Complete nuclear ribosomal DNA sequence analyses of the black-footed abalone Haliotis iris

The black-footed abalone Haliotis iris is an economically important shellfish species in New Zealand. We successfully amplified, sequenced and analysed the complete nuclear ribosomal DNA (nrDNA) of H. iris. The length of the nrDNA was determined to be around 9.6?kb and included, in order, small subunit ribosomal RNA (nrSSU, 1858bp), internal transcribed spacer (ITS, 749?bp), large subunit ribosomal RNA (nrLSU, 3412bp) and an intergenic spacer (IGS, 3560–3662?bp). The nrLSU genes were identical in two individuals, whereas the nrSSU and ITS regions existed at three and four base differences, respectively. The IGS was more variable than the other nrDNA regions. A phylogenetic tree was constructed based on the ITS sequence datasets, which revealed that Haliotidae has two major subclades, mainly distributed in the North Pacific, Europe and Australia. The complete nrDNA sequence will be useful for the classification, phylogeny and breeding of this shellfish.     

龙须菜5.8S rRNA和ITS区的克隆与系统学分析

研究青岛产龙须菜居群内的遗传多样性和系统学分类,通过对龙须菜居群内不同个体的5.8S rRNA-ITS区(包括ITS1-5.8S rRNA-ITS2)进行PCR扩增和序列分析,得到扩增DNA片段长度均为1 066 bp,包含完整的ITS1(250 bp)、5.8S(159 bp)和ITS2(657 bp);利用GenBank数据库对Cracilaria(江蓠属)和Gracilariopsis属共20个物种的rRNA-ITS相应序列进行了比较和系统进化分析.结果表明,龙须莱和江蓠科19个物种中rRNA的5.8S区的长度和序列非常保守,而ITS区的长度和序列则变异较大,20种江蓠科物种的遗传距离在0.013～0.596之间,龙须菜在5.8S rRNA-ITS区特性上相比江蓠属物种与Gracilariopsis属物种更为相似;采用UPGMA法构建了这20种江蓠科物种的系统发育树;分析结果表明青岛产龙须菜的居群内不存在遗传差异,且应属于Gracilariopsis属,并且在江蓠科中较早分化,而龙须菜处于Gracilariopsis属中比较古老的位置.     

Molecular analysis of complete ssu to lsu rdna sequence in the harmful dinoflagellatealexandrium tamarense (korean isolate, HY970328M)

New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellateAlexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report inAlexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison withProrocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton,A tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities ofAlexandrium genus, particularly of the tamarensis complex.     

烟台海域暴发浒苔ITS及5.8S rDNA的克隆及序列分析

以烟台海域引发"绿潮"的浒苔为研究对象,采用PCR技术扩增出浒苔的ITS-1、5.8SrDNA及ITS-2片段,将扩增出的片段纯化后克隆至pGEM-TEasy载体,筛选阳性克隆进行序列测定。结果表明,浒苔的ITS-1序列长度为195bp,5.8S序列为155bp,ITS-2序列为181bp,该序列与浒苔属的多种物种ITS序列具有很高的同源性,在ITS-1区、5.8SrDNA区和ITS-2区仅存在4个转换/颠换位点。结合GenBank注册序列和本研究的结果发现,单纯依靠ITS序列并不能对浒苔属种类进行有效的分类鉴定。     

Molecular phylogenetic analyses of threeSynechococcus strains isolated from seawater near the ieodo ocean research station

ThreeSynechococcus strains were isolated from seawater near the Ieodo Ocean Research Station (IORS), and their 16S rDNA genes and the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes were sequenced to investigate their phylogenetic relationships. Phylogenetic trees based on the 16S rDNA and ITS sequences showed that they clustered in the main MC-ASynechococcus group (subcluster 5.1), but formed branches differentiating them from the described clades. As the IORS is located in an area affected by diverse water masses, highSynechococcus diversity is expected in the area. Therefore, the IORS might be a good site to study the diversity, physiology, and distribution of theSynechococcus group. Key words —Synechococcus, Phylogeney, 16S rRNA, ITS gene, Ieodo     

利用rDNA和ITS序列对1株裸甲藻的初步鉴定

测定了 1株分离自青岛胶州湾海水样品、从形态上初步确定为裸甲藻属 (Gymnodiniumsp,编号为 GYN- 1 5)的核糖体基因 (r DNA)和转录单元内间隔区 (Internal Transcribed Spacer,ITS)序列 ,并利用该序列对该藻进行了初步鉴定。测定的序列长 2 658bp,涵盖了小亚基 (smallsubunit,SSU) r DNA基因 3'端 1 747bp,ITS1 - 5.8S r DNA- ITS2全长和大亚基 (large subunit,LSU) r DNA基因 5'端 337bp。同源性分析该序列发现 GYN- 1 5与共生甲藻属 (Symbiodinium)中的 2个种 (Symbiodinium californium和 Gymnodinium varians)的对应序列具有很高的相似性 ;3株藻的各段 r DNA和 ITS序列的相似性均为 99%以上 ;以 SSU r DNA序列中的 3个可变区 (V1 V2 V3)和邻接法构建的系统树表明 ,GYN- 1 5与 S.californium和 G.varians构成 1个独立的新的子类群 ,该子类群属于共生甲藻属 ,而与各种裸甲藻的亲缘关系较远。根据这些结果可将GYN- 1 5初步鉴定为属于共生甲藻属。鉴于 GYN- 1 5和其它 2个种所构成的分支明显与 5个已知的子类群 (A,B,C,D,E)不同 ,因此将该分支命名为子类群 F。     

缘管浒苔5.8SrDNA及其转录间隔区的序列测定及分析

利用PCR扩增、克隆和测序的方法对青岛市红岛虾池生长的缘管浒苔(Enteromorpha linza)的5.8S rD-NA及其转录间隔区ITS序列进行了分析,并依据ITS序列构建了浒苔属不同种之间的进化树。结果表明,获得了核糖体DNA完整的ITS和5.8S rDNA序列,ITS1为195 bp,5.8S rDNA为155 bp,ITS2为184 bp,总长度GC含量为63%。缘管浒苔与浒苔和曲浒苔的序列差异百分率较小,分别为2.4%和4.6%,与(U.olivascens)的序列差异百分率最大,为16.7%。Kimura-2参数遗传距离结果表明缘管浒苔和浒苔的遗传进化关系较近。     

三株赤潮硅藻5.8S rDNA及转录间隔区(ITS)的克隆及序列分析

对引发赤潮的3株硅藻——1株尖刺拟菱形藻(Pseudo-nitzshia pungens)和2株中肋骨条藻(Skeletonema costatum)的5.8SrDNA和ITS(internal transcribed spacers)序列进行了PCR扩增、克隆和序列测定,并分析了硅藻门10株赤潮藻(7株从GenBank获得)的系统进化关系.研究结果表明,尖刺拟菱形藻的ITS和5.8SrDNA的长度为693bp,SK-1(分离自东海赤潮暴发区)测序得到715bp,除ITS和5.8SrDNA外,还包含部分18SrDNA和28SrDNA;SK-2(分离自青岛养殖场)的ITS和5.8SrDNA的长度为331bp,尖刺拟菱形藻与从GenBank中获得的2株尖刺拟菱形藻相似程度最高,为100%,与该属的多列拟菱形藻相似程度稍低,为82.9%.SK-2的ITS序列与SK-1的相似程度很低,只有51%,但与拟中肋骨条藻的ITS序列相似程度高,为95.5%.SK-1的ITS序列与拟中肋骨条藻的相似程度也低,为56.7%.系统进化树反映的结果与相似性反映的结果一致.研究的该株尖刺拟菱形藻从根据ITS序列研究的结果与形态鉴定的结果看是一致的;SK-2可能属于拟中肋骨条藻;SK-1比较特殊,有待于用其他的方法进一步研究确定其分类地位.     

北部湾海域球形棕囊藻遗传多样性分析

南海北部湾海域近年来连年暴发球形棕囊藻赤潮,严重威胁近海生态安全,亟待探明赤潮成因。球形棕囊藻是一种广泛分布于温带至热带海域的有害藻华微藻,具有明显的种下遗传分化。为了解北部湾海域球形棕囊藻的遗传多样性及其对赤潮藻种来源的指示意义,本研究针对分离于北部湾海域赤潮发生期间的4株棕囊藻,以核糖体28S大亚基rRNA基因（28S rDNA）D1-D2区和转录间隔区（ITS区）为靶区构建克隆文库,通过测序分析了北部湾海域球形棕囊藻的遗传多样性。结果表明,从北部湾分离的4株棕囊藻均为球形棕囊藻,但不同年份分离的球形棕囊藻藻株之间存在明显的遗传差异。部分球形棕囊藻藻株（PG2015和PG2017）的不同克隆间也存在遗传差异,这种遗传差异在由单一藻细胞建立的克隆培养系中仍存在,表明遗传差异是来自细胞内多拷贝的基因序列,可能源自不同地理种群球形棕囊藻之间的基因交流。与ITS区相比,28S rDNA D1-D2区能更好地反映球形棕囊藻遗传多样性状况,具有作为分子标记指示球形棕囊藻地理种群的潜力,但仍需深入研究。研究结果深化了对北部湾球形棕囊藻遗传多样性的认识,有望为进一步解析北部湾海域球形棕囊藻赤潮原因种来源提供判据。     

黄海西部细弱刚毛藻（刚毛藻目，绿藻门）的表型可塑性与分类——暗示基因序列作为DNA条形码的可行性

由于广泛的形态可塑性,导致刚毛藻属物种的分类仍存在不确定性。作为分布较广的物种之一,细弱刚毛藻已报道了许多变异类型,这为对其鉴定造成了困难。针对这个问题,本研究通过采集于黄海西部相似于细弱刚毛藻的9个样品,分析了它们的形态多样性。一些样品具有明显的可变鉴定特征,难于利用形态分类准确鉴定。因此,采用18S rDNA和 ITS序列对它们进行了分子鉴定。其中,样品的18S rDNA序列相似度为99.6%-100%,而ITS的为98.7% -100%。分子数据强烈地支持了形态可变的样品可准确定位为细弱刚毛藻。通过对样品分类特征的比较分析发现,作为分类标准的分枝方式和密度、藻体颜色、藻体高度和质地受环境影响和藻体成熟度而发生较大变化。此外,作为相对稳定的特征,细胞大小在种内水平上也常发生变化。18S rDNA和ITS序列在本种鉴定上的成功应用,表明以DNA条形码为基础的基因序列分析可作为传统形态分类学强有力的辅助方法。     

象山港养殖大黄鱼寄生新贝尼登虫成虫形态学和28S rDNA,ITS1分子鉴定

贝尼登类单殖吸虫是象山港海水养殖鱼类中一类危害严重的寄生虫。应用PCR扩增及DNA序列分析的分子生物学手段,并结合对成虫的形态学分析,对象山港养殖大黄鱼体表寄生的贝尼登类单殖吸虫(记作XSp)进行了种类鉴定,结果表明XSp从形态特征上属于新贝尼登虫属,与梅氏新贝尼登虫高度相似。扩增得到XSp的28S rDNA和ITS1序列长度分别为393和427 bp,与梅氏新贝尼登虫和鱾新贝尼登虫的5个28S rDNA 序列、2个ITS1序列的比对分析显示相似性除1个为97.4%外其余均大于99%,提示XSp与这几个鱾新贝尼登虫和梅氏新贝尼登虫为种内关系,而XSp与贝尼登虫的3个28S rDNA 和1个ITS1序列相似性分别为84.3%~89.5%和60.2%。系统进化树显示该吸虫与梅氏新贝尼登虫和鱾新贝尼登虫形成一个紧密的簇,而与贝尼登虫亲缘关系较远。根据普通生物学和序列特征分析,将XSp定种为梅氏新贝尼登虫,并且支持Whittington和Horton (1996)提出的梅氏新贝尼登虫和鱾新贝尼登虫为同种异名的分类观点。     

以16S和16S—23S rDNA间区为靶区建立副溶血弧菌(Vibrio parahaemolyticus) PCR快速检测技术

提要应用BLAST程序和DNAstar软件,比较分析了5株副溶血弧菌和其他细菌的16S—23S rDNA间区序列,选取IGSIA作为扩增靶区,结合相邻的16S rDNA序列,设计合成了一对特异性引物,通过优化扩增条件,建立了快速检测副溶血弧菌的PCR方法,对其特异性和敏感性进行了探讨,并初步进行了应用研究。结果表明,新建的PCR方法能特异性地扩增出大小为306bp和552bp的2条带,分别对应于副溶血弧菌的IGS0和IGSIA,检测灵敏度为5.6×102CFU/ml,半个工作日即可得到准确的结果,能有效检测出在杂色鲍、凡纳滨对虾和各种水质中的副溶血弧菌,适用于海洋水产动物副溶血弧菌病的早期快速诊断、水产品的检疫及水质环境的监测。     

裂殖壶菌OUC88 及10 个派生菌株18S rDNA 基因克隆和分析

用PCR方法从裂殖壶菌Schizochytrium limacinum OUC88及以其为出发菌株经紫外诱变筛选的10个菌株中扩增出18S rDNA基因序列(1751bp到1758bp)进行序列测定,以上序列已登录GenBank(HM042904-HM042914)并与已登录的裂殖壶菌属5条18S rDNA序列比对分析。结果表明,S.limacinum OUC88以及10个派生菌株间18S rDNA的遗传距离是0.000~0.013,与Schizochytrium sp.FJU-512 18S rDNA(GenBank No.AY758384)的同源性最高,为98%~99%;与S.limacinum(GenBank No.AB022107)的同源性为96%;与同属异种S.mangrovei(GenBank No.DQ100293)只有93%的同源性。并运用序列比对分析和MEGA4.0系统进化树,结果显示种内诱变产生的细微变异小于同属内不同物种之间的变异。本研究除了为裂殖壶菌这种重要的经济海洋真菌提供分子生物学资料以外,同时表明18S rDNA序列不仅在分子分类上是一个重要的标志,也可分析由突变引起的物种内细微的遗传变异。     

北部湾涠洲岛红色赤潮藻的分子鉴定

为了弄清北部湾涠洲岛6株疑似红色赤潮藻（Akashiwo sanguinea）株的种类,作者采用光学显微镜和荧光显微镜对其进行形态学初步鉴定;并对藻株SSU rDNA、LSU rDNA和ITS序列测序,利用最大似然法构建系统发育树。结果表明,北部湾涠洲岛6株藻与红色赤潮藻形态特征基本符合;3种序列系统发育树分析均发现北部湾涠洲岛6株藻与不同海域来源的红色赤潮藻聚在同一大分支上,自展值高达99、100、100;与来自亚洲海域的韩国安山株、新加坡株、中国厦门港株序列差异最小,亲缘关系最近,而与其他地理来源的红色赤潮藻序列差异大,亲缘关系较远。红色赤潮藻SSU、LSU、ITS种内遗传距离分别为0.001~0.008、0.003~0.025和0.045~0.406,明显小于该藻与其他裸甲藻科（Gymnodiniaceae）藻属下的种间遗传距离0.032~0.072、0.165~0.222和0.589~1.559,因此可确定北部湾涠洲岛6株藻均为红色赤潮藻。研究结果将为北部湾海域赤潮生物来源与组成、赤潮的发生与管理提供基础信息。     

基于18S和ITS-5.8S rDNA基因序列的白色霞水母(Cyanea nozakii)的分子鉴定与检测

采用通用引物PCR扩增法,测定了辽东湾海域的白色霞水母(Cyanea nozakii)螅状体、碟状体及水母体的18S以及ITS-5.8S r DNA序列,同时利用Gene Bank数据库中已有同源序列对其进行序列分析及系统分析。结果显示,白色霞水母3个个体的18S和ITS-5.8S r DNA序列完全一致。白色霞水母样品的ITS-5.8S r DNA序列与Gen Bank中未知真核生物的序列高度相似(≥99%),推测该物种可能是早期发育阶段(卵、浮浪幼虫或碟状体)的白色霞水母样品。霞水母属不同种间18S r DNA序列经比对后同源序列长度为1709bp,多态位点数33个;比对后ITS1同源序列长度为368bp,其中变异位点203个,简约信息位点数178个,单变异位点21个。基于18S r DNA基因序列的霞水母属种内和种间平均遗传距离分别为0、0.008,而基于ITS1序列的霞水母属种内和种间平均遗传距离分别为0.019、0.284。基于ITS1的种间遗传距离是种内遗传距离的15倍,适合于进行物种鉴定。NJ系统树的结果也表明同种的不同个体各自聚枝,其聚类结果大致与形态分类吻合。研究表明,ITS基因片段在霞水母不同种间变异较大,更适于大型水母种类鉴定、检测及属内种间水平的系统进化研究。     

有棘无棘两种表型栉江珧28S和COI基因序列差异的比较

在形态学分析的基础上,对有棘和无棘两种不同表型栉江珧(Atrina pectinata)的28S rDNA和COI基因片段进行序列分析比较.结果显示这两种表型的DNA序列差异很小,28S rDNA(1 075 bp)无差异,而COI(659 bp)碱基差异最大为1.5%,不能提供这两种表型的栉江珧划分为两个种的证据.     

文蛤属(Meretrix)16S rRNA基因及ITS1序列的系统学分析

利用线粒体16SrRNA基因片段及核糖体DNA转录间隔区ITS1序列分析的方法,以近缘属的青蛤(Cyclinasinensis)为外群,对文蛤属的文蛤(M.meretrix)、丽文蛤(M.lusoria)、帘文蛤(M.lyrata)和斧文蛤(M.lamarckii)4种贝类进行了系统学研究。经ClustalX多重比对及DNAsp软件分析后,用PAUP4.0分析软件,计算出种间序列的碱基转换/颠换频率,利用邻接法NJ构建系统发育树。结果表明,帘文蛤与该属其他三种的分歧时间较早,两类序列与其他种类间的相对遗传距离分别在0.25866—0.28218和0.15644—0.20104之间。文蛤与丽文蛤间的16SrDNA及ITS1序列间的遗传距离仅为0.04805和0.04201,拓扑结构图显示关系很近,并且二者的分布区大面积重叠,其序列间的转换/颠换频率接近种内单元型(haplotype)间的序列变化,鉴于它们的贝壳形态有一定差别,应归为同一个种内的不同地理亚种比较恰当。     

Effects of kelp Ecklonia maxima inclusion in formulated feed on the growth,feed utilisation and gut microbiota of South African abalone Haliotis midae

The effect of kelp Ecklonia maxima inclusion in formulated feeds on abalone growth and gut bacterial communities has not been previously investigated in South Africa. An eight-month on-farm growth trial was conducted with sub-adult Haliotis midae (～43 mm shell length) fed graded levels of kelp in formulated feeds. Kelp inclusion (0.44–3.54% of pellet dry mass) promoted faster growth (65.7–74.5% total mass gain), with better feed and protein conversions (apparent feed conversion ratio [FCR] 1.4–1.8, apparent protein efficiency ratio [PER] 2.3–2.7), as compared with the non-supplemented feed (52.3% total mass gain, FCR 2.1, PER 1.9; p < 0.001). Abalone-gut bacterial DNA was sequenced using 16S rRNA gene pyrosequencing and the sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity level. A supplementary 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) analysis was employed. The dominant OTUs differed in terms of their relative abundances, with an autochthonous Mycoplasma strain being significantly more abundant (p = 0.03) in the gut of abalone fed a kelp-supplemented feed. The DGGE band patterns displayed higher within-group variability for abalone fed the control diet, suggesting that dietary kelp inclusion promotes gut-bacteria homeostasis. This may contribute to better feed utilisation and growth in abalone fed kelp-supplemented feeds.     

日本绒螯蟹放流群体12S rRNA序列研究

参考果蝇与蚤状汊序列进行了日本绒螯蟹放流群体的线粒体ＤＮＡ１２Ｓ ｒＲＮＡ基因片段的引物设计、ＰＣＲ扩增及序列测定,得到４５７ｂｐ的碱基序列,其Ａ、Ｔ、Ｇ、Ｃ含量分别为１５８ｂｐ（３４．５７％）、１７８ｂｐ（３８．９５％）、５１ｂｐ（１１．１６％）、７０ｂｐ（１５．３２％）,与果蝇与蚤状相同基因片段序列含量相似。     

Molecular identification of bloom-forming species Phaeocystis globosa (Pryninesiophyta) and its dispersal based on rDNA ITS sequence analysis

The colony-forming Phaeocystis species are causative agents of dense bloom occurrences in coastal waters worldwide. It is difficult to separate them because of the different morphologies associated with their colonial stages. In this study we applied molecular approaches to analyze the genetic variation of Phaeocystis globosa and Phaeocystis pouchetii from several geographic regions, and to assist in tracing the dispersal of bloom-forming Phaeocystis species in coastal waters of China. The sequences of the internal transcribed spacers (ITS1 and ITS2) of rDNA and the 5.8S ribosomal RNA gene of Phaeocystis strains were determined. Sequence comparison shows that P.globosa was the most divergent to P. pouchetii, exhibiting sequence divergence higher than 0.08. However, lower genetic divergences existed between strains of P.globosa. The sequence comparison of the Phaeocystis rDNA ITS clearly shows that the species isolated from the southeast coast of China is identified as P.globosa rather than P. cf. pouchetii or other species. Furthermore, the significance of rDNA variation in distinct global populations of P.globosa suggested it might have had sufficient time to accumulate detectable mutations at the rDNA locus, supporting the hypothesis of ancient dispersal of P.globosa to many areas, meaning that P.globosa blooms in the coastal waters of China are endemic rather than a newly introduced species or a foreign source. Finally, based on the high divergent region of rDNA ITS, a pair of species-specific primers for P.globosa were designed, they could be useful to detect the presence of this species in mixed plankton assemblages or flagellate stages that are recognized with diffculties by means of conventional microscopy.