2种亚洲龙鱼的D—Loop序列结构分析

采用PCR技术对2种亚洲龙鱼的mtDNAD-Loop全序列进行扩增和测序，序列结构分析和序列同源性比对结果表明，2种亚洲龙鱼的mtDNAD-Loop在靠近5’端有3个终止相关序列TAS（Ⅰ、Ⅱ、Ⅲ），靠近D-Loop的3’端有4个保守区域CSB1、CSB2、CSB3、CSB—D。在终止相关序列和保守区域之间是连续重复区域。经DNASP4．0软件分析，全序列中检测出多态位点数（S）为26，其中有17个转换，核苷酸多样性（Pi）为0．013，平均核苷酸差异数（K）为17．333。     

湛江海域文昌鱼(Branchiostoma)线粒体Cyt b基因的全序列分析

测定了邻近湛江的茂名海区和硇洲岛海域文昌鱼各2个样本线粒体Cytb基因全序列,以尾索动物亚门的2种海鞘(Ciona spp.)和脊椎动物亚门的七鳃鳗(Petromyzon marinus)作为外群,利用分子系统学方法与GenBank收录的14个文昌鱼(Branchiostoma)的Cytb基因全序列进行了比较,UPGMA法构建了系统发生树。全序列分析表明:主产于大西洋(批针文昌鱼B.lanceolatum和佛罗里达文昌鱼B.floridae)、厦门文昌鱼(B.belcheri belcheri Gray)和湛江文昌鱼线粒体Cytb基因全序列长度为1143bp,终止密码子同为UAG;主产于青岛文昌鱼(B.belcheri tsingtaunese)和日本文昌鱼为1141bp,以T结尾,转录过程中构成UAA终止密码子。系统分析表明:16个文昌鱼样品体现出太平洋和大西洋、太平洋南北明显分化。确认采自茂名和硇洲岛的文昌鱼为厦门文昌鱼(B.belcheribelcheriGray);支持将白氏文昌鱼青岛亚种(B.belcheri tsingtaunese)提升为种,命名为日本文昌鱼(B.japonicus)。     

基于18S-ITS1-5.8S序列的吉富罗非鱼群体遗传多样性分析

运用PCR技术扩增吉富罗非鱼选育群体第20和21世代18S-ITS1-5.8S序列,分析二序列的遗传变异。结果表明,18S-ITS1-5.8S序列中,18S、内转录间隔区(ITS)1和5.8S序列分别为156、483~540、64 bp,主要变异位点位于ITS1中;基于ITS1序列的第20代吉富罗非鱼(17尾)群体内遗传距离为0.001,保守位点530个,变异位点10个,单倍型4个,单倍型多样性为0.331±0.143,核苷酸多样性为0.002,平均核苷酸差异为1.279;基于ITS1的第21代吉富罗非鱼(42尾)群体内遗传距离为0.015,6个个体存在严重碱基缺失现象,保守位点492个,变异位点49个,单倍型12个,单倍型多样性为0.638±0.083,核苷酸多样性为0.014,平均核苷酸差异为6.945。ITS序列在该两吉富罗非鱼群体中变异较小,基于ITS1序列分析的两代群体遗传多样性水平较低。     

五种鲤科鱼类生长激素cDNA的克隆和序列分析

通过RT-PCR方法,以草鱼、鳙鱼、鲫鱼、鲤鱼、齐口裂腹鱼等5种鲤科重要经济鱼类的垂体总RNA为模板扩增出其生长激素(fish growth hormone,fGH)的完整ORF序列,并克隆到pMD18-T载体上,命名为pMD-1(草鱼)、pMD-2(鳙鱼)、pMD-3(鲫鱼)、pMD-4(鲤鱼)、pMD-5(齐口裂腹鱼)。测序结果显示,ORF序列其长度均为633 bp。序列分析表明,所有ORF序列均以ATG为起始密码,以TAG为终止密码,编码210个氨基酸残基,推导的生长激素前体由22个氨基酸的信号肽和188个氨基酸的成熟肽组成。同源性分析表明,草鱼、鳙鱼、鲤鱼、鲫鱼和齐口裂腹鱼的fGH同源性在93.3%~99.5%之间。     

大口黑鲈抗菌肽hepcidin cDNA序列和结构分析

以大口黑鲈为材料,提取肝脏总RNA,经RT-PCR扩增出hepcidin cDNA的开放阅读框(ORF)及3′端非编码区序列,应用5′RACE方法得到大口黑鲈hepcidin cDNA5′末端。将所获得的两个片段分别克隆到T载体后进行测序,并拼接成大口黑鲈hepcidin全长cDNA。序列分析表明:大口黑鲈hepcidin全长cDNA为564bp,含有一个258bp的ORF,编码86个氨基酸残基,由信号肽(24个残基)、前肽(42个残基)和成熟肽(20个残基)3部分组成hepcidin前体。在前肽部分具有前肽转化酶典型的RX(K/R)R基元,成熟肽部分含有8个保守的半胱氨酸残基,可形成四个链内二硫桥,使β-折叠结构保持稳定。大口黑鲈Hepcidin与其他鱼类的同源性在29.7%~90.5%间,尤其是信号肽区域,与鳜、尼罗罗非鱼、真鲷、花鲈、黑鯛、金眼狼鲈仅有2~3个氨基酸的差别。     

金钱鱼IGF-1和IGF-2的克隆及其在胚胎发育过程的表达

【目的】克隆金钱鱼IGF-1和IGF-2基因,研究二基因在金钱鱼胚胎发育过程中的表达。【方法】采用实时荧光定量PCR,以不同发育时间点的金钱鱼(Scatophagus argus)胚胎为实验材料,分析IGF-1和IGF-2基因在鱼类胚胎发育过程中的表达图式。【结果】金钱鱼IGF-1和IGF-2的蛋白全序列分别为186和215个氨基酸。序列分析表明,IGF-1和IGF-2均有胰岛素样生长因子蛋白典型结构,含有N端信号肽、B、C、A、D和E结构域,且有6个保守的半胱氨酸,与其他鲈形目鱼类花鲈的IGF-1和IGF-2相似性较高(95%和91%)。IGF-1从受精卵到出膜的胚胎发育过程中表达量逐渐上调;IGF-2在受精后至囊胚期表达量极低,而在原肠胚期和神经胚期表达量显著上调至最高水平,在神经胚期后表达量显著降低并维持稳定。【结论】IGF-1和IGF-2序列保守性低,与鲈形目同源性高,在金钱鱼胚胎发育过程中起不同调控作用。     

蛤蜊科3种贝类16S rRNA基因片段及ITS2核苷酸序列分析

利用PCR技术分别扩增连云港及启东沿海蛤蜊科的西施舌(Coelomactra antiquata)、中国蛤蜊(Mactrachinensis)和四角蛤蜊(Mactra veneriformis)3种双壳贝的16S rRNA基因片段和ITS2核苷酸序列,测序后用DNA star软件分析了核苷酸差异。结果显示:三种贝类16S rRNA基因片段长度相同,均为306bp(去除引物),核苷酸存在多态性,共有45个变异位点,54个核苷酸发生了变异,全部为碱基置换。西施舌与中国蛤蜊此片段核苷酸的同源性为88.9%,与四角蛤蜊的同源性为88.6%,中国蛤蜊与四角蛤蜊的同源性为90.6%。三种蛤蜊ITS2序列分别为390 bp(西施舌)4、41 bp(四角蛤蜊)和466 bp(中国蛤蜊),存在长度多态性,ITS2核苷酸差异分析结果显示,西施舌与中国蛤蜊的同源性为70.9%-71.1%,西施舌与四角蛤蜊的为70.5%-71.0%,中国蛤蜊与四角蛤蜊的同源性为88.1%-88.8%。ITS2序列分析结果与16S rRNA基因片段分析结果一致,2种分子分析法均显示中国蛤蜊与四角蛤蜊的亲缘关系近。     

加州鲈生长激素和胰岛素样生长因子I cDNA的克隆及序列分析

GH-IGF-I轴是鱼体体内一个重要的内分泌生理轴,主要调控鱼体的生长发育。用RT-PCR方法从加州鲈脑垂体和肝脏组织中分别扩增出加州鲈GH和IGF-I cDNA,克隆到pMD19 T-Vector上进行序列测定和分析。结果表明:1)加州鲈GH cDNA开放阅读框长为615 bp,编码204个氨基酸,其中信号肽17个氨基酸,成熟肽187个氨基酸。成熟肽中有四个保守的半胱氨酸残基(分别位于69,177,193,202),可形成两对二硫键。加州鲈GH氨基酸序列与蓝太阳鱼、斜带石斑鱼、金头鲷、虹鳟、鲤鱼、斑马鱼相比较,同源性分别为100%、97%、94%、66%、56%、53%。2)加州鲈IGF-I cDNA开放阅读框长为561bp,编码包括信号肽和B、C、A、D、E五个区域的186个氨基酸,形成成熟肽时,信号肽和E区域被切除。成熟蛋白的氨基酸序列与GenBank中已知的河鲈、三角鲂、金头鲷、舌齿鲈、斑马鱼的相比较,结果发现四个区域的保守性有差异,A区和B区保守性较高,C区和D区保守性较差。加州鲈GH和IGF-I cDNA的获得为进一步研究鱼体GH-IGF-I轴对生长发育的调控机制奠定了基础。     

粤西镇海湾近江牡蛎线粒体16S rRNA基因序列变异分析

采用聚合酶链式反应 (PCR)技术对粤西镇海湾水域的近江牡蛎Crassostrearivularis(Gould)群体 2 7个个体的线粒体DNA16SrRNA基因序列片段进行扩增 ,获得大约 5 0 0bp的扩增产物。PCR产物经纯化后进行序列测定 ,经ClustalX同源排序 ,除去引物及部分端部序列 ,得到4 14bp的核苷酸片段。 2 7个个体共检测到 2个变异位点 ,均为颠换位点 ,没发现碱基位点插入、缺失及转换位点 ,共 3种单倍型 ,每个单倍型只有一个碱基的差异。运用DNASP软件计算得该群体的核苷酸多样性和平均核苷酸差异数分别为 0 .0 0 0 36和 0 .14 815。此结果提示镇海湾近江牡蛎群体遗传多样性已很低 ,很有必要从其他分布区引进近江牡蛎亲贝来扩大该种群的遗传多样性     

剑尾鱼IgM基因的克隆及免疫对其组织表达的影响

以免疫两周的剑尾鱼为材料,提取脾脏总RNA,通过逆转录-聚合酶链式反应(RT-PCR)扩增出分泌型免疫球蛋白M(secretory immunoglobulin M,sIgM)重链基因的核心序列,再应用3’和5’快速扩增cDNA末端(RACE)方法扩增其末端序列,拼接后获得剑尾鱼sIgM重链全长cDNA序列。全长序列含有免疫球蛋白的信号序列:FKCIANH,得到的5个可变区序列可能来源于4个不同的VH家族。经用半定量RT-PCR分析sIgM在脾脏和头肾的表达变化,初步认为利用剑尾鱼头肾sIgM的mRNA表达变化替代检测血清抗体效价具有可能性。     

Structure of mitochondrial DNA control region of Pholis fangi and its phylogenetic implication

In this study, the entire mitochondrial DNA （mtDNA） control region （CR） of Pholis fangi was amplified via polymerase chain reaction followed by direct sequencing. The length of the mtDNA CR consensus sequence of P. fangi was 853 bp in length. In accordance with the recognition sites as were previously reported in fish species, the mtDNA CR sequence of P. fangi can be divided into 3 domains, i.e., the extended terminal associated sequence （ETAS）, the central conserved sequence block （CSB）, and the CSB domain. In addition, the following structures were identified in the mtDNA CR sequence of P. fangi：2 ETASs in the ETAS domain （TAS and cTAS）, 6 CSBs in the central CSB domain （CSB-F to CSB-A）, and 3 CSBs in the CSB domain （CSB-1 to CSB-3）. These demonstrated that the structure of the mtDNA CR of P. fangi was substantially different from those of most other fish species. The mtDNA CR sequence of P. fangi contained one conserved region from 656 bp to 815 bp. Similar to most other fish species, P. fangi has no tandem repeat sequences in its mtDNA CR sequence. Phylogenetic analysis based on the complete mtDNA CR sequences showed that there were no genetic differences within P. fangi populations of the same geographical origin and between P. fangi populations of different geographical origins.     

Structure Analysis of the Complete Mitochondrial Genome in Cultivation Variety 'Rongfu'

In this report,complete mitochondrial genome sequences of Laminaria cultivation variety 'Rongfu' were obtained.The results showed the length of circular molecule of mtDNA was 37 638 bp(64.7% A+T),encoding three rRNAs(23S,16S and 5S),25 tRNAs,35 known mitochondrial proteins and 3 ORFs.Sequence alignment indicated its mtDNA genome was very similar to that of Laminaria japonica.Phylogenetic trees inferred from concatenated 30 mitochondrial genes showed that 'Rongfu',Laminaria japon-ica,Laminaria longipedalis,L...     

3种星虫线粒体16S rRNA、COI和Cyt b基因片段的序列比较

对裸体方格星虫(Sipunculus nudus)、可口革囊星虫(Phascolosoma esculenta)和澳洲管体星虫(Siphonosoma australe)的线粒体16S rRNA、COI和细胞色素b(Cytb)基因片段序列进行比较,并对其系统发生进行了初步探讨。采用PCR方法得到总长度分别为531～544bp(16S)、652～675bp(COI)和406～453bp(Cytb)的线粒体片段。片段碱基A+T比例较高(16S rRNA基因58.3%,COI基因56.9%,Cytb基因59.5%)。16S rRNA片段存在169个碱基变异位点(其中包括167个简约信息位点)和44个碱基插入/缺失,种内个体间变异较小;COI片段有512个碱基(333个简约信息位点)存在变异,79个碱基插入/缺失;Cytb片段存在347个碱基(318个简约信息位点)变异位点,16个碱基插入/缺失。数据分析结果支持3种星虫和环节动物的分类地位较近,与软体动物较远的分类观点。此外,裸体方格星虫与澳洲管体星虫之间亲缘关系较近(D=0.3159、0.3156、0.2361)。认为3种星虫线粒体16S rRNA、COI和Cytb基因在种间存在明显的多态性,证实了三种基因序列均普遍适用于星虫种及以上阶元的系统学分析。     

Bacterial diversity, composition and temporal-spatial variation in the sediment of Jiaozhou Bay, China

Studies on the diversity and distribution of bacterial populations will improve the overall understanding of the global patterns of marine bacteria and help to comprehend local biochemical processes and environments. We evaluated the composition and the dynamics of bacterial communities in the sediment of Jiaozhou Bay (China) using PCR-denaturing gradient gel electrophoresis (DGGE). Sediment samples were collected from 10 different sites in May, August, and November 2008 and in February 2009. There was significant temporal variation in bacterial community composition at all sites. However, the spatial variation was very small. The DGGE analyses of bacterial communities were used to divide the 10 stations into three types. Canonical correspondence analysis (CCA) revealed that the changes in bacterial communities were driven by sediment properties. Sequence analysis of DGGE band-derived 16S rRNA gene fragments revealed that the dominant bacterial groups in the sediment were of the classes γ-proteobacteria and δ-proteobacteria and phyla Bacteroidetes and Nitrospirae. Our results provide considerable insight into the bacterial community structure in Jiaozhou Bay, China.     

Genetic diversity analysis with ISSR PCR on green algae Chlorella vulgaris and Chlorella pyrenoidosa

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR (ISSR PCR). Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity (h *) and Shannon index of diversity (I *) in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones (0.190 3 and 0.274 8), which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.     

Genetic diversity analysis with ISSR PCR on green algae <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR （ISSR PCR）. Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity （h^＊） and Shannon index of diversity （I^＊） in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones （0.190 3 and 0.274 8）, which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

1　Introduction　PorphyraisthemainobjectforalgafarmingandplaysaveryimportantroleinChinesemarineindus tries.Recently ,therehasbeenagreatlossinPor phyracultivationduetothedegenerationoftheculti var,sothereisanincreasingdemandforgoodPor phyracultivars .Theprerequisiteofthetraditionalbreed ingandbioengineeringresearchofPorphyraistheconstructionofpurelines .Traditionally ,theclassifi cationofPorphyrawasaccordingtotheirmorphologi calcharacteristics .However ,mostmorphologicalfea turesofPorphyraar…     

Taxonomic and genetic status of lancelet in Weihai coastal waters based on mitochondrial DNA sequence

Lancelets (subphylum Cephalochordata) are a transitional species between invertebrates and vertebrates. They are currently listed in the Second Order of Protected Animals in China. Lancelets were first documented in the waters around the city of Weihai (Shandong, China) in 2002. However, little is known about the phylogeny of this population. We analyzed the sequences of cytochrome b (Cyt b) and cytochrome oxidase c subunit I (CO I) genes from samples collected from coastal waters in the cities of Weihai and Qingdao (~150 km to the south). We analyzed 176 sequences, of which 150 were novel sequences and 26 were obtained from GenBank. Our results suggest that (1) lancelets in the two cities belong to the species Branchiostoma japonicus and have a high level of genetic diversity; (2) there is a high level of gene flow and low level of genetic differentiation between lancelets from the two cities; (3) demographic expansion occurred an estimated 1.1 million years (Ma) ago (mid Pleistocene) for lancelets in Weihai-Qingdao; and (4) the divergence between B. belcheri and B. japonicus was estimated at between 37.75 Ma (early Oligocene)-46.5 Ma (late Eocene).     

Seasonal variation in imposex intensity of Thais clavigera

Imposex, specifically caused by TBT pollution, refers to the superimposition of male sexual characteristics in gastropod females. Seasonal variation of imposex intensity in Thais clavigera from both slightly and severely contaminated sites in Hong Kong waters was studied from 1988 to 1999. The male penis length showed significant difference between both sites and seasons. It was shortest during late autumn and early winter (October to December) and longest during spring and early summer (February to June). Female penis length also showed significant difference between sites. It did not change seasonally, however. The RPS (Relative Penis Size) index was the highest during autumn and early winter, and the lowest during spring and early summer. The VDS (Vas Deferens Sequence) index remained stable throughout the sampling period. This study showed that VDS index is a better indicator when we compare relative intensity of imposex. The comparison can only be meaningful provided the samples from different locations are taken during the same season.