Microbial Diversity in Nankai Trough Sediments at a Depth of 3,843 m

Dense populations of bivalves, primarily Calyptogena sp., were observed at cold seeps of the Nankai Trough. Bacterial input to the sediment was estimated through determination of phospholipid ester-linked fatty acid (PLFA) and DNA profiles. Results indicated a bacterial biomass of 10⁹ cells (g dry wt)^-1 while individual fatty acid profiles revealed a predominance of monounsaturated fatty acids, mainly 18:1 isomers. The presence of these fatty acids can be interpreted to reflect a response to low temperature and a predominance of psychrophilic bacteria. DNA fragments encoding bacterial ribosomal RNA small-subunit sequences (16S rDNA) were amplified by the polymerase chain reaction method using DNA extracted directly from the sediment samples. From the sequencing results, at least 19 kinds of bacterial 16S rDNAs related to mostly the Proteobacteria and a few gram-positive bacteria were identified. These results suggest that the bacterial community in the Nankai Trough sediments consists of mainly bacteria belonging to the Proteobacteria , , and subdivisions. Bacteria belonging to the and subdivisions, which are known to include epibiont and sulfate reducing bacteria, respectively, were mostly detected in the sediment obtained from inside the area of the Calyptogena community, and the -Proteobacteria may function to supply reduced sulfur to bacterial endosymbionts of Calyptogena.     

Phylogenetic analysis of Pectinidae(Bivalvia) based on the ribosomal DNA internal transcribed spacer region

The ribosomal DNA internal transcribed spacer (ITS) region is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, the molecular phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) was performed using the nucleotide sequences of the nuclear ITS region in nine species of this family. The sequences were obtained from the scallop species Argopecten irradians, Mizuhopecten yessoensis, Amusium pleuronectes and Mimachlamys nobilis, and compared with the published sequences of Aequipecten opercularis, Chlamys farreri, C. distorta, M. varia, Pecten maximus, and an outgroup species Perna viridis. The molecular phylogenetic tree was constructed by the neighbor-joining and maximum parsimony methods. Phylogenetic analysis based on ITS1, ITS2, or their combination always yielded trees of similar topology. The results support the morphological classifications of bivalve and are nearly consistent with classification of two subfamilies (Chlamydinae and Pectininae) formulated by Waller. However, A. irradians, together with A. opercularis made up of genera Amusium, evidences that they may belong to the subfamily Pectinidae. The data are incompatible with the conclusion of Waller who placed them in Chlamydinae by morphological characteristics. These results provide new insights into the evolutionary relationships among scallop species and contribute to the improvement of existing classification systems.     

基于16S rRNA 部分序列探讨部分鳚亚目鱼类的分子系统进化关系

通过PCR扩增获得了3科5属6种中国黄渤海海域的鳚亚目(Blennioidei)鱼类的线粒体16S rRNA基因序列片段约669bp碱基,结合来自 GenBank的5种鳚亚目其他科鱼类的相应基因片段,并以眼斑雪冰鱼(Chionodraco rastrospinosus)为外群,生成供系统发育分析的序列矩阵,利用MEGA 4.0软件分析序列的碱基组成、差异百分比、转换/颠换值等,应用最大简约法(MP)和邻接法(NJ)构建系统发育树.结果显示:在鳚亚目鱼类的16S rRNA 片段生成的序列矩阵中发现有碱基的插入缺失现象,共有207 bp变异位点,转换/颠换值为0.8,碱基平均差异为3.36;支持绵鳚(Enchelyopus elongates)归于鳚亚目绵鳚科(Zoarcidae),鳚(Azuma emmnion)归于鳚亚目线鳚科(Stichaeidae);方氏云鳚(Enedrias fangi)和云鳚(Enedrias nebulosus)种间遗传距离只有0.01,亲缘关系最近.     

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.     

Sensitive and rapid detection of two toxic microalgae Alexandrium by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was designed and evaluated for rapid detection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning (PSP). Two sets of four specific primers targeting these two species were derived from the sequence of internal transcribed spacer (ITS) of ribosomal DNA. The method worked well in less than an hour under isothermal conditions of 65℃. LAMP specificity was validated in closely related algae as a comparison, suggesting the strict specificity of the LAMP primers. Two visual inspection approaches were feasible to interpret the positive or negative results. The detection limits of A. catenella and A. minutum samples using the LAMP assay were found to be 5.6 and 4.5 pg DNA, respectively. The sensitivity of this LAMP assay was 10 or 100-fold higher than Polymerase Chain Reaction (PCR) method in detecting the two microalgae. These characteristics of species specificity, sensitivity, and rapidity suggest that this method has the potentiality in the monitoring of red tide caused by A. catenella and A. minutum.     

条斑紫菜SSU 中内含子分析

根据GenBank中已知的条斑紫菜(Porphyra yezoensis)SSU和LSU r RNA基因序列分别设计引物,分别以混合个体和单个个体的基因组DNA以及c DNA为模板进行扩增,测序分析后推测SSU r RNA基因中的内含子转录后加工可能仍被保留,而在LSU r RNA基因上游序列中没有发现内含子。长511bp的内含子位于SSU r RNA基因序列的上游,其插入位点为TCTGGTG-CCAGCAGCC,内含子5′和3′端的核苷酸序列分别为AAC-和-ATGG。该内含子的剪接位点以及保守区P,Q,R,S与I型内含子是不同的。研究发现,在同种紫菜中,不同个体SSU基因中内含子的有无、分布及结构特征是不同的,说明内含子对于分类是不太适合的。但是内含子的长度以及核苷酸序列在种间又具有一定的特异性,可以为红藻种间以及种内亲缘关系较远的亚种和不同地理群的区分和鉴定提供参考。     

巨蛎属牡蛎DNA序列的比较及鉴定

【目的】了解巨砺属牡蛎的系统关系和种类区分。【方法】对我国沿海分布的7种巨砺属牡蛎(香港牡蛎、有明牡蛎、熊本牡蛎、岩牡蛎、艾氏牡蛎、太平洋牡蛎、葡萄牙牡蛎)的线粒体DNA片段(COI、12S、16S和tRNAs),以及核糖体RNA基因转录间隔子序列(ITS-1、ITS-2和ITS)进行分子系统学研究。【结果】无论种间或种内,tRNAs序列差异最大,亚种内TS1序列差异最大;tRNAs和COI序列较其它序列变异更快,种间变异与种内变异之间有明显的条形码间隙;12S、16S、ITS和ITS2的种间变异与种内变异无重叠及条形码间隙;ITS1的种间变异与种内变异出现重叠。在亚种层面,12S、ITS、ITS1和ITS2的亚种间变异与亚种内变异出现重叠;16S的亚种间变异与亚种内变异无重叠及条形码间隙;tRNAs与COI序列亚种间变异与亚种内变异之间有明显的条形码间隙,有利于区分亚种。【结论】tRNAs和COI序列可用于种及亚种的鉴定。     

胶州湾浮游桡足类18S核糖体RNA基因序列变异分析

设计了桡足类特异引物,从胶州湾浮游生物混合DNA中选择性扩增了浮游桡足类18S核糖体RNA基因片段,建立了该基因片段变异类型文库。从文库中随机选择83个克隆,对所含的18S核糖体RNA基因片段进行了V,sp I和Bsh NI RFLP分析,发现5种操作分类单元(OTU),并对这些OTU的代表克隆进行了测序。分析发现获得的克隆均属于桡足亚纲。根据各OTU覆盖的克隆数计算的胶州湾浮游桡足类遗传多样性指数为0．96。特定基因序列分析能提供易整合、易比较的多样性数据,也是建立浮游生物群落结构分子生物学剖分方法的基础和形态分类的补充。