南海沉积物细菌胞外蛋白酶在家族水平上的多样性

海洋产蛋白酶细菌及其分泌的胞外蛋白酶在降解有机氮以推动海洋氮循环进行方面发挥着重要作用，但目前对这二者多样性的认识非常有限。本研究自南海沉积物中筛选得到90株产蛋白酶细菌，并通过N-端氨基酸序列，分析了其胞外蛋白酶在家族水平上的多样性。16S rRNA基因序列分析的结果表明，筛选的产蛋白酶细菌均属于Gammaproteobacteria纲，且大多数属于Alteromonadales目和Vibrionales目的不同属。对其中14株菌株的14个胞外蛋白酶的N-端氨基酸序列分析表明，所有这些蛋白酶属于金属蛋白酶的M4家族或丝氨酸蛋白酶的S8家族。本研究提供了海洋沉积物产蛋白酶细菌在类群及其胞外蛋白酶在类型上的新细节，这将有助于全面了解微生物酶促降解海洋沉积物有机氮的过程和机理。     

大黄鱼EBI3基因的克隆与表达分析

通过克隆大黄鱼(Larimichthys crocea)的EBI3(Epstein-Barr virus-induced gene 3)基因并对其进行了序列分析.大黄鱼EBI3(Lyc EBI3)基因开放阅读框长747bp,编码248个氨基酸,存在1个信号肽序列和2个FN3结构域(37~130,145~230).多序列比对发现Lyc EBI3分子与其他已知EBI3氨基酸水平一致性为27.68%~57.53%.Real-time PCR结果表明,Lyc EBI3基因在脾脏和血液中表达量最高,且溶藻弧菌(Vibrio alginolyticus)刺激后,大黄鱼头肾和脾脏中Lyc EBI3基因的转录水平会显著升高,说明Lyc EBI3基因可能参与了抑制由细菌引起的炎症反应.     

大黄鱼(Larimichthys crocea)IL-10基因的克隆与表达分析

白细胞介素10(Interleukin 10,IL-10)是一种抗炎细胞因子,可以抑制机体免疫反应。本研究经过分析大黄鱼基因组数据库发现了IL-10同源基因,并对其cDNA编码区序列和基因组DNA序列进行了克隆分析。大黄鱼IL-10(LycIL-10)基因由5个外显子和4个内含子构成,其序列全长1 869bp,其开放阅读框(ORF)长555bp,编码184个氨基酸,其N端的22个氨基酸残基为预测的信号肽,成熟肽由162个氨基酸残基组成,包含了脊椎动物IL-10标志性保守序列。LycIL-10的氨基酸序列同其他已知物种的IL-10氨基酸序列的一致性为26.49%~77.01%。Real-time PCR分析发现LycIL-10在检测的组织中为组成型表达,在脾脏和肌肉中转录水平相对较高。三联灭活细菌疫苗和聚肌苷酸胞苷酸(poly(I∶C))刺激后,大黄鱼头肾和脾脏中LycIL-10mRNA的转录水平会显著升高,表明LycIL-10可能参与抑制大黄鱼由细菌和病毒引起的炎症反应。     

长牡蛎两种engrailed同源基因的鉴定及其与贝壳发育相关性的研究

engrailed基因属于同源异形基因家族成员,在许多动物的分节、附肢发育、神经系统发育和贝壳形成过程中发挥作用。本研究克隆了长牡蛎两个engrailed同源基因,命名为cgi-eng1和cgi-eng2。序列分析表明,两个基因均具备典型engrailed基因保守的5个EH结构域。利用整装原位杂交技术检测了cgi-eng1和cgi-eng2在贝壳形成的关键时期早期D形幼虫时期的表达情况。结果显示,cgi-eng1和cgi-eng2 m RNA高表达于贝壳外缘,可能与早期贝壳形成过程有关。此外,两个基因的在贝壳外缘的表达模式亦有区别,提示两个基因的功能可能存在一定程度的分化。本研究首次系统鉴定了长牡蛎engrailed基因的成员,并发现它们可能均参与幼虫贝壳形成,研究结果有助于加深对贝类早期发育及贝壳形成的理解。     

淋巴囊肿病毒mcp基因变异特征及其基因型分析

淋巴囊肿病毒是引起多种海、淡水鱼淋巴囊肿病的病原,且不同淋巴囊肿病毒分离株间存在一定的差异。本文对25株淋巴囊肿病毒分离株mcp基因的变异特征进行分析,结果表明:分离自同一宿主的淋巴囊肿病毒mcp基因序列并非完全一致,有些同宿主分离株间存在着较少的变异,其MCP蛋白序列间的变异位点多位于不规则结构域、属于非蛋白相互作用位点;分属不同基因型淋巴囊肿病毒分离株MCP蛋白序列间的变异位点也多位于不规则结构域,变异位点为蛋白相互作用位点的比率仅占21.98%。金头鲷淋巴囊肿病毒分离株LCDV-sa是一种新的淋巴囊肿病毒基因型,此25株淋巴囊肿病毒可分为7个基因型。对淋巴囊肿病毒变异的研究,可加深对其感染机制的认识,有助于了解其传播途径,可为淋巴囊肿病的防控提供重要的理论依据。     

Epsilon-变形菌趋化信号通路中偶联蛋白CheV与趋化受体蛋白的CZB结构域的功能研究

细菌通过趋化系统感知和响应外界环境变化并进行趋利避害, 因此很多细菌进化出了非常复杂多样的趋化系统以适应不同的生态位。Epsilon-变形菌广泛存在于自然界中, 能适应不同的生态环境, 特别是深海Epsilon-变形菌能适应深海热液口和冷泉等极端环境, 其趋化系统可能有其独特之处。通过BlastP和MIST数据库分析, 我们发现大部分深海Epsilon-变形菌拥有F3类型趋化系统且具有单拷贝的双功能域融合蛋白CheV。此外, 一种特殊结构域CZB (C-terminal Zinc-Binding) 存在于深海Epsilon-变形菌的趋化受体蛋白中, 而且在部分深海Epsilon-变形菌的趋化受体中还发现并鉴定了一种新的结构域CZB-like结构域。文章以Epsilon-变形菌的模式菌株空肠弯曲杆菌(Campylobacter jejuni 81-176)为研究对象, 用细菌双杂交试验证实了CheV能与所有含MA结构域的趋化受体相互作用。用电感耦合等离子体质谱(ICP-Mass)技术证实了CZB-like结构域不能结合Zn离子, 但细菌双杂交试验证实它能介导趋化受体Tlp9与CheV的互作。     

4株海洋红球菌产类胡萝卜素分析及其全基因组测序

以源于东太平洋海水的4株红球菌分离菌Rhodococcussp. EPR-134、Rhodococcus sp. EPR-147、Rhodococcus sp. EPR-157和Rhodococcus sp. EPR-279为研究对象,开展了菌株的色素提取和色素全波长扫描,并基于全基因组测序分析了4株细菌类胡萝卜素代谢通路中的相关基因。色素全波长扫描结果显示,菌株EPR-134不具备类胡萝卜素产生能力,而其它3株红球菌能够产生类胡萝卜素,且所产类胡萝卜素组分不同。基因组分析表明,4个细菌基因组中存在较为完整的促使类胡萝卜素形成的基因簇。对菌株参与番茄红素形成的3个关键基因crt E、crt B和crt I的氨基酸序列同源性两两比对分析表明,菌株EPR-134 3个基因氨基酸序列与其它3个菌株相应基因氨基酸序列同源性最低,这可能是导致该菌株不产类胡萝卜素的关键原因。该研究结果为产类胡萝卜素红球菌的遗传改造,以及为产类胡萝卜素工程菌的构建奠定了前期基础。     

瘤背石磺表皮生长因子基因的克隆、结构及进化分析

首次在瘤背石磺(Onchidium struma)中克隆得到一种新的表皮生长因子(EGF)的cDNA序列全长。EGF基因cDNA的全长为1158bp,命名为Os-egf1,其中开放阅读框长度为846 bp,编码一条包含281个氨基酸残基的多肽链。根据氨基酸序列比对和结构域分析结果发现其含有2个保守的EGF结构域和1个EGF-like结构域,每个结构域中均包含至少6个半胱氨酸残基,且形成CX7 CX4-5 CX10-13CXCX8 C结构,其结构域由C1~C3、C2~C4和C5~C6之间形成的3个二硫键维持,符合表皮生长因子家族及其相关蛋白的特征结构域,但其余氨基酸序列与现有相关基因差异较大,推测可能是一种新的EGF-like基因。利用MEGA6.0软件构建Os-egf1与EGF家族相关蛋白的系统进化树,表明EGF家族蛋白具有一定的物种特异性。     

海带CRY-DASH基因的克隆与转录表达分析

利用cDNA末端快速克隆(RACE)技术,获得海带(Saccharina japonica) CRY-DASH基因(SjCRYDASH)全长序列,结构分析发现,其ORF区长1779 bp,编码592个氨基酸。进行氨基酸同源序列比对,其与其他藻类和高等植物间存在两个重要辅基MTHF和FAD结合的保守域。通过不同光质照射诱导海带幼孢子体,发现蓝光、白光诱导1h后,均能使SjCRY-DASH转录水平上升,且SjCRY-DASH对蓝光的响应更强烈。本研究结果为研究大型褐藻-海带CRY-DASH受光诱导调控功能打下基础。     

日本囊对虾Cathepsin L基因的鉴定及其在幼体发育中的表达研究

本研究从日本囊对虾（Marsupenaeus japonica）中获得半胱氨酸蛋白酶家族中的cathepsin L基因,命名为Mj-Cathepsin L。Mj-Cathepsin L基因cDNA序列全长1191bp,其开放阅读框编码的Cathepsin L蛋白前体包含327个氨基酸,由信号肽,蛋白前体和成熟蛋白三部分组成。同源序列比对发现日本囊对虾Cathepsin L蛋白与其他已知物种的cahtepsin L蛋白具有较高的同源性。对Mj-Cathepsin L基因在不同组织以及早期幼体幼体发育阶段的研究发现,Mj-Cathepsin L主要在肝胰腺中大量表达,且在幼体发育不同阶段,其表达水平受发育阶段调控,且Mj-Cathepsin L基因显示出与其五个蜕皮时期相关的表达变化,说明Mj-Cathepsin L有可能在日本囊对虾个体发育过程中扮演不同的重要作用。并通过Western Blot方法在日本囊对虾肝胰腺,胃和肠中检测到Cathepsin L酶原和成熟Cathepsin L两种蛋白存在形式。     

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.     

A Review of Some Environmental Issues Affecting Marine Mining

This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested.     

Increased Particle Fluxes at the INDEX Site Attributable to Simulated Benthic Disturbance

Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement.     

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。     

Determination of In Situ Sediment Shear Strength from Advanced Piston Corer Pullout Forces

The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments.     

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.