Depth matters: Microbial eukaryote diversity and community structure in the eastern North Pacific revealed through environmental gene libraries

Protistan community structure was examined from 6 depths (1.5, 20, 42, 150, 500, 880 m) at a coastal ocean site in the San Pedro Channel, California. A total of 856 partial length 18S rDNA protistan sequences from the six clone libraries were analyzed to characterize diversity present at each depth. The sequences were grouped into a total of 259 Operational Taxonomic Units (OTUs) that were inferred using an automated OTU calling program that formed OTUs with approximately species-level distinction (95% sequence similarity). Most OTUs (194 out of 259) were observed at only one specific depth, and only two were present in clone libraries from all depths. OTUs were obtained from 21 major protistan taxonomic groups determined by their closest BLAST matches to identified protists in the NCBI database. Approximately 74% of the detected OTUs belonged to the Chromalveolates, with Group II alveolates making up the largest single group. Protistan assemblages at euphotic depths (1.5, 20 and 42 m) were characterized by the presence of clades that contained phototrophic species (stramenopiles, chlorophytes and haptophytes) as well as consumers (especially ciliates). Assemblages in the lower water column (150, 500 and 800 m) were distinct from communities at shallow depths because of strong contributions from taxa belonging to euglenozoans, acantharians, polycystines and Taxopodida (Sticholonche spp. and close relatives). Species richness (Chao I estimate) and diversity (Shannon index) were highest within the euphotic zone and at 150 m, and lowest for protistan assemblages located in the oxygen minimum zone (500 and 880 m). Multivariate analyses (Bray-Curtis coefficient) confirmed that protistan assemblage composition differed significantly when samples were grouped into shallow (≤150 m) and deep water assemblages (≥150 m).     

厚壳贻贝(Mytilus coruscus)组织及其海水环境中真菌群落结构差异分析

海洋真菌广泛参与近海生态系统的物质循环和能量流动, 同时与海洋动物之间存在复杂的相互作用。贝类是我国主要的海水养殖生物, 为深入了解海洋真菌与贝类养殖的潜在关系, 选择厚壳贻贝养殖区海水及8种组织真菌为研究对象, 利用荧光定量PCR以及ITS rDNA高通量测序解析养殖厚壳贻贝各组织及所处海水环境的真菌群落丰度和结构特征。结果显示厚壳贻贝养殖区内和边缘海域的真菌丰度显著高于养殖区外围海域; 从贻贝养殖区和组织中共获得1 409个OTUs, 其中粪壳菌纲(Sordariomycetes) 在海水真菌群落为优势纲; 而在贻贝组织中, 锤舌菌纲(Leotiomycetes, 足20.13%、肾脏14.72%)、座囊菌纲(Dothideomycetes, 鳃2.89%、后闭壳肌1.92%、血淋巴1.36%)、散囊菌纲(Eurotiomycetes, 性腺3.59%、足1.57%)和伞菌纲(Agaricomycetes, 性腺3.09%、消化腺2.71%、鳃2.50%)占据优势地位。多样性分析显示厚壳贻贝和海水间真菌群落存在显著性差异; Bray-Curtis相似距离分析显示贻贝真菌群落与养殖区内海水更为相似, 而与边缘和外围海水差距较大。厚壳贻贝不同组织间、不同区域海水间的Beta多样性差异主要来自物种替换; 贻贝与海水间真菌Beta多样性的差异主要来自丰富度差异。综上所述, 厚壳贻贝体内真菌具有组织差异性, 并且养殖活动改变了养殖区海水的真菌群落。研究结果将为贝类真菌资源、贝类-真菌相互作用及生态影响提供基础。     

马氏珠母贝Su(H)基因克隆与组织特异性表达

Su(H)基因(Suppressor of Hairless gene)对生物体细胞的分化、增殖和凋亡起重要的调控作用。根据马氏珠母贝(Pinctada martensii)转录组数据库中注释为Su(H)的unigene序列设计基因特异性引物,应用cDNA末端快速扩增(RACE)技术克隆获得了马氏珠母贝Su(H)[pm-Su(H)]基因cDNA全长序列。结果表明:pm-Su(H)基因全长3 830 bp,其中开放阅读框含有1 920 bp,编码640个氨基酸残基,5'UTR为1 568bp,3'UTR为342 bp,含有27 bp polyA;预测其分子质量为70.6 ku,等电点为7.38;多序列比对显示,pm-Su(H)与其他物种的Su(H)有较高的保守性,与牡蛎(Crassostrea gigas)的同源序列高达80%;荧光定量PCR分析表明,pm-Su(H)在马氏珠母贝闭壳肌、鳃、珍珠囊、外套膜、肝胰脏、性腺、足、血淋巴等8组织中均有表达,其中以性腺中表达量最高,其次是珍珠囊和外套膜。     

草鱼白细胞介素6基因克隆与表达分析

白细胞介素6(IL-6)是一个多效的细胞因子,在机体免疫应答、急性期反应以及造血调控等过程中发挥着重要作用。以草鱼(Ctenopharyngodon idella)为研究对象,采用RT-PCR和Smart RACE技术克隆获得草鱼IL-6(CiIL-6)cDNA序列,CiIL-6的cDNA全长为1 145 bp,包含一个长为702 bp的开放阅读框,能编码233个氨基酸,预测CiIL-6的蛋白质分子质量为26.74 ku,等电点为8.72。其中5'和3'非编码区(UTR)分别为86 bp和357bp。氨基酸同源性分析显示,草鱼和斑马鱼(Danio rerio)的亲缘关系最近,它们的CiIL-6氨基酸同源性高达76%,而与其他物种的同源性均低于30%。RT-PCR的结果显示,CiIL-6在健康草鱼的胸腺、头肾和脾脏表达最高,而在鳃、胃和心脏中的表达量最低。     

10种蟹类18S rDNA 和COⅠ基因序列分析及其分子系统发育研究

应用分子系统发育学的方法,以蟹类18S rDNA和线粒体细胞色素C氧化酶亚单位Ⅰ(COⅠ)基因序列片段为分子标记,结合形态学特征对10种蟹类的分类地位进行探讨。实验共获得10 条18S rDNA序列,长度为1780~1787 bp,其中A、T, G,C平均含量分别为23.72%, 24.58%,24.52%和27.17%;通过序列对比,发现18S rDNA序列相对保守,只含有1个从88 bp 到130 bp 约 50 bp的高可变区;物种间碱基距离比较小,从0.001到0.017。18S rDNA系统发育树为方蟹总科、沙蟹总科和梭子蟹总科起源于同-海洋蟹类祖先提供了分于生物q证惦,开上火亚N内尔量分别为26.88%.弓蟹科。获得的5条CO Ⅰ基因序列,长度均为709 bp,A、T,G、C平均含量分别为26.88%,37.62%,17.50%和18.00%;COⅠ基因片段变异性高,物种间碱基距离从0.016到0.138。COⅠ基因系统发育树真实地反映了住属各物种之间的进化关系,和传统分类非常吻合,为形态特征相似的姆类鉴定提供了-种快速准确的方法。     

不同养殖区红藻表面假交替单胞菌多样性分析

利用2216E培养基从我国沿海6种养殖红藻的20个样品表面分离了327 株假交替单胞菌。经过16S rDNA序列鉴定, 分别隶属于Pseudoalteromonas carrageenovora、P. haloplanktis、P. marina、P. agarivorans、P. elyakovii和P. lipolytica 6个种。其中P. carrageenovora数量最多, 总共211株, 在14个样品上均有发现。     

缩骨鲫(Carassius auratus sogu var.)MC4R克隆、组织分布以及与生长相关SNPs的筛选

黑色素皮质素受体-4(Melanocortin reporter-4,MC4R)是G蛋白偶联受体超家族成员,在能量调控中发挥重要作用。本实验采用同源克隆以及染色体步移对缩骨鲫(Carassius auratus sogu var.)MC4R基因进行克隆,对其核苷酸序列和推测的氨基酸序列进行生物信息学分析,通过Real-time PCR对其m RNA组织分布进行研究,并对缩骨鲫MC4R编码区潜在的与生长相关的SNPs进行筛选。本实验克隆MC4R基因组基因长度为2854bp,只包含一个外显子,长度为981bp,共编码326个氨基酸,MC4R共有七个跨膜结构,是较为稳定的疏水蛋白。缩骨鲫MC4R与鲫鱼的MC4R氨基酸序列的同源度最高达到99.3%,与MC3R和MC5R同源度相对其他MCRs成员较高。缩骨鲫MC4R m RNA在脑中表达量最为显著,眼中次之,肌肉,卵巢和肾脏中表达较少,在鳃、肠、心脏和肝胰腺中微量表达。在MC4R的CDs区内检测到6个与生长潜在相关的SNPs:两个错义突变(A199G和A881C),4个同义突变(A123G,C231T,A444G和C480T)。本实验可为缩骨鲫生长和发育的分子机制研究以及遗传育种提供基础资料。     

南黄海和渤海近海海域中华哲水蚤的现场摄食研究

桡足类是海洋生态系统中初级生产者和较高营养级消费者之间的关键联系环节,掌握桡足类的现场食物组成对于准确评估海洋食物网中的营养关系和能量转移至关重要。本研究中,我们以中华哲水蚤这一在中国、日本以及韩国近海具有重要生态地位的大型哲水蚤属桡足类为研究对象,应用之前开发的基于PCR的克隆技术,通过分析中华哲水蚤所摄食生物的18S rDNA序列,研究了中华哲水蚤的现场食物组成。结果揭示了南黄海（Y19站位）和渤海（B49站位）中华哲水蚤食物组成的多样性。共检测出43个操作分类单元（OTUs）,分别隶属于13个类群：硅藻（Bacillariophyta）、甲藻（Dinoflagellata）、硅鞭藻（Dictyochophyceae）、金藻（Chrysophyta）、Katablepharidophyta、浮生藻（Pelagophyceae）、无根虫（Apusozoa）、水螅水母（Hydrozoa）、栉水母（Ctenophora）、棘皮动物（Echinodermata）、被囊动物（Tunicata）、毛颚动物（Chaetognatha）以及海洋真菌。结果还表明,当发生藻类暴发时,中华哲水蚤可以摄食引发藻类暴发的藻种。当周围海域浮游植物的丰度相对较低时,中华哲水蚤可以选择摄食各种后生动物尤其是水螅水母和栉水母的卵、幼虫或者有机碎屑。我们的研究结果表明中华哲水蚤是一种杂食性桡足类,它对食物的选择依赖其生活海域中食物的可获得性。     

三十烷醇对海带三个品系雌配子体克隆生长和发育的影响

以海带雌配子休克隆为实验材料,通过施用三十烷醇处理。结果表明,三个品系在供试条件下,LH36生长最好,LH30发育率最高。它们对TA的反应为浓度在0．5～1．0×10-5mg／L时,都有促生长作用,其中LH30反应最敏感,LH10次之。浓度在1．0×10-5mg／L时,发育率最高,其中LH30发育率为51．5％,LH10为18．0％。LH36对各浓度处理的发育反应,差异都不明显。     

Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences

Vibrios are widespread in the marine environment and a few pathogenic species are known to be commonly associated with outbreaks of diarrheal diseases in humans due to the consumption of raw or improperly cooked seafood. However, there are also many Vibrio species which are potentially pathogenic to vertebrate and invertebrate aquatic animals, and of which little is known. In an attempt to develop rapid PCR detection methods for these latter class of vibrios, we have examined the 16S-23S intergenic spacers (IGSs) of 10 lesser-known Vibrio species and successfully developed species-specific primers for eight of them--Vibrio costicola, V. diazotrophicus, V. fluvialis, V. nigripulchritudo, V. proteolyticus, V. salmonicida, V. splendidus and V. tubiashii. The IGS amplicons were amplified using primers complementary to conserved regions of the 16S and 23S rRNA genes, and cloned into plasmid vectors and sequenced. Analysis of the IGS sequences showed that 37 ribosomal RNA (rrn) operons representing seven different IGS types have been cloned from the 10 vibrios. The three IGS types--IGS(0), IGS(IA) and IGS(Glu)--were the most prevalent forms detected. Multiple alignment of representative sequences of these three IGS types from different Vibrio species revealed several domains of high sequence variability, which were used to design species-specific primers for PCR. The specificity of the primers were evaluated using total DNA prepared from different Vibrio species and bacterial genera. The results showed that the PCR method can be used to reliably detect eight of the 10 Vibrio species in marine waters in this study.