条斑紫菜类囊体膜蛋白质组双向电泳研究方法

以蓝绿温和胶电泳为工具,首次在国内用于条斑紫菜(Porphyra yezoensis)类囊体膜色素蛋白质复合物的研究。结果显示:(1)利用非离子型去污剂十二烷基麦芽糖苷(DM)增溶条斑紫菜类囊体膜,DM/Chla(w/w)15︰1产生的效果较好,在BN-PAGE胶中可以分离到较多较清晰的条带。(2)选取蔗糖密度层50%条带制备得到的类囊体膜样品进行第一向BN-PAGE和第二向SDS-Urea-PAGE电泳实验。第一向电泳分离出4个蛋白复合物,对第二向电泳胶上的15个蛋白点切取做质谱鉴定,检测到PSⅡ47ku,PSⅡ44ku,cytochrome f,PSⅡD2,PSⅡD1等蛋白。本实验证实了温和胶电泳与SDS电泳结合对于条斑紫菜这种原始红藻的类囊体膜研究方面应用的可行性。     

温度、光强和pH对条斑紫菜孢子体和配子体类囊体膜上光系统活性的影响

光合作用研究中,对于个体发育过程中类囊体膜蛋白结构和功能的变化所知甚少,其中的一个限制因素是能否纯化得到大量高活性的稳定且均一的膜蛋白。作者以条斑紫菜（Porphyra yezoensis）2个不同发育阶段（孢子体和配子体）作为研究对象,分离得到了孢子体和配子体的类囊体膜．在不同的温度、光强和pH下,对其类囊体膜上的光系统活性进行了研究。结果表明,孢子体和配子体的PSⅠ活性均随温度和光强的升高而呈现一个先升高后降低的趋势,在温度18℃,光强为2320lx时具有最高的PSⅠ活性。而孢子体乖配子体的PSll活性随温度和光强的变化趋势不明显,孢子体PSⅡ活性在温度18℃,光强为2320lx时最高,配子体PSⅡ活性在温度13℃,光强为2800lx时最高。孢子体PSⅠ在酸性条件下较活跃,在pH5．4时具有最高的PSⅠ活性,配子体PSⅠ在碱性条件下较活跃,PSⅠ活性在pH10．4时最高;孢子体PSⅡ活性随pH的升高呈现一个先升高后降低的趋势,配子体PSⅡ活性随pH变化趋势不明显,但都在pH8．0处活性最高。     

条斑紫菜孢子体与配子体光合系统Ⅱ(PSⅡ)稳定性研究

膜蛋白是生物领域研究中的热点和难点。在光合作用研究中,对于藻类个体发育过程中光合膜蛋白结构和功能的变化所知甚少,其中的一个限制因素是能否纯化得到大量高活性的稳定且均一的光合膜蛋白。作者从条斑紫菜(Porphyra yezoensis)孢子体和配子体中分离纯化得到光合系统(PSⅡ)复合物,并研究了其完整性和放氧活性。结果表明,孢子体和配子体的PSⅡ复合物,在4℃条件下保存比在-80℃下保存放氧活性高,稳定性高。配子体PSⅡ复合物,在-80℃保存第6天就已经没有放氧活性,而孢子体PSⅡ复合物仍有放氧活性。对4℃下保存的PSII复合物进行分子筛柱层析,室温吸收光谱测定以及放氧活性测定,发现随着放氧活性逐渐降低,蛋白大分子有聚合现象。室温吸收光谱表明经过长期的保存,吸收峰向短波长方向偏移,叶绿素易降解成为脱镁叶绿素     

裙带菜(Undaria pinnitifida)色素蛋白复合物的分离及光谱特性的初步研究

以褐藻裙带菜(Undaria pinnatifida)为实验材料，采用蔗糖密度梯度超速离心的方法纯化类囊体膜；以去污剂sDs为增溶剂(SDS：Chl=20：1，4℃增溶20min)，再用蔗糖密度为60％、50％、40％、30％、20％、15％和10％的梯度离心法，从裙带菜中成功地分离出5条含色素的蛋白质复合物带。离心结果显示在离心管顶部没有任何颜色，表明经过SDS增溶没有产生游离色素，纯化的类囊体膜和5条色素条带进行吸收光谱分析，显示了属于叶绿素n(Chl-a)的吸收峰：418、436和667到672nm。叶绿素c(Chl-c)的吸收峰：615和620nm。荧光发射最高峰位于682到690nm的区域。荧光激发光谱显示有Chl-a和Chl-c的激发峰，40％层带所含有的色素复合物只有属于Chl．n的吸收峰。分析结果表明：10％、15％层带所含有的色素复合物是褐藻的捕光色素复合物；20％、30％层带所含有的色素复合物是浅绿色的PSⅡ复合体(仍然含有捕光色素)，40％层带所含有的色素复合物是深绿色PSⅠ复合体，50％层带所含有的色素复合物是没有被增溶的类囊体膜。     

条斑紫菜光系统Ⅱ反应中心蛋白编码psbA和psbD 的表达分析

从条斑紫菜（Porphyra yezoensis Ueda）的3个不同的生长阶段：壳孢子、孢子体和配子体中提取总RNA，并用相应基因的引物扩增序列，用荧光定量PCR技术检测条斑紫菜3个生长阶段中psbA和psbD的转录水平变化。结果表明，psbA和psbD的变化趋势不一致，并且，在壳孢子内基因表达量相对较高。这说明在细胞内，D1蛋白和D2蛋白并非同比例合成。壳孢子成熟发育成叶状体的过程要求光合作用提供更多的能量。     

不同去污剂对裙带菜和石莼色素蛋白复合物分离的影响

以褐藻裙带菜(Undaria pinnatifida)和绿藻石莼(Ulva pertusa)为实验材料,利用SDS,Dig,Triton X-100三种去污剂,分别与叶绿素按不同比例增溶类囊体膜,采用蔗糖密度梯度超速离心的方法分离色素蛋白复合物,蔗糖密度梯度为60%,50%,40%,30%,20%,15%和10%。实验结果表明:当SDS与叶绿素质量比为20∶1时,能够从裙带菜类囊体膜中分离得到5条含蛋白质复合物带。对于石莼类囊体膜的增溶,当SDS与叶绿素的质量比为10∶1,Triton与叶绿素质量为20∶1时,同样能够分离得到5条含蛋白质复合物带。褐藻和绿藻色素蛋白复合物对去污剂的敏感程度有很大差别。     

RSAP标记技术在紫菜遗传多样性检测及种质鉴定中的应用

限制性位点扩增多态性（restriction site amplification polymorphism，RSAP）是根据基因组内普遍存在的酶切位点来设计特殊的引物，通过简单的梯度PCR反应来产生多态性。本研究首次将RSAP标记技术成功应用于紫菜遗传多样性检测和种质鉴定。利用所建立的适合于紫菜RSAP分析的PCR反应体系和反应条件，使用30对引物对15个紫菜系DNA进行了PCR扩增，经过3次重复验证，有12对引物能够扩增出清晰且稳定的条带。这12对引物共扩增出413条带，其中408条为多态性条带，多态性比例96％，平均每对引物产生34条多态性条带，片段大小在50-500bp之间。利用这413条带进行聚类分析，产生了这15个紫菜系的进化树。15个紫菜系在0.69相似系数水平上分为两大类：一类包括坛紫菜；另一类包括条斑紫菜和少精紫菜。选2对引物R1／R6和R3／R4所扩增的10条稳定且重复性好的条带，构建了这15个紫菜系的RSAP-DNA指纹图谱。在该指纹图谱中，每个紫菜系都有其独一无二的指纹模式，能够很容易地与其它紫菜系相区分。     

基于LC-MS/MS 技术对蓝藻Gloeobacter violaceus PCC 7421细胞膜的蛋白质组学研究

Gloeobacter violaceus PCC 7421是一种在进化上很古老的无类囊体蓝藻,由于光合作用电子传递链与呼吸系统在其细胞质膜上共存,这两个系统可能会共享一些元件,为研究其光合作用系统结构的独特之处,应用高效液相色谱-串联质谱联用(LC-MS/MS)技术对其细胞膜蛋白进行蛋白质组学分析.经鉴定发现, G. violaceus的细胞质膜上具有PSⅠ、PSⅡ和细胞色素b6等光合作用关键蛋白,同时具有呼吸作用过程中的关键酶1,5-二磷酸羧化酶/加氧酶、F0F1- ATP酶α和β亚基、细胞分裂蛋白、硝酸盐/亚硝酸盐转运蛋白、青霉素结合蛋白以及多种药物受体ABC转运蛋白.由此可知, G. violaceus的细胞质膜不仅能够同时进行光合作用和呼吸作用,且营养盐运输等转运功能也同时存在.G. violaceus的全基因数据分析显示其基因序列具有独特性,其蛋白注释相对比较少,因此其蛋白功能的研究需要更多数据的支持.     

别藻蓝蛋白β亚基基因在莱茵衣藻中重组表达及其对光能传递的影响研究

为了探究别藻蓝蛋白与类囊体膜光合系统之间的光能传递规律,及其与异源类囊体膜光合系统之间光能转移的可能性,本研究构建了含有钝顶节旋藻(Arthrospira platensis)的别藻蓝蛋白β亚基基因(apcB),及合成藻蓝胆素的血红素氧化酶基因(ho)和铁氧还蛋白氧化还原酶基因(pcyA)的转化载体pHyg3-apcB-ho-pcyA,利用玻璃珠转化法将其导入莱茵衣藻中。聚合酶链式反应筛选验证阳性转化藻株,Southern Blot结果表明基因apcB、ho、pcyA成功转化入莱茵衣藻基因组中。而后将转化藻株与对照藻株均置于高光(50μmol·m^-2·s^-1)和低光(25μmol·m^-2·s^-1)条件、白光和绿光条件中培养,并检测别藻蓝蛋白β亚基的表达、总叶绿素含量、77 K低温荧光、光系统表观电子传递效率、生物量变化。结果显示：实时定量PCR分析和Western Blot检测表明外源基因apcB,pcyA和ho均在转基因藻株Cr-ApcBHP中得到了转录表达。高光和低光条件下光系统Ⅱ(PSⅡ)和光...     

坛紫菜耐低盐品系的选育与特性分析

本实验室采用人工诱变技术获得一个坛紫菜耐低盐品系(YZ-7),为了验证该品系的耐低盐性状能否遗传,本文对其壳孢子和F1叶状体的低盐耐受性分别进行了测试,并以坛紫菜野生品系(WT)作为对照,结果表明:在不同盐度下培养2周的YZ-7和WT的壳孢子,在26盐度组存活率无显著差异;在15盐度组,YZ-7的壳孢子存活率和假根发生率分别比WT提高了34%和35%;在8和5盐度组,YZ-7的壳孢子存活率、分裂率及假根发生率分别比WT提高了65%、32%和62%,以及137%、80%和154%;在3盐度组,YZ-7的壳孢子存活率和分裂率分别比WT提高了458%和171%,其中约44.9%的YZ-7壳孢子萌发体能形成假根,而WT的壳孢子萌发体基本上不长假根。在26盐度中培养35d后的F1叶状体再分别在26、15、8、5和3盐度下培养30d,YZ-7叶状体的特定生长率分别为WT的1.7、1.9、1.5、1.6和1.6倍,绝对生长率分别是WT的3.4、4.1、1.8、2.0和1.6倍。此外,YZ-7叶状体的叶绿素荧光活性(PSⅡ最大光量子产量、实际光量子产量)及三种主要光合色素(叶绿素a、藻红蛋白和藻蓝蛋白)的含量也明显高于WT。上述结果证实,坛紫菜选育品系YZ-7是一个耐低盐且生长快的优良品系,其优良性状可稳定传递给下一代,有望被培育成适宜栽培的新品种。     

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.     

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.     

Phylogenetic diversity and phenotypic characterization of cultivable bacterioplankton isolated from polar oceans

A set of 27 marine planktonic bacteria isolated from the polar regions was characterized by 16S rDNA sequencing and physiological and biochemical testing. More than half of these bacteria were positive for caseinase, gelatinase and 13-glucosidase, and could utilize glucose, maltose or malic acid as carbon source for cell growth. Twelve isolates expressed nitrate reduction activities. Except for one antarctic isolate BSwlO175 belonging to Actinobacteria phylum, these isolates were classified as γ-Proteobacteria, suggesting that γ-Proteobacteria dominated in cultivable marine bacterioplankton at both poles. Genus Pseudoalteromonas was the predominant group in the Chukchi Sea and the Bering Sea, and genus ShewaneUa dominated in cultivable bacterioplankton in the Prydz Bay. With sequence similarities above 97%, genus Psychrobacter was found at both poles. These 27 isolates were psychrotolerant, and significant 16S rDNA sequence similarities were found not only between arctic and antarctic marine bacteria （ 〉 99% ）, but also between polar marine bacteria and bacteria from other aquatic environments （ ≥ 98.8% ）, including temperate ocean, deep sea, pond and lake, suggesting that in the polar oceans less temperature-sensitive bacteria may be cosmopolitan and have a bipolar, even global, distribution at the species level.     

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,     

Estimation of Sediment Properties during Benthic Impact Experiments

Sediment properties, such as water content and density, have been used to estimate the dry and wet weights, as well as the volume of sediment recovered and discharged, during benthic impact experiments conducted in the Pacific and Indian Oceans. The estimates show that the weights of dry (355-1332 t) and wet sediment (1651-4888 t) recovered during the experiments, are not only a function of the total duration and distance covered during the experiment, but also depend on water content and density of the sediment. Estimation of dry sediment and its volumetric ratio in the discharge, are key parameters for calculating the discharged volume. The estimated volume of wet sediment recovered (1427-4049 m 3 ) as well as discharged (2693-6951 m 3 ) during the experiments, provide important inputs to evaluate the sediment resettlement and migration. Using these estimates, the average depth of excavation on the seafloor, can also been calculated. However, the stages of sediment recovery and discharge are expressed differently in some of the experiments. In order to standardize these, different stages have been identified, and definitions of certain terms have been suggested, for use in the future. The methods of calculating different properties, as well as weight and volume of discharged sediment are described in the paper for use in other applications concerning deep-sea discharges.