基于激光共聚焦扫描显微术和16SrRNA基因序列探讨一株水华蓝藻的分类地位

首次从福州市一重要供水水库中分离到一株体型微小的丝状蓝藻,该藻喜粘附在微囊藻胶被内外。为了确定该蓝藻的种类和分类学地位,通过形态特征分析和16S rRNA序列分析的方法鉴定其种类,并利用激光共聚焦扫描显微镜的荧光光谱以及色素提取物的吸收光谱分析其色素组成。结果表明,该藻株为粘伪鱼腥藻(Pseudanabaena mucicola)(Gen Bank序列登录号为KR912197),黏伪鱼腥藻不仅含有藻蓝蛋白,同时也含藻红蛋白。此结果为该藻株分类位置的确定提供了有利佐证。     

Cyanovirin-N蓝藻的筛选及其分子鉴定

目的:从实验室培养的自牛蓝藻中筛选出含有能编码抗病毒蛋白(cyanovirin-N,CV-N)基因的藻株并对其进行分子鉴定.方法:应用PCR技术和自行设计的特异性引物从80株蓝藻中筛选目标藻株,通过克隆、测序后,进行blast比对,经在线生物信息学软件预测该抗病毒蛋白CV-N的一级,二级,三级结构,并推测该基因序列编码的蛋白的抗病毒潜能.结果:筛选出藻株RZHA4.其所含的CV-N基因与已报道的CV-N基因有97%的相似性.结论:经16S rRNA-PCR和序列分析,该藻株与蓝藻Nostocaceae有99%的相似性,为一在实验室常规培养条件下生长良好的念珠藻(Nostoc),有可能成为新的CV-N生产藻株.     

16S rRNA及rpoC1基因用于螺旋藻、节旋藻系统发育的比较北大核心CSCD

【目的】利用16S rRNA和rpoC1基因分子标记研究螺旋藻、节旋藻的系统发育关系,并对其区分能力进行比较。【方法】以84株螺旋藻、节旋藻为研究对象,对其进行16S rRNA、rpoC1基因序列的扩增、测序及分析,并对构建的系统发育树进行对比。【结果】rpoC1基因序列保守位点所占比例49.7%、平均G+C百分含量47.7%和序列相似度76%–100%明显低于16S rRNA基因序列的79.4%、55.6%和91%–100%,其变异程度高于16S rRNA基因;基于16S rRNA、rpoC1基因构建的系统发育NJ树拓扑结构基本一致,84株实验藻株分为2个属3个类群,其中仅F-351、F-904-2、F-1070和TJBC14-1藻株为螺旋藻,其余均为节旋藻;虽然2个基因都不能区分形态种和地理种,但rpoC1基因NJ树的置信度(100%)高于16S rRNA基因(99%),属内分群效果也明显优于16S rRNA基因。【结论】支持了螺旋藻、节旋藻为两个不同属的结论,且在属内分类时rpoC1基因比16S rRNA基因具有更高的区分度。     

全细胞多重PCR检测蓝藻、微囊藻及产毒微囊藻方法初探

选取三对分别针对微囊藻、蓝藻16S rDNA及微囊藻毒素合成酶基因mcyB的保守序列的特异性引物209F/409R、27F1/409R、MTR/MTF,其中409R为一条共用引物。设计并优化了一种可以同时检测蓝藻和微囊藻的两重全细胞PCR方法和一种可以同时检测蓝藻、微囊藻和可产毒微囊藻的三重全细胞PCR方法,并且测试了这两种PCR反应的灵敏度区间,分别为10⁵～10³cell·mL^-1、10⁵～10²cell·mL^-1。对采集水库水样检测结果表明双重全细胞PCR方法可以直接应用于对天然水样的检测,三重全细胞PCR方法可用于实验室培养藻细胞的筛查。全细胞多重PCR方法具有快速、简便、准确等特点,在水体微囊藻毒素检测预警方面具有应用价值。     

发菜藻蓝蛋白基因克隆及原核表达

发菜（Nostoc flagelliforme）是一种陆生固氮蓝藻,具有重要的经济和生态价值。运用双向电泳技术、MALDI-TOF-TOF/MS鉴定和数据库检索,获得藻蓝蛋白部分氨基酸序列并设计简并性引物,克隆藻蓝蛋白基因并研究其表达。结果表明,发菜藻蓝蛋白α和β亚基两个基因的编码序列及两者之间的间隔序列全长为1097bp,编码β亚基和α亚基的基因序列全长分别为519bp和489bp,β亚基基因序列位于α亚基基因序列上游,两者之间通过89bp的基因片段连接,GenBank登录号为GU549478,并对推译的α和β亚基三维结构进行了预测。将藻蓝蛋白的α和β亚基基因在大肠杆菌中表达,获得了符合预期的外源重组蛋白。研究结果为进一步研究发菜藻蓝蛋白的分子结构及生物学功能奠定了基础。     

基于克隆文库法研究古尔班通古特沙漠蓝藻多样性

该研究采用克隆文库法研究古尔班通古特沙漠藻类生物结皮中蓝藻多样性及分布。在古尔班通古特沙漠不同区域采集10份藻类结皮代表性土样,分别构建古尔班通古特沙漠蓝藻16S rRNA和psbA基因克隆文库,并进行系统发育分析,对蓝藻多样性和丰度与环境因子进行关联分析,研究蓝藻分布特点及影响因子。结果显示:(1)16S rRNA基因系统发育树中包括具有明确分类地位的蓝藻有6科10属(占总克隆文库的94.85%)和一个未分类蓝藻属,其中颤藻属(Oscillatoria)和微鞘藻属(Microcoleus)分别占克隆文库的42.54%和37.16%,为古尔班通古特沙漠蓝藻优势属;psbA基因系统发育树中仅鉴定有蓝藻类群4科4属,但优势属与前者结果一致。(2)10个样点藻类结皮中所含蓝藻种类不尽相同,但每个采样点都出现颤藻属和微鞘藻属,证明这二者是古尔班通古特沙漠藻类结皮中的优势属;且样点Gur2和Gur17中蓝藻种类较多,Gur3、Gur5和Gur9中种类较少,但Gur2、Gur3、Gur5和Gur17相对地理位置较近,表明地理位置不是影响蓝藻分布的主要因素。(3)RDA(Redundancy analysis)分析结果显示,微生物量氮(MBN)和土壤有机碳(SOC)对蓝藻多样性影响程度最大,其次是硝态氮(NO~-_3-N)、微生物量碳(MBC),全磷(TP)和全钾(TK)对其影响程度最小。研究表明,古尔班通古特沙漠不同区域沙漠蓝藻多样性和土壤理化性质具有空间异质性,综合分析可得沙漠中南部藻类结皮土壤营养最为丰富,蓝藻多样性较高,而东部和西部土壤营养较为贫瘠,蓝藻丰富度和多样性较低。     

通过同源重组在聚球藻7002中表达小鼠金属硫蛋白-Ⅰ的初步研究

用PCR方法从海洋单细胞蓝藻聚球藻7002(Synechococcus sp. PCC 7002)基因组DNA中扩增得到藻蓝蛋白β亚基基因(cpcβ)的上游序列(Pcpcβ),及编码谷氨酰胺合成酶的glnA基因片段.以Pcpcβ作为启动子、以glnA基因片段作为整合平台,构建含有小鼠金属硫蛋白-Ⅰ(mMT-Ⅰ)cDNA的同源整合表达载体pKGC-MT.通过自然转化法将整合表达载体导入聚球藻7002中,经氨苄青霉素筛选,得到遗传性状稳定的转基因藻.PCR检测证明mMT-Ⅰ基因已整合到蓝藻基因组DNA上;蛋白质印迹表明mMT-Ⅰ已在蓝藻中表达;ELISA结果显示mMT-Ⅰ在蓝藻中的表达量约为800 μg/g.     

蓝藻的起源和演化

蓝藻的学名为Ｃｙａｎｏｐｈｙｔａ,最初由Ｓａｃｈｓ（１８７４）建议采用的。蓝藻体内含有特殊的藻蓝蛋白和别藻蓝蛋白,这两种蛋白加上叶绿素蛋白而使蓝藻藻体呈现蓝绿色。蓝藻又名蓝绿藻、蓝细菌、蓝菌等。蓝藻的主要特征是原核（原始核）、以细胞分裂的方式进行繁殖和体内含有藻胆素（藻蓝蛋白和藻红蛋白的总称）。蓝藻的研究,尤其是蓝藻的起源和演化是生物学家关注的热点问题之一。在解开生命起源之谜如生命起源的时间、真核细胞的起源和机理等问题上,蓝藻的研究会提供有价值的研究资料。本文就蓝藻的起源和演化作一简介。１　蓝藻…     

鄱阳湖的中国水华蓝藻新记录属气丝藻属

2012年4月和10月在江西省鄱阳湖进行野外调查时, 发现一种浮游的丝状蓝藻。通过分离纯培养, 获得了6个纯化藻株。根据藻株主要形态学特征及其16S rRNA基因序列与比较, 鄱阳湖这些藻株与老挝的2株Aerosakkonema funiforme较为相近, 其中16S rRNA基因序列的相似度达到98%。鉴于这些藻株不同于颤藻目中其他属的藻株, 因此确定为我国一水华蓝藻新记录属气丝藻属Aerosakkonema Nanda Watanabe 2012, 模式种为索状气丝藻Aerosakkonema funiforme Nanda Watanabe 2012。       

螺旋藻藻蓝蛋白光敏作用的研究进展

螺旋藻是一种广泛养殖的丝状体蓝藻。从螺旋藻中提取的藻蓝蛋白具有抗肿瘤和增强免疫等多种生物学功能,作为光敏剂,藻蓝蛋白可应用于肿瘤治疗。本文阐述了藻蓝蛋白的结构、光动力疗法的原理和藻蓝蛋白光敏作用的研究进展,并介绍了藻蓝蛋白在光动力疗法中的应用现状与前景。     

Simultaneous quantification of cyanobacteria and Microcystis spp. using real-time PCR

In order to develop a protocol to quantify cyanobacteria and Microcystis simultaneously, the primers and probe were designed from the conserved regions of 16S rRNA gene sequences of cyanobacteria and Microcystis, respectively. Probe match analysis of the Ribosomal Database Project showed that the primers matched with over 97% of cyanobacterial 16S rRNA genes, indicating these can be used to amplify cyanobacteria specifically. The TaqMan probe, which is located between two primers, matched with 98.2% of sequences in genus GpXI, in which most Microcystis strains are included. The numbers of cyanobacterial genes were estimated with the emission of SYBR Green from the amplicons with two primers, whereas those of Microcystis spp. were measured from the fluorescence of CAL Fluor Gold 540 emitted by exonuclease activity of Taq DNA polymerase in amplification. It is expected that this method enhances the accuracy and reduces the time to count cyanobacteria and potential toxigenic Microcystis spp. in aquatic environmental samples.     

High-resolution differentiation of Cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis: Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

Application of sequence-specific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization

DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (Nostoc, Microcystis, and Planktothrix species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats.     

Endolithic photosynthetic communities within ancient and recent travertine deposits in Yellowstone National Park

Molecular and culture based methods were used to survey endolithic, photosynthetic communities from hot spring-formed travertine rocks of various ages, ranging from<10 to greater than 300,000 years. Much of this travertine contained a 1-3-mm-thick greenish band composed mainly of cyanobacteria 1-5 mm below the rock surface. The travertine rocks experienced desiccation in summer and freezing in winter. A total of 83 environmental 16S rRNA gene sequences were obtained from clone libraries and denaturing gradient gel electrophoresis. Small subunit rRNA gene sequences and cell morphology were determined for 36 cyanobacterial culture isolates from these samples. Phylogenetic analysis showed that the 16S rRNA gene sequences fell into 15 distinct clusters, including several novel lineages of cyanobacteria.     

High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

An early origin of plastids within the cyanobacterial divergence is suggested by evolutionary trees based on complete 16S rRNA sequences

It is generally accepted that the plastids arose from a cyanobacterial ancestor, but the exact phylogenetic relationships between cyanobacteria and plastids are still controversial. Most studies based on partial 16S rRNA sequences suggested a relatively late origin of plastids within the cyanobacterial divergence. In order to clarify the exact relationship and divergence order of cyanobacteria and plastids, we studied their phylogeny on the basis of nearly complete 16S rRNA gene sequences. The data set comprised 15 strains of cyanobacteria from different morphological groups, 1 prochlorophyte, and plastids belonging to 8 species of plants and 12 species of diverse algae. This set included three cyanobacterial sequences determined in this study. This is the most comprehensive set of complete cyanobacterial and plastidial 16S rRNA sequences used so far. Phylogenetic trees were constructed using neighbor joining and maximum parsimony, and the reliability of the tree topologies was tested by different methods. Our results suggest an early origin of plastids within the cyanobacterial divergence, preceded only by the divergence of two cyanobacterial genera, Gloeobacter and Pseudanabaena.      

Gene copy number variation and its significance in cyanobacterial phylogeny

ABSTRACT: BACKGROUND: In eukaryotes, variation in gene copy numbers is often associated with deleterious effects, but may also have positive effects. For prokaryotes, studies on gene copy number variation are rare. Previous studies have suggested that high numbers of rRNA gene copies can be advantageous in environments with changing resource availability, but further association of gene copies and phenotypic traits are not documented. We used one of the morphologically most diverse prokaryotic phyla to test whether numbers of gene copies are associated with levels of cell differentiation. RESULTS: We implemented a search algorithm that identified 44 genes with highly conserved copies across 22 fully sequenced cyanobacterial taxa. For two very basal cyanobacterial species, Gloeobacter violaceus and a thermophilic Synechococcus species, distinct phylogenetic positions previously found were supported by identical protein coding gene copy numbers. Furthermore, we found that increased ribosomal gene copy numbers showed a strong correlation to cyanobacteria capable of terminal cell differentiation. Additionally, we detected extremely low variation of 16S rRNA sequence copies within the cyanobacteria. We compared our results for 16S rRNA to three other eubacterial phyla (Chroroflexi, Spirochaetes and Bacteroidetes). Based on Bayesian phylogenetic inference and the comparisons of genetic istances, we could confirm that cyanobacterial 16S rRNA paralogs and orthologs show significantly stronger conservation than found in other eubacterial phyla. Conclusions: A higher number of ribosomal operons could potentially provide an advantage to terminally differentiated cyanobacteria. Furthermore, we suggest that 16S rRNA gene copies in cyanobacteria are homogenized by both concerted evolution and purifying selection. In addition, the small ribosomal subunit in cyanobacteria appears to evolve at extraordinary slow evolutionary rates, an observation that has been made previously for morphological characteristics of cyanobacteria.     

Cloning vectors for Streptococcus thermophilus derived from a native plasmid

Marine sponges frequently contain a complex mixture of bacteria, fungi, unicellular algae and cyanobacteria. Epifluorescent microscopy showed that Mycale (Carmia) hentscheli contained coccoid cyanobacteria. The 16S rRNA gene was amplified, fragments cloned and analysed using amplified rRNA gene restriction analysis. The nearly complete 16S rRNA gene of distinct clones was sequenced and aligned using ARB. The phylogenetic analysis indicated the presence of four closely related clones which have a high (8%) sequence divergence from known cyanobacteria, Cyanobacterium stanieri being the closest, followed by Prochloron sp. and Synechocystis sp. All belong to the order Chroococcales. The lack of non-molecular evidence prevents us from proposing a new genus.     

基于多位点序列分型方法的于桥水库微囊藻遗传多样性分析

为了研究天津于桥水库微囊藻(Microcystis)的遗传多样性及其系统发育关系,将分离自于桥水库的10株微囊藻的7个管家基因(ftsZ、glnA、gltX、gyrB、pgi、recA和tpi)构建了微囊藻的多位点序列分型(MLST)基因库,与20个鄱阳湖序列型(STs)和237个日本湖泊的序列型进行比对,结果发现于桥...