Detection of Seven Major Evolutionary Lineages in Cyanobacteria Based on the 16S rRNA Gene Sequence Analysis with New Sequences of Five Marine Synechococcus Strains

Although molecular phylogenetic studies of cyanobacteria on the basis of the 16S rRNA gene sequence have been reported, the topologies were unstable, especially in the inner branchings. Our analysis of 16S rRNA gene phylogeny by the maximum-likelihood and neighbor-joining methods combined with rate homogeneous and heterogeneous models revealed seven major evolutionary lineages of the cyanobacteria, including prochlorophycean organisms. These seven lineages are always stable on any combination of these methods and models, fundamentally corresponding to phylogenetic relationships based on other genes, e.g., psbA, rbcL, rnpB, rpoC, and tufA. Moreover, although known genotypic and phenotypic characters sometimes appear paralleled in independent lineages, many characters are not contradictory within each group. Therefore we propose seven evolutionary groups as a working hypothesis for successive taxonomic reconstruction. New 16S rRNA sequences of five unicellular cyanobacterial strains, PCC 7001, PCC 7003, PCC 73109, PCC 7117, and PCC 7335 of Synechococcus sp., were determined in this study. Although all these strains have been assigned to ``marine clusters B and C,' they were separated into three lineages. This suggests that the organisms classified in the genus Synechococcus evolved diversely and should be reclassified in several independent taxonomic units. Moreover, Synechococcus strains and filamentous cyanobacteria make a monophyletic group supported by a comparatively high statistical confidence value (80 to 100%) in each of the two independent lineages; therefore, these monophylies probably reflect the convergent evolution of a multicellular organization. Received: 3 September 1998 / Accepted: 30 November 1998     

大肠杆菌O141 O-抗原基因簇的破译及进化分析

对大肠杆菌O141 O-抗原基因簇进行测序,序列全长15601bp,用生物信息学的方法进行序列分析,共发现12个基因：鼠李糖合成酶基因(rmlB,rmlD,rmlA,rmlC)、甘露糖合成酶基因(manB,manC),糖基转移酶基因(orf6,orf7,orf9,orf10)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy)。用PCR的方法筛选出了针对大肠杆菌O141的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O141的快速检测。通过对大肠杆菌O141的O-抗原基因簇及甘露糖和鼠李糖合成酶基因的进化分析发现：大肠杆菌O141 O-抗原基因簇是低GC含量的片段,仅O-抗原特异的基因才出现在O-抗原基因簇;并且这些基因可能介导了O-抗原基因簇间的重组及以O141 O-抗原基因簇的形成。     

五种裂腹鱼亚科鱼类外胚层发育不良因子A受体基因Edar的克隆和序列分析

裂腹鱼亚科鱼类具有丰富的体表附属器官变异,而Edar是控制这类结构发育的关键基因。以5种裂腹鱼的代表物种为材料克隆其Edar基因的编码区片段,序列分析表明:该基因编码的蛋白在裂腹鱼中存在长度不同的组氨酸重复序列,串联组氨酸插入可能与蛋白定位有关;基因树支持裂腹鱼与金线鲃具有最近的亲缘关系;分子进化分析未检测到正选择信号,裂腹鱼Edar基因的低复杂区可能是受选择区域。以上结果说明裂腹鱼Edar基因的结构变异可能与裂腹鱼表型变异和演化有关。     

乙型流感病毒河北株的HA1基因序列及种系发生的分析

对１９８９年春在河北保定分离的两株乙型流感病毒进行了抗原性、ＨＡ＿１基因序列和种系发生分析,与不同期的国内外代表株比较结果表明,自１９８８年以来乙型流感病毒变异较快,Ｂ／河北／５３／８９株的ＨＡ＿１基因序列与Ｂ／挪威／１／８５株相比,其氨基酸的同源性为９４．５２％,与同期的Ｂ／香港／２０／８９株同源性为９７．１２％。种系发生分析结果,从１９８８至１９８９年乙型流感病毒出现了５个支系,同期在保定分离的两株病毒,在同源替代中分别属于两个支系。日本国基本每隔两年出现一次乙型流感的流行优势型,其发生频度与甲型流感相似。国内少见乙型的流行优势型,可能和使用的分离病毒材料有关,日本用ＭＤＣＫ细胞比国内用鸡胚对乙型流感病毒的分离阳性率高。     

对虾白斑综合征病毒的细胞因子受体基因的分析与表达

在对虾白斑综合征病毒(White spot syndrome virus,WSSV)的基因组中发现一个具有细胞因子受体特征的开放阅读框,该阅读框全长2022个核苷酸,编码674个氨基酸,蛋白质理论分子量为76kDa。该基因含有真核生物细胞因子gp130受体特征序列。为了研究该基因的功能,采用PCR方法从病毒基因组中扩增出基因片段,克隆到pGEM-T Easy载体中,经BamH I和Sal I双酶切后插入pET28b表达载体中。重组质粒转化到大肠杆菌BL21中,IPTG诱导后,经SDS-PAGE电泳表明在。76kDa处有目的蛋白表达。用冰浴超声波对诱导后的菌液进行处理以获得初步纯化的蛋白,作为抗原人工免疫实验兔子以获得含特异性抗体的抗血清。该基因的表达成功,为其功能的进一步深入研究奠定了基础。     

五株登革病毒的分离鉴定及E基因序列分析

登革病毒作为一种重要的蚊媒传播病毒,威胁全球人类健康,及时甚至实时了解病毒的变异和流行病学规律,对科学防控登革热具有重要意义.因此,本研究利用广东省深圳市发现的登革热患者血清进行病毒分离、鉴定及囊膜蛋白E基因的扩增、测序及生物信息学分析病毒E基因进化特点.结果显示,应用Vero和C6/36细胞培养法从登革热病患血清中分离获得5株登革病毒,利用MEGA7软件对囊膜蛋白E的核苷酸和氨基酸序列进行分析显示,5株均属血清Ⅱ型登革病毒,其中输入性SZ926株和本地株SZ38株同源性为99.5％,均与泰国16681经典毒株关系密切,可能衍生于同一祖先;SZ868株和SZ871株为境外输入性病患分离株,两株病毒核苷酸同源性达到99.9％,说明两株病毒可能源于同一毒株;SZ29株与新几内亚的New Guinea C毒株同源性为99.8％,显示出进化保守、稳定传播的特点.基因型分析显示,SZ926和SZ38为基因型Ⅲ型,SZ868和SZ871为基因型Ⅳ型,SZ29为基因型Ⅰ型.本研究成功分离获得5株血清Ⅱ型登革病毒,分属于3个基因型,且不同基因型的E基因整体变异较低,而且我国存在多种基因型感染的潜在威胁,必须加强防范意识和研究力度.     

不同葡萄品种CBF1基因的克隆及序列分析

根据欧洲葡萄CBF1基因序列设计1对特异引物,采用PCR方法从山葡萄、贝达、SO4、Ln33和黑比诺的基因组中各扩增出1个DNA片段并克隆到pMD18-T栽体中.经序列测定和分析,各片段均长756 bp,与欧洲葡萄CBF1基因的核苷酸序列及其推导的氨基酸序列分别具有98％和99％的同源性,而且推导的氨基酸序列中包含有同源性更高的AP2/EREBP DNA结合域,以及CBFs蛋白的两段特征序列(PKK/RPAGRxKFxETRHP和DSAWR).结果表明从5种葡萄中均可以克隆出CBF1基因.     

5种寄生蚌螨COI基因片段序列及系统发育分析

采用PCR技术对5种寄生蚌螨COI基因片段进行序列测定.序列分析的结果表明,比对后的序列总长度均为658 bp;其中A、G、C、T 4种碱基的平均含量分别为34.6%、20.2%、14.3%、30.9%,平均嘧啶含量（65.5%）明显高于嘌呤含量（34.5%）,说明碱基存在偏向性.弯弓蚌螨Unionicola arcuata与Y纹蚌螨U.ypsilophora 的种间遗传差异为26.4%,达到属间分类水平,结果支持Vidrine将沃蚌螨亚属从寄蚌螨亚属中分离的修订;螫爪蚌螨U.chelata与敏捷蚌螨U.agilex之间遗传差异为23.3%,达到属间分类水平,结果支持文春根等将敏捷蚌螨与其姊妹种腰蚌螨U.lumbaria放入韦蚌螨亚属中的修订.以营自由生活的厚蚌螨U.crassipes作为外类群,使用PAUP4.0b 10软件中的最大似然法（ML）和邻接法（NJ）构建系统树,结果显示：在5种寄生蚌螨中敏捷蚌螨可能是最早从祖先种分化出来的种;弯弓蚌螨与Y纹蚌螨可能起源于蚌螨属的同一个祖先.     

基于功率谱分析的基因编码序列单碱基突变研究

针对DNA序列单碱基的不同类型突变,利用数字信号处理方法,研究了单碱基替换突变、删除突变、插入突变对DNA序列三周期功率谱的影响。研究结果表明:对于不同长度的编码序列,替换突变对序列功率谱的影响较小,删除突变和插入突变对序列功率谱的影响较大;随着序列编码区长度的减小,替换、删除、插入突变对序列编码区的功率谱影响会越来越大。对于中等长度外显子,插入突变对序列三周期功率谱影响最大,对于短外显子,删除突变对序列三周期功率谱的影响最大。研究结果可为含突变基因编码区的识别与检测提供参考。     

基于Bioperl的基因序列获取的程序设计与实现

Bioperl在序列操作和数据库的远程获取方面有独特的优势。为了从Genbank获取大量硫酸盐还原菌的16S rRNA基因序列,进一步研究硫酸盐还原菌的分子进化及系统发育规律,该文基于bioperl设计了远程获取大量基因序列的程序,并成功地实现了硫酸盐还原菌的16S rRNA基因序列的批量获取。该程序小巧灵活方便快捷,perl语言强大的字符串操作和模式匹配功能使程序能实现不同类型基因序列的远程获取,具有很好的通用性和实用性。     

Recent Evolutionary History of Human Immunodeficiency Virus Type 1 Subtype B: Reconstruction of Epidemic Onset Based on Sequence Distances to the Common Ancestor

We obtained and studied HIV-1 sequences with a known sampling year from three outbreaks of the HIV-1 epidemic: 141 env V3 (270 nt) sampled between 1984 and 1992 and 117 pol prot/RT (804 nt) sequences sampled between 1986 and 1999 from Dutch homosexual men and injecting drug users (IDUs), as well as 77 env V3 sequences sampled between 1983 and 1994 in the United States. Since retrospective serological and/or epidemiological data on these populations are available, providing estimates of the dates of the onset of the HIV-1 epidemics, we had the opportunity to test different phylogenetic models for their accuracy in deriving the recent evolutionary history of HIV-1 subtype B and the onset date of the HIV-1 epidemic. We observed that, in any given year, individual sequences vary widely in their distances to the common ancestor, and sequences close to the ancestors were found decades after the onset of the epidemic. Nevertheless, the mean evolutionary distances of virus strains to ancestors were increasing significantly during the course of the studied epidemics, which indicates that the molecular clock is operational in the recent evolution of HIV-1. When the relationship between the sampling years of sequences and their nucleotide distances to the common ancestor was extrapolated to the past, analysis of pol sequences provided accurate estimates of the onset years of the epidemics, whereas analysis of V3 sequences by the maximum-likelihood or neighbor-joining methods led to an overestimation of the age of the epidemics. Separate analysis of nonsynonymous and synonymous distances revealed that this overestimation results from nonsynonymous substitutions, whose numbers were not increasing significantly in all three virus populations over the observation period. In contrast, analysis of synonymous env V3 distances provided accurate estimates of the onset years for the outbreaks we studied. Received: 26 October 2001 / Accepted: 8 November 2001     

基于线粒体Cytb基因全序列探讨两爪鳖和山瑞鳖的系统进化关系

采用PCR方法对两爪鳖(Carettochelys insculpta)和山瑞鳖(Pdea steindachneri)mtDNA的细胞色素b基因(Cytb)进行了扩增和测序,并结合GenBank中已公布的其它15种龟鳖类和两种鳄类的同源序列,进行了序列变异比较和系统发生分析.以凯门鳄和扬子鳄为外群,采用邻接(neighbor ioining,NJ)法、最大简约(maximum likelihood,ML)法和最大似然(maximum parsimony,MP)法重建分子系统树.结果均支持将龟鳖目分为3支:侧颈龟科(Pelomedusidae)(非洲侧颈龟Pelomedusa subrufa),两爪鳖科(Carettochelyidae)(两爪鳖Carettochelys insculpta)和一支由其它15种现生龟鳘类组成的进化支.在这里两爪鳘科被认为可能代表一新的亚目;而山瑞鳖聚于鳖科(Trionychidae)中,与中华鳌(Pelodiscus sinensis)最先聚成一支,再依次与马来鳖(Dogania subplana)、非洲鳖(Trionyx triunguis)、印度箱鳖(Lissemys punctata)聚成一组.     

Sequence Analysis of Hypothetical Lysine Exporter Genes of Rhizobium leguminosarum bv. trifolii from Calamine Old Waste Heaps and Their Evolutionary History

The aim of this study was to identify heavy metal detoxification system in Rhizobium leguminosarum bv. trifolii isolated from Trifolium repens inhabiting old (70–100 years) Zn–Pb waste heaps in Poland by PCR reaction with czcD1 and czcD2 primers. By sequence analysis, four different genotypes of obtained amplicons were identified among eight examined isolates. Their sequence similarity ranged 91–99 %. They indicated the highest sequence identity to the hypothetical lysine exporter gene of R. leguminosarum bv. trifolii WSM1325 (91–97 %) and 76–81 % sequence similarity to hypothetical lysine exporter genes of R. leguminosarum bv. trifolii WSM2304 and R. etli CFN42 and CIAT652. On phylogenetic tree of obtained amplicons, all four studied R. leguminosarum bv. trifolii genotypes formed common monophyletic cluster with R. leguminosarum bv. trifolii WSM1325 at 100 % bootstrap support showing that all four amplicons obtained in PCR with czcD1 and czcD2 primers are fragments of hypothetical lysine exporter gene (lysE). We also suggest that Lys efflux exporter may participate in heavy metal transport out of R. leguminosarum bv. trifolii cells.     

Inferring Species Trees from Gene Trees: A Phylogenetic Analysis of the Elapidae (Serpentes) Based on the Amino Acid Sequences of Venom Proteins

Toward the goal of recovering the phylogenetic relationships among elapid snakes, we separately found the shortest trees from the amino acid sequences for the venom proteins phospholipase A₂and the short neurotoxin, collectively representing 32 species in 16 genera. We then applied a method we term gene tree parsimony for inferring species trees from gene trees that works by finding the species tree which minimizes the number of deep coalescences or gene duplications plus unsampled sequences necessary to fit each gene tree to the species tree. This procedure, which is both logical and generally applicable, avoids many of the problems of previous approaches for inferring species trees from gene trees. The results support a division of the elapids examined into sister groups of the Australian and marine (laticaudines and hydrophiines) species, and the African and Asian species. Within the former clade, the sea snakes are shown to be diphyletic, with the laticaudines and hydrophiines having separate origins. This finding is corroborated by previous studies, which provide support for the usefulness of gene tree parsimony.     

Phylogenetic Analysis of Isolates of Pasteurella pneumotropica from Laboratory Animals Based on the gyrB Gene Sequence.

Phylogenetic analysis using the gyrB sequence was performed to investigate the genetic relevance among 49 isolates of P. pneumotropica. In the phylogeny, the isolates were clearly classified into three groups as follows: group A for the isolates of biotype Jawetz derived from mice, group B for the isolates of biotype Jawetz derived from rats, and group C for the isolates of biotype Heyl. These results suggest that the gyrB sequence of P. pneumotropica differs between the isolates of two biotypes, and also between the isolates derived from mice and rats in the biotype Jawetz.