东寨港红树林土壤芽胞杆菌分离及其多样性分析

采用热处理法从海南省东寨港红树林海漆林区土壤中分离到276株芽胞杆菌,利用PCR-RFLP与序列分析技术对其16S rDNA遗传多样性进行了研究。16S rDNA PCR-RFLP酶切图谱的聚类分析表明,在100%的相似性水平上,分离的276株芽胞杆菌分属于15个遗传类群,表明存在较为丰富的遗传多样性。15种遗传类型的26株代表芽胞杆菌的16S rDNA序列分析可知,这些芽胞杆菌主要分布于Bacillus(69.2%)、Halobacillus(3.8%)、Virgibacillus(7.7%)、Gracilibacillus(3.8%)、Oceanobacillus(7.7%)和Lysinibacillus(7.7%)6个属,其中Bacillus为优势属。有3株芽胞杆菌的16S rDNA序列与数据库中相应模式菌株的最大相似性在98.O%~98.9%之间,可能为潜在的新分类单元。     

八门湾红树林土壤芽胞杆菌分离与多样性分析

【目的】了解八门湾红树林海漆林区土壤中可培养芽胞杆菌资源的多样性。【方法】采用水浴处理与直接涂布相结合的方法选择性分离土壤中的芽胞杆菌;利用16S rDNA PCR-RFLP与16S rDNA序列分析技术研究可培养芽胞杆菌资源的遗传多样性和系统发育关系。【结果】16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析表明,在100%的相似性水平上,分离的155株芽胞杆菌分属21个遗传类群,显示了较为丰富的遗传多样性;由21种遗传类型代表菌株的16S rDNA序列分析结果得知,这些芽胞杆菌主要分布在Bacillaceae和Paenibacillaceae科下的Bacillus、Halobacillus、Virgibacillus和Paenibacillus 4个属,其中Bacillus为优势属;有8株芽胞杆菌的16S rDNA序列与数据库中相应模式菌株的最大相似性在95.1%-99.0%之间。【结论】八门湾红树林土壤可培养芽胞杆菌有着较为丰富的遗传多样性,并存在新的芽胞杆菌物种资源。     

新疆泥火山细菌遗传多样性

为了解新疆乌苏泥火山细菌多样性,从泥火山泥浆样品中直接提取总DNA,构建了含150个有效转化子的泥火山细菌16S rDNA基因文库,转化子经菌液PCR及HaeⅢ酶切后获得16个不同带型,克隆测序结果表明,其分属于16个不同的分类单元.一部分序列与已知细菌类群的16S rDNA序列相似性较高,归属变形菌门(Proteobacteria),厚壁菌门(Firmicutes),梭杆菌门(Fusobacteria),放线菌门(Actinobacteria);另外一部分序列与已知细菌类群的16S rDNA序列同源性较低,可能代表新的分类单位.研究结果显示,泥火山环境中微生物种群丰富,值得进一步研究.     

佳西热带雨林土壤芽胞杆菌分离与多样性分析

采用热处理法从海南省佳西热带雨林土壤中分离到147株芽胞杆菌,并利用16S rDNA PCR-RFLP与序列分析技术对其遗传多样性进行了研究。16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析结果表明,在100%的相似性水平上,这些芽胞杆菌分属13个遗传类群。不同遗传类型代表菌株的16S rRNA基因序列分析结果显示,它们分布在Bacillaceae、Planococcaceae和Paenibacillaceae科的Bacillus、Lysinibacillus、Paucisalibacillus、Bhargavaea和Paenibacillus五个属,其中Bacillus为优势属(占50%);有3株芽胞杆菌的16S rRNA基因序列与数据库中相应模式菌株的最大相似性在98.3%~98.9%之间。结果表明,佳西热带雨林土壤中芽胞杆菌有着较为丰富的遗传多样性。     

红豆杉免培养内生细菌多样性的初步研究

【目的】了解红豆杉(Taxus chinensis)内生细菌的组成及多样性。【方法】提取红豆杉组织总DNA,选用细菌通用引物799F和1492R对总DNA进行16S rDNA特异性扩增,构建红豆杉内生细菌16S rDNA克隆文库,对阳性克隆进行PCR-RFLP(限制性内切酶片段长度多态性)分析,并对酶切带谱不同的菌液进行测序,构建系统发育树。【结果】根据酶切带谱分析和测序结果的不同,将随机挑取的158个阳性克隆归为26个不同的可操作分类单元(OUTs),系统发育分析表明这些克隆序列分别属于变形菌门(Proteobacteria,包含Alpha、Beta、Gamma、Delta亚群)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)4个门。其中,变形菌门(Proteobacteria,占克隆总数的58.86%)为最优势类群。序列比对结果表明这些克隆序列分别与已报道的20个属具有较高的相似性。此外,还有一个OUTs在系统发育树上形成独立分支且未能确定其分类。【结论】红豆杉内生细菌多样性丰富,并且可能存在新的分类单元。     

西藏扎布耶茶卡盐碱湖古菌多样性的非培养技术分析

采用非培养技术,直接从西藏扎布耶茶卡盐碱湖样品中提取微生物总DNA。以样品总DNA为模板,PCR扩增湖中古菌的16S rDNA序列。扩增产物经过克隆并随机挑选60个克隆进行测序得到它们的16S rDNA部分序列,大部分序列与嗜盐碱古菌的16S rDNA相近。在系统发育树上,部分克隆与已知古菌属归于同一分支,主要分布在嗜盐菌科的Natronococcus、Natronorubrum、Natronobacterium、Natronomonas、Natrinema、Halorubrum、Haloterrigena和Halorhabdus等8个嗜盐古菌属中, 也有一些克隆形成了独立的分支。它们共同显示出扎布耶茶卡湖中的古菌具有丰富的多样性。     

云南省德宏州含羞草β-根瘤菌多样性及系统发育研究

【目的】通过对分离自云南德宏州的含羞草β-根瘤菌进行遗传与表型多样性研究,揭示我国含羞草β-根瘤菌的物种多样性。【方法】应用16S rDNA PCR-RFLP、全细胞蛋白SDS-PAGE电泳及16S rDNA全序列分析对分离得到的60株含羞草根瘤菌进行多样性研究。【结果】16S rDNA PCR-RFLP及全细胞蛋白SDS-PAGE图谱分析将供试菌株分为2个遗传型群和2个表型群,分别与贪铜菌属(Cupriavidus)和伯克霍尔德菌属(Burkholderia)参比菌株聚群。经16S rDNA全序列分析,供试菌株被归到台湾贪铜菌(Cupriavidus taiwanensis)、含羞草伯克霍尔德菌(Burkholderia mimosarum)及结瘤伯克霍尔德菌(Burkholderia phymatum)等3个种群。【结论】云南德宏州的含羞草β-根瘤菌主要为贪铜菌及伯克霍尔德菌类群,其中贪铜菌占绝对优势,且存在遗传和表型的丰富多样性,该研究揭示了含羞草β-根瘤菌的物种多样性并丰富了我国β-根瘤菌菌种资源。     

应用16SrDNA-RFLP方法分析宁夏地区稻田土壤细菌的多样性

水稻是宁夏地区主要粮食作物, 水稻种植也具有维持生态系统平衡, 防止土地荒漠化等重要的生态功能。而稻田土壤细菌是维持土壤生态功能的基础。但长期以来缺乏对干旱地区稻田土壤细菌多样性的认识。本研究采用非培养技术提取稻田土壤样品总DNA, 构建其16S rDNA克隆文库, 用PCR-RFLP分析进一步测序后聚类分析细菌群落的多样性。从稻田土样中分离获得了大于23 kb的DNA片段。PCR-RFLP共得到74种酶切带型, 序列分析发现77.3%的克隆序列与环境中未培养细菌的16S rDNA序列有较高的相似性, 仅有22.7%的克隆序列与数据库中可培养细菌有较高的相似性, 表明宁夏稻区土壤中的多数细菌尚未培养。系统发育研究发现74个序列分属于12个类群, 其中变形细菌所占比例最大(37.8%), 依次为酸杆菌(16.2%)、放线菌(12.2%)、拟杆菌(10.8%)、绿屈挠菌(10.8%)、浮游霉菌(8.1%), 另外有少量厚壁菌门、芽单胞菌门和疣微菌门细菌克隆。在变形细菌的序列中包括、、γ和δ 4个类型, 比例分别为13.5%、5.4%、12.2%和6.8%。表明宁夏稻区土壤中优势细菌类群为变形杆菌和酸杆菌, 且土壤细菌类群具有丰富的多样性。     

海绵Pachychalina sp.体内细菌多样性的研究

通过非分离培养分析方法,直接从海绵体内提取细菌总DNA。以样品总DNA为模板进行PCR扩增获得细菌16S rDNA。用16S rDNA限制性酶切片段长度多态性(ARDRA)和测序方法对南海湛江海域海绵Pachychalina sp．体内的细菌多样性进行了研究。在细菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱间存在差异;随机挑选22个克隆进行测序得到它们的16S rDNA部分序列,大部分序列属于γ-proteobacterium和α-proteobacterium,但有少数克隆序列与RDP数据库中收录的16S rDNA序列间的相似性极小,不参与系统发育树的构建。研究结果表明海绵Pachychalina sp.体内细菌组成具有丰富的多样性。     

16S rDNA克隆文库方法分析太岁样品中细菌的多样性

通过构建16S rDNA克隆文库的方法,分析太岁样品中细菌的群落结构及多样性。太岁样品中的细菌归属于4个门9个目,优势类群依次是芽胞杆菌目(Bacillales,33.01%)、柄杆菌目(Caulobacterales,32.04%)和伯克霍尔德氏菌目(Burkholderiales,12.62%);优势属为短波单胞菌属(Brevundimonas,30.10%)、葡萄球菌属(Staphylococcus,29.13%)和食酸菌属(Acidovorax,7.77%)。并且其中的5个目中含有未培养的细菌,红杆菌目(Rhodobacterales)、伯克霍尔德氏菌目和红环菌目(Rhodocyclales)的11个克隆子的细菌16S rDNA序列同源性低于97%。研究表明太岁样品中细菌多样性较丰富,且蕴藏着许多未知的微生物资源。     

Clonal polymerase chain reaction-single-strand conformational polymorphism analysis: an effective approach for identifying cloned sequences

The amplification of target sequences from genomic DNA can result in more than one amplicon sequence being produced even when highly specific primers are used. Here we present a clonal polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) approach for screening cloned amplicons and identifying particular clones prior to sequence determination. Comparison of the PCR-SSCP patterns of the cloned amplicons with the PCR-SSCP patterns observed for the DNA templates from which the clones were derived allows PCR artifacts, different alleles, and even different loci to be differentiated prior to sequencing. Using this approach, the number of clones required for reliable sequence determination is minimized, and complex “mixed” amplicons can be resolved easily, cost-effectively, and reliably.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Rapid Identification of Rhizobia by Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes

Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa.     

Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification

A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling.     

Effective molecular examination of eukaryotic plankton species diversity in environmental seawater using environmental PCR,PCR-RFLP,and sequencing

Phytoplankton are primary producers and can be important indicators of environmental change. To monitor the plankton species composition of environmental seawater samples, we developed a molecular method composed of colony polymerase chain reaction (PCR), polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and sequencing. A clone library of the ribosomal small subunit RNA gene (18S rDNA) in the nuclear genome was constructed by environmental PCR using a newly designed primer set and clones were directly amplified by colony PCR. To select unique putative clones, we choose a PCR-RFLP method that employed two restriction enzymes (MseI and Tsp509I). After the PCR-RFLP pattern was evaluated, selected clones were sequenced and analyzed. In this study, we revealed the hidden biodiversity in environmental seawater containing a wide range of taxonomic groups in the Alveolata (Ciliphora and Dinophyceae), Euglenozoa, Stramenopiles (Bacillariophyta), and Viridiplantae (Chlorophyta) without the need to conduct extensive colony isolation techniques. Moreover, we found species of fungi and Metazoa (Arthropoda, Annelida, and Mollusca). Therefore, this improved molecular method can be used to generate a robust database describing the species diversity of environmental samples and provide useful information regarding the dynamics of the eukaryotic plankton community structure.     

Geographic variation and diversity of the cytochrome b gene in Japanese wild populations of medaka, Oryzias latipes

We conducted a polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome b gene to elucidate the detailed genetic population structure of Japanese wild populations of medaka, Oryzias latipes. The analysis of 1,225 specimens collected from 303 sites identified 67 mitotypes. Subsequently we determined the nucleotide sequences of the complete cytochrome b gene (1141-bp) to clarify the phylogenetic relationships among mitotypes. The phylogenetic tree based on nucleotide sequences indicated three major clades (A, B and C) that differed by 11.3-11.8%, corresponding to three clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of mitotypes in clades A and B was fully concordant with the Northern and Southern Populations defined by allozymes. Clade A could be subdivided into three subclades and clade B into eleven, with sequence divergences among subclades of 1.3-5.8%. Each distribution of mitotypes in subclades roughly corresponded to that of mtDNA haplotypes in subclusters previously identified. Mitotypes in clade C were found only in the Kanto district. The phylogenetic relationships and the estimated divergence times suggest that three Japanese clades originated from a common ancestor and were separated during the Pliocene, and that the regional differentiation of subclades was closely connected with the geological history of the Quaternary. This study has also demonstrated the possibility of artificial disturbance of natural distribution especially in the Kanto district and the superior efficacy of PCR-RFLP analysis as a simple method for detecting genetic variation and artificial gene flow of medaka.     

Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism

Accurate analysis of DNA sequence variation in not only humans and animals but also other organisms has played a significant role in expanding our knowledge about genetic variety and diversity in a number of different biological areas. The search for an understanding of the causes of genetic variants and mutations has resulted in the development of a simple laboratory technique, known as the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, for the detection of single nucleotide polymorphisms (SNPs). PCR-RFLP allows rapid detection of point mutations after the genomic sequences are amplified by PCR. The mutation is discriminated by digestion with specific restriction endonucleases and is identified by gel electrophoresis after staining with ethidium bromide (EtBr). This convenient and simple method is inexpensive and accurate for SNP genotyping and especially useful in small basic research studies of complex genetic diseases. The whole protocol takes only a day to carry out.     

Application of a Novel <Emphasis Type="Italic">rpoC1</Emphasis>-RFLP Approach Reveals that Marine <Emphasis Type="Italic">Prochlorococcus</Emphasis> Populations in the Atlantic Gyres are Composed of Greater Microdiversity than Previously Described

To elucidate the degree of microdiversity within the genus Prochlorococcus, novel Prochlorococcus-specific polymerase chain reaction (PCR) primers were developed for the rpoC1 gene, which encodes the ribonucleic acid (RNA) polymerase core subunit. The size of the PCR fragment (925 bp) coupled with high sequence variation within the rpoC1 fragments (70–99% sequence similarity, 16S ribosomal RNA sequences show greater than 97% sequence similarity) meant that it was possible to distinguish Prochlorococcus strains by restriction fragment length polymorphism (RFLP) analysis. Clone libraries were constructed from environmental deoxyribonucleic acid samples from two stations, one in the northern and one in the southern oligotrophic gyre of the Atlantic Ocean. These were screened to determine the microdiversity of Prochlorococcus populations using this high-resolution high-throughput analysis approach. RFLP analysis of the clone libraries from the two gyre sites revealed that the two Prochlorococcus populations had a high degree of microdiversity with 40 and 52 different RFLP-type clones among the 143 clones tested for both the northern and southern gyres, respectively. Phylogenetic analysis of the nucleotide sequences of the RFLP types not only showed that it contained representatives of each of the currently recognized Prochlorococcus clades (based on the internal transcribed spacer region as molecular marker) but also led to the discovery of a previously unseen genetic microdiversity. This level of diversity was greater at the southern gyre site compared to the northern gyre site. Moreover, the high genetic resolution approach also revealed that there are two putative novel lineages within the HL I clade. Analyses of further samples by producing clone libraries from different geographic origins is likely to reveal further diversity and novel lineages within Prochlorococcus.     

Fifty microsatellite markers for Japanese quail

A Japanese quail genomic library enriched for (CA/GT)n simple sequence repeats was screened and positive clones were sequenced. Fifty original microsatellite sequences were isolated that consisted mainly of perfect repeats of the dinucleotide (CA/GT)n motif and a corresponding number of polymerase chain reaction (PCR) primer pairs complementary to unique DNA sequences flanking the microsatellite repeats were designed to detect the repeats. Forty-six percent (23 of 50) of the markers revealed polymorphism in two unrelated quail individuals (one male and one female) randomly sampled from a population of wild quail origin. All 50 primer pairs were tested in the PCR for their ability to amplify chicken genomic DNA. Amplification products were obtained for 14 (28.0%) of the markers at the annealing temperature optimized for quail. These results provide an opportunity to begin characterizing the quail genome for the development of a genetic map for this economically valuable species and the eventual construction of a comparative genetic map in Phasianidae, which comprises a number of agriculturally important species of poultry.     

PCR-Mediated Recombination between Cryptosporidium spp. of Lizards and Snakes

ABSTRACT: The presence or absence of genetic recombination has often been used as one of the criteria for Cryptosporidium species designation and population structure delineation. During a recent study of cryptosporidiosis in reptiles that were housed in the same room, 4 lizards were found to have concurrent infections of C. serpentis (a gastric parasite) and C. saurophilum (an intestinal parasite), and 6 snakes were concurrently infected with C. serpentis, C. sattrophitmm and a new Cryptosporidium as indicated by PCR-RFLP analysis of the SSU rRNA gene. DNA sequence analysis of cloned PCR products confirmed the diagnosis of mixed infections. Surprisingly, it appeared that 11 of the 22 clones (8 and 14 clones from a lizard and a snake, respectively) had chimeric sequences of two Cryptosporidium spp. BootScan analysis indicated the existence of recombinants among the cloned sequences and detection of the informative sites confirmed the BootScan results. Because the probability for genetic recombination between gastric and intestinal parasites is small, these hybrid sequences were likely results of PCR artifacts due to the presence of multiple templates. This was confirmed by PCR-sequencing analysis of single-copy templates using diluted DNA samples. Direct sequencing of 69 PCR products from 100-to 1.000-fold diluted DNAs from the same snake and lizard produced only sequences of C. serpentis, C. saurophilum and the unnamed Cryntosnoridium. , sp. Thus, care should be taken to eliminate PCR artifacts when determining the presence of genetic recombination or interpreting results of population genetic studies.