青蛤(Cyclina sinensis)cDNA文库构建及免疫相关因子基因的筛选

在鳗弧菌侵染下,利用SMART方法构建了青蛤的cDNA文库,并采用高通量测序方法和BLASTX比对筛选出ESTs及免疫相关因子的基因.结果表明,所构建的cDNA文库的库容量为1.12×106,插入片段均在500bp以上;大于100bp的有效ESTs序列1113条,平均长度725bp,拼接后获得一致性序列420条,其中包括126个叠联群和294个单拷贝EST,BLASTX比对后获得注释序列271条,进一步筛选获得青蛤免疫相关因子基因序列45条,为克隆其免疫防御相关因子的基因提供了重要的基础.     

耳鲍(Haliotis asinina)核糖体小亚基(18S rRNA)编码基因的克隆与序列分析

采用分子生物学的方法,对南海不同海区的两个地理群体耳鲍(Haliotis asinina)18S rRNA基因全长进行了克隆和序列分析,并将耳鲍18S rRNA基因的序列与NCBI数据库中已收录鲍的18SrRNA基因进行了比较。结果发现,南海耳鲍核糖体18S rRNA基因与耳鲍H.asinina isolate H11核糖体18S rRNA基因序列的相同率高达98%;同一地理群体内耳鲍核糖体18S rRNA基因序列完全一致;不同地理群体间耳鲍核糖体18S rRNA基因在碱基组成上的相似率为99%,仅在某些位点处发生了碱基替换,即腺嘌呤(T)被鸟嘌呤(G)替换;同时,将这两个不同群体中耳鲍的18S rRNA基因与泰国耳鲍18S rRNA基因序列进行比较分析发现,它们之间也只是发生了碱基替换。     

刺参(Apostichopus japonicus)EST序列中微卫星分布分析及其标记的筛选

利用微卫星查找软件对现已公布的6632条刺参ESTs的数据库中2—6个碱基重复单元组成的简单序列重复进行筛选,进而对其微卫星的丰度和分布进行比较研究。共发现微卫星序列416个,占整个ESTs数据库的6.48%;其中含双碱基重复序列146个,数量最多,占ESTs数据库中发现微卫星序列总数的65.86%;三、四、五、六碱基重复序列分别占微卫星序列总数的26.68%、1.68%、0.24%、5.53%。对含有SSR位点符合微卫星引物设计的EST序列利用Primer软件结合人工方法设计合成引物21对,其中13对有扩增产物且均为多态位点。扩增产物变性聚丙烯酰胺胶电泳,统计每个微卫星标记等位基因数目,计算等位基因频率、观测杂合度、期望杂合度等统计学指标的评价。结果表明,在13个微卫星位点上,等位基因的数目从3到8不等,等位基因长度从175—382bp,观测杂合度和期望杂合度分别为0.158—0.650和0.198—0.841,所筛选微卫星标记可于遗传分析。     

日本绒螯蟹线粒体DNA序列研究Ⅱ.16S rRNA

参考果蝇、卤虫与锯缘青蟹的线粒体 DNA16 S r RNA基因序列进行了日本绒螯蟹相同基因片段的引物设计、PCR扩增及序列测定 ,得到 56 7bp的碱基序列 ,其 A、T、G、C含量分别为194 bp(34.2 2 % )、2 16 bp(38.10 % )、98bp(17.2 8% )、59bp(10 .4 1% ) ,与果蝇、卤虫及锯缘青蟹类的 16 S r RNA基因片段序列含量相似。     

深海化能极端环境中劳盆拟刺铠虾(Munidopsis lauensis)的全长转录组测序分析

劳盆拟刺铠虾(Munidopsis lauensis)是一种十足目铠甲虾, 能够适应深海化能合成生态系统的极端环境, 但是其已知基因序列信息却很少。为此, 采用PacBio平台的SMRT测序技术对来自南海台西南冷泉劳盆拟刺铠虾的肝胰腺、鳃、肌肉和肠混合组织进行了三代全长转录组测序。通过聚类、校正、去冗余之后共获得28 811条高质量isoform, 平均长度为2 086 bp, N50长度为2 275 bp。使用Nr、SwissPort、KEGG、KOG数据库对转录本序列进行功能注释, 共有20 616 (71.56%)条isoform得到注释。进一步高级注释共获得21 848个蛋白编码序列, 共预测到537个转录因子、6 430个长链非编码RNA和10 060个SSRs位点。KEGG通路分析发现两条可能与劳盆拟刺铠虾适应深海化能极端生境相关的通路, 分别为过氧化物酶体通路和谷胱甘肽代谢通路。其中, 共有180条isoform被发现参与编码过氧化物酶体通路中的31个关键酶, 213条isoform参与谷胱甘肽代谢通路中19个关键酶的编码。基于全长转录组数据, 利用同源比对、功能结构域预测及系统发育树构建等方法对劳盆拟刺铠虾谷胱甘肽S-转移酶(GST)基因家族进行挖掘与鉴定, 共发现属于theta、delta、mu、kappa四种亚家族的20条GST候选序列。通过对劳盆拟刺铠虾全长转录组测序、功能注释和环境适应相关的重要基因及通路的分析, 不仅丰富了深海组学数据资源, 也为进一步揭示甲壳动物适应深海化能极端环境的分子机制奠定了基础。     

长牡蛎(Crassostrea gigas)Wnt4基因cDNA克隆与表达分析

Wnt4作为Wnt基因家族的重要成员,在动物的生长发育过程中起重要调控作用。本文利用RACE技术克隆了长牡蛎Wnt4基因c DNA全长序列,该序列全长1999bp,开放阅读框为1068bp,编码355个氨基酸,该蛋白序列与人(Homo sapiens)、沙蚕(Platynereis dumerilii)和栉孔扇贝(Chlamys farreri)Wnt4蛋白的相似性分别为44%、48%和46%。通过荧光定量RT-PCR分析长牡蛎Wnt4基因在成体不同组织和不同发育阶段的表达情况,发现长牡蛎的Wnt4基因具有广泛的组织表达特点,在所检测的多种组织中(外套膜、鳃、唇瓣、消化腺、雄性性腺、雌性性腺)均有表达,推测长牡蛎的Wnt4以信号分子的形式参与多种组织细胞的生命过程;长牡蛎个体发育过程中Wnt4基因的高表达主要集中在胚胎发育的早期(桑葚期最高,原肠胚期次之),幼贝期该基因的表达量很低,说明Wnt4基因可能在早期发育阶段参与了某些器官的形成。     

中国卤虫早期胚胎发育相关基因的研究——卤虫幼体trh基因的整体荧光原位杂交技术的建立

中国卤虫(Artemia sinica)是重要的饵料生物,是当前海洋生物学研究的热点生物和重要的生物模型。本文报告了卤虫无节幼体整体荧光原位杂交(WFISH,whole-mount fluorescent in situ hybridization)技术,用于快速对卤虫早期发育相关基因的特定时空表达图式进行分析,有助于对卤虫早期发育相关基因功能进行鉴定。人工合成带有FITC标记的卤虫trh基因(trachealess gene)寡核苷酸探针,检测到trh基因在卤虫无节幼体的头部背面盐腺中表达。     

低盐胁迫下凡纳滨对虾鳃丝抑制性消减杂交cDNA文库构建及差异ESTs分析

本研究以凡纳滨对虾(Litopenaeus vannamei)为对象,分别以低盐度胁迫下(4 ppt)凡纳滨对虾鳃丝cDNA为检测子,正常海水中凡纳滨对虾鳃丝cDNA为驱动予,采用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建了急性低盐胁迫诱导下凡纳滨对虾鳃丝消减cDNA文库。文库的重组率均高于95%,插入片段集中在250~750 bp之间,随机测序后在线拼接得到15组片段重叠群和37个单一序列共52个非重复序列。同源检索发现30个非重复序列与GenBank中的已知基因序列有较高的相似性。按差异表达基因编码的蛋白具体功能可分为6小类,依次为:(1)离子通道及转运蛋白;(2)蛋白合成翻译及转录因子;(3)应激抗氧化因子;(4)能量代谢;(5)信号受体和转导;(6)细胞纤维及骨架蛋白,所占比例分别为23.3%、20.0%、20.0%、16.7%、10.0%和10.0%。经功能聚类分析发现文库中含有大量离子通道蛋白及功能转运酶、胞内信号传导分子、细胞骨架蛋白等渗透调节和应激适应性相关基因,表明急性低盐胁迫诱导凡纳滨鳃丝抑制性消减杂交cDNA文库构建成功。     

大黄鱼(Pseudosciaena crocea) leptin和cholecystokinin基因的克隆和表达特性研究

本文克隆了两种大黄鱼(Pseudosciaena crocea)摄食调控因子胆囊收缩素(CCK)、瘦素(LEP)基因的全长序列,并对其表达特性进行了研究。结果表明,克隆得到CCK基因属硬骨鱼类的CCK1亚家族,全长900nt,编码137个氨基酸,与其他鱼类的CCK1相比序列组成非常保守,特别是在20氨基酸的信号肽和8个氨基酸的CCK-8活性结构区域;而克隆得到LEP基因属硬骨鱼类的LEPA亚家族,全长1290nt,编码161个氨基酸,与其他鱼类LEP相比基因同源性较差,但在3D结构上却保留了LEP经典的4个α螺旋特征。两种因子在所检测的所有组织中均有表达,但尤以脑、胃、肠消化道、肝脏等摄食及能量平衡相关组织中表达活性最高。8天的饥饿能使大黄鱼脑、消化道组织的CCK和肝脏、脂肪组织中的LEP表达显著降低(P0.05)。该结果说明CCK和LEP可能在大黄鱼的摄食生理和能量平衡中起着重要的调控作用;本研究将为深入了解鱼类食欲调控的神经内分泌机理提供基础。     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

Assessing the Validity of Seismo-Acoustic Predictor Equations for Obtaining Seabed and Sub-Surface Sediment Physical Properties

The interdependence between the seismo-acoustic properties of a marine sediment and its geotechnical/physical parameters has been known for many years, and it has been postulated that this should allow the extraction of geotechnical information from seismic data. Though in the literature many correlations have been published for the surficial layer, there is a lack of information for greater sediment depths. In this article, a desktop study on a synthetic seafloor model illustrates how the application of published near-surface prediction equations to subsurface sediments (up to several tens of meters burial depth) can lead to spurious predictions. To test this further, acoustic and geotechnical properties were measured on a number of sediment core samples, some of which were subjected to loading in acoustically-equipped consolidation cells (oedometers) to simulate greater burial depth conditions. For low effective pressures (representing small burial depths extending to around 10 meters subsurface), the general applicability of established relationships was confirmed: the prediction of porosity, bulk density, and mean grain size from acoustic velocity and impedance appears generally possible for the investigated sedimentary environments. As effective pressure increases through, the observed relationships deviate more and more from the established ones for the near-surface area. For the samples tested in this study, in some instances increasing pressure even resulted in decreasing velocities. There are several possible explanations for this abnormal behavior, including the presence of gas, overconsolidation, or bimodal grain size distribution. The results indicate that an appropriate depth correction must be introduced into the published prediction equations in order to obtain reliable estimates of physical sediment properties for greater subsurface depths.     

Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification

An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm.     

海上大直径钢管桩打桩过程中桩周土强度弱化研究

海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。     

Interannual Sea Level Variations and Annual Tides in the Northwestern Pacific

Specific properties of the interannual sea level variations and annual tides in the Northwestern Pacific were studied. Several tide stations were monitored. The monthly mean sea level for the year of 1995 was analyzed at each tide station. A seismic event in 1995, some tectonic activity around the subject area, and the Kuroshio (the oceanic western boundary current) may possibly contaminate results which would have occurred from the astronomical annual tide alone.     

Flow Cytometric Analysis of Coastal Lagoon Bacterioplankton and Picophytoplankton: Fixation and Storage Effects

The effect of different fixation and storage protocols on the flow cytometric (FCM) simultaneous analysis of bacterioplankton and phytoplankton in coastal seawater samples (Mediterranean coastal lagoons) was investigated. FCM measurements (cell number, fluorescence and scatter characteristics) were obtained through DAPI staining. Three fixatives [glutaraldehyde (GA), formaldehyde (FA) and paraformaldehyde (PFA)] and two storage (3 months duration) methods (5 °C and −196 °C) were tested. Two dominant populations were detected in studied samples: bacteria and eukaryotic picophytoplankton. Adding fixatives (2% final concentration) appears necessary to obtain FCM exhaustive counts of all the bacteria and phytoplanktonic cells. This was related to the permeation effect of fixatives which allowed a better DAPI staining of the cells. Maximum fluorescence, i.e. optimal staining of the cells was obtained with FA or PFA, and significant lower fluorescences with GA. Fixed samples stored at 5 °C induced rapid cell loss. Only storage in liquid nitrogen of samples fixed with FA or PFA, allows mid-term (≥4 months) preservation of bacteria or picophytoplankton cell numbers, and limited evolution of DAPI-induced fluorescence and scatter characteristics.     

INFORMATION FOR AUTHORS

<正>Acta Oceanologica Sinica(AOS)is a comprehensive academic journal edited by the Editorial Committee of Acta Oceanologica Sinica and is designed to provide a forum for important research papers of the marine scientific community which reflect the information on a worldwide basis.The journal publishes scholarly papers on marine science and technology,including physics,chemistry,biology,     

Deep-Sea Sediment Compression Curves: Some Controlling Factors, Spurious Overconsolidation, Predictions, and Geophysical Reproduction

Oedometer tests have been carried out on 70 undisturbed surficial clays (at approximately 250 mm below the mudline), mostly collected by free-fall corers from sites widely scattered throughout the deep-sea North Atlantic. Acoustic measurements were also made, initially on contiguous samples and ultimately on the same sample using a geophysically instrumented oedometer which also collected electrical resistivity data. Apart from those quiescent areas below the carbonate compensation depth, such as north of the West Indies where very fine clays exist, most of the samples are silty clays whose geotechnical-geophysical properties are dependent on the type of clay minerals present (and their ability to take in moisture), the sand-size fraction, and the quantity of carbonate present. Thus the pure clays have high compressibilities which decrease on the addition of coarse particles, while the converse is true for the acoustic parameters, these increasing with the sand fraction. Using the notion of the intrinsic compression line for all samples, and comparison to it of the measured compression curves, it is clear that, contrary to some previously held ideas, most deep-sea clays are normally consolidated; the addition of carbonate has the effect of creating an open, stronger sediment skeleton. Interestingly, where information is available, the variation with depth of a sample's acoustic velocity follows the void ratio pressure relationship of the compression curve. This allows the construction of an in-situ sediment compression curve using the in-situ geophysical observations.