated Sludge Microbial Community

We used a culture-independent approach, namely, thermal gradient gel electrophoresis (TGGE) analysis of ribosomal sequences amplified directly from community DNA, to determine changes in the structure of the microbial community following phenol shocks in the highly complex activated sludge ecosystem. Parallel experimental model sewage plants were given shock loads of chlorinated and methylated phenols and simultaneously were inoculated (i) with a genetically engineered microorganism (GEM) able to degrade the added substituted phenols or (ii) with the nonengineered parental strain. The sludge community DNA was extracted, and 16S rDNA was amplified and analyzed by TGGE. To allow quantitative analysis of TGGE banding patterns, they were normalized to an external standard. The samples were then compared with each other for similarity by using the coefficient of Dice. The Shannon index of diversity, H, was calculated for each sludge sample, which made it possible to determine changes in community diversity. We observed a breakdown in community structure following shock loads of phenols by a decrease in the Shannon index of diversity from 1.13 to 0.22 in the noninoculated system. Inoculation with the GEM (Pseudomonas sp. strain B13 SN45RE) effectively protected the microbial community, as indicated by the maintenance of a high diversity throughout the shock load experiment (H decreased from 1.03 to only 0.82). Inoculation with the nonengineered parental strain, Pseudomonas sp. strain B13, did not protect the microbial community from being severely disturbed; H decreased from 1.22 to 0.46 for a 3-chlorophenol–4-methylphenol shock and from 1.03 to 0.70 for a 4-chlorophenol–4-methylphenol shock. The catabolic trait present in the GEM allowed for bioprotection of the activated sludge community from breakdown caused by toxic shock loading. In-depth TGGE analysis with similarity and diversity algorithms proved to be a very sensitive tool to monitor changes in the structure of the activated sludge microbial community, ranging from subtle shifts during adaptation to laboratory conditions to complete collapse following pollutant shocks.     

簇毛麦端体6VS的显微切割及其专化DNA序列的克隆和分析

从簇毛麦(Haynaldia villosa (L.) Schur.)组合CA9211/RW15(6D/6V异代换系)幼胚培养SC2后代中,用原位杂交方法鉴定出T240-6为6VS端体异代换系. 以此为材料,采用微细玻璃针切割法及"单管反应"技术体系,对6VS进行切割分离及LA (Linker adaptor)-PCR扩增.扩增带在100～3 000 bp 之间,大部分集中在600～1 500 bp.利用32P标记的簇毛麦基因组为探针进行Southern杂交,证实扩增产物来源于簇毛麦.扩增产物纯化后,连接到pGEM-T载体上,构建了6VS DNA质粒文库.对文库的分析表明,文库大约有17 000个白色克隆;插入片段分布在100～1 500 bp,平均600 bp.点杂交结果表明,37%克隆有中度到强烈的杂交信号,证明含有中度或高度重复序列;63%克隆有较弱的信号或没有信号,证明为单/低拷贝序列克隆.从文库中获得8个簇毛麦特异克隆,对其中两个克隆pHVMK22和 pHVMK134进行了RFLP分析和序列分析,并利用该探针对小麦抗白粉病基因Pm21进行了检测.RFLP 结果表明,两个克隆一个为低拷贝序列克隆(pHVMK22),另一个为高度重复序列克隆,均为簇毛麦专化DNA序列.以pHVMK22为探针对抗、感病小麦(Triticum aestivum L.)品系的Southern杂交发现抗病品系有一条2 kb的特征带, 该探针可能作为检测抗病基因Pm21的探针.     

An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process.

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter-1 day-1 and then to 1.5 g liter-1 day-1. After the loading rate was increased to 1.5 g liter-1 day-1, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter-1 day-1. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter-1 day-1. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.     

代表活性污泥中苯酚降解菌群的ERIC-PCR产物片段的多态性

对可能代表活性污泥中两个时期的主要苯酚降解菌群的：ERIC-PCR指纹图谱中的两条2．1kb的条带(P1和P8)中的DNA片段进行克隆、转化,得到193个转化子。．HinfⅠ物理图谱分析得到35种酶切类型,将两端各进行1次测序后,同源性大于90％的克隆归为一类,共得到10种序列类型。对各类型的代表克隆进行了全长测序,5种类型为P1条带所有,3种类型为P8所有,二者共享的类型为2种,丰度最高的片段为S3,P1条带中76．3％的转化子和P8条带中66．7％转化子属该类型。对所得序列进行检索分析,它们与GenBank中已知基因序列均无显著同源性。用S3特异性引物对活性污泥样品及其它在LB、dCGY、MP和FWM培养基上的回收菌进：行扩增,除LB上的回收菌没有显示目的条带外,活性污泥和其回收菌中均检测到带有该基因组片段的菌种的存在。研究为专一性分离降酚活性污泥中的优势菌提供了分子探针。     

应用TGGE技术分析人肠道中双歧杆菌的组成

用温度梯度凝胶电泳(TGGE)技术结合16SrDNA克隆、测序,对健康人肠道中双歧杆菌的组成进行了分析。10例健康人肠道双歧杆菌的TGGE分析显示:人肠道内双歧杆菌的组成具有宿主特异性,不同人肠道双歧杆菌种的多样性和种类不同。通过对一健康儿童肠道双歧杆菌属特异性PCR扩增产物的测序及TGGE电泳行为的分析发现,该个体双歧杆菌TGGE图谱中的条带分别代表Bifidobacteriumbifidum、B.infantis、B.longum、B.adolescentis、B.pseudocatenulatum等种和一新种,其中B.pseudocatenulatum(假小链双歧杆菌)是大多数个体共有且较优势的种类。用传统培养方法只检出B.pseudocatenulatum和B.longum两种。基于双歧杆菌属特异性PCR基础上的TGGE方法结合16SrDNA克隆文库分析可较灵敏、直观地反映人肠道中双歧杆菌的组成。     

Identification and diversity of putative aminoglycoside-biosynthetic aminotransferase genes from deep-sea environmental DNA

We obtained DNA fragments encoding putative aminotransferases possibly involved in the biosynthesis of aminoglycoside antibiotics from deep-sea sediments of the northwest Pacific Ocean by nested PCR, and 34 individual genes (total 89 clones) were identified. About half of the deep-sea sequences showed similarity with genes of known aminoglycoside-producers, but others were deep-sea specific genes. Furthermore, we found that temperature-gradient gel electrophoresis (TGGE) can be an effective tool in the analysis of these DNA fragments.