以rDNA为同源重组位点酵母表达鲑鱼降钙素基因多拷贝整合载体的构建

根据酵母整合质粒的设计要求,PCR扩增2.2 kb rDNA片段,导入酿酒酵母(Saccharomyces cerevisiae)整合载体pYD1中,设计并合成适合在酵母中表达的鲑鱼降钙素(Salmon Calcitonin),以此为目标基因,构建适合酿酒酵母多拷贝整合的表达载体p-r-sCT.通过载体p-r-sCT引入到酿酒酵母菌株EBY100中,得到酵母重组菌株yAGA2-r-sCT.流式细胞检测结果表明,目标基因鲑鱼降钙素在重组菌株的yAGA2-r-sCT中得到了表达,实现了外源基因在酿酒酵母菌株中的稳定表达.     

大黄鱼肌肉生长抑制素基因外显子Ⅱ遗传多态性分析

肌肉生长抑制素是控制动物骨骼肌生长发育的重要细胞因子,采用PCR-SSCP和测序方法分析了150尾大黄鱼样本肌肉生长抑制素基因(myostatin)外显子Ⅱ的单核苷酸多态性,结果发现,外显子Ⅱ中有10个多态位点,分别是29、131、206、261、231、243、265、273、297和300位。其中131位点T→A突变引起相应的氨基酸由L→Q,265位点T→C突变引起相应氨基酸由C→R,261位点A→G突变引起相应氨基酸由S→G,231位点的T→C突变引起相应氨基酸由W→S,273位点的C→T突变引起相应的氨基酸由R→W,297位点的A→G突变引起相应氨基酸由I→V,243位点的G→A突变引起相应氨基酸由D→N,300位点的G→A突变引起相应氨基酸由E→K。29位点A→G和206位点C→A均属于同义突变。存在6种基因型,他们的基因频率分别是0.57、0.23、0.10、0.04、0.02、0.04,该基因位点的杂合度为0.33。     

龙须菜血红素加氧酶138位亮氨酸突变对藻红胆素合成的影响

藻红胆素是一个开链四吡咯结构的发色团,其与脱辅基藻红蛋白结合使藻红蛋白具有光学活性,而血红素加氧酶基因ho对于藻红胆素的合成是至关重要的。本研究对实验室前期已获得的龙须菜中的血红素加氧酶基因ho-1的基因序列进行分析发现,其在藻类中比较保守,存在6个高度保守结构域;结构功能分析显示其具有15个可能的血红素(Heme)结合位点,其中第138位亮氨酸(L)既位于118～145位HemeO的扭结螺旋结构内,又在115～142位的高度保守的结构域内,而且经预测是可能的血红素(Heme)结合位点之一。为了进一步验证该位点的作用,我们拟对其进行定点突变,将第413位碱基由T突变成C,得到ho(M)基因,碱基突变后所对应编码的第138位氨基酸由亮氨酸(L,非极性氨基酸)突变为丝氨酸(S,极性氨基酸)。在此基础上,我们构建了重组质粒pET-ho-1、pET-ho-1-pebS、pET-ho-1-pebA-pebB及pET-ho(M)、pET-ho(M)-pebS、pET-ho(M)-pebA-pebB,将其转化到E.coli BL21中构建重组菌株E.coli ph、E.coli phS、E.coli phAB、E.coli ph(M)、E.coli ph(M)S和E.coli ph(M)AB进行表达,检测重组菌株的荧光发射光谱。结果表明,E.coli phAB和E.coli phS在595 nm处检测到藻红胆素的荧光特征峰,表明ho-1基因编码的血红素加氧酶可以参与催化藻红胆素的合成;E.coli ph(M)、E.coli ph(M)S和E.coli ph(M)AB与E.coli BL21一样无任何特征峰出现,这表明ho(M)中第413位碱基对应编码的第138位亮氨酸是血红素的结合位点,直接影响藻红胆素的合成,该位点的突变使得PebS和PebB/A两条催化途径均无法合成藻红胆素。本研究为阐明血红素加氧酶基因在合成藻红胆素过程中的重要性及龙须菜中藻红蛋白的合成机制,进而进行藻红蛋白的重组表达奠定了基础。     

转鲑鱼降钙素基因酵母及RANKL对大鼠血管平滑肌细胞体外钙化的影响

观察核因子kB受体激活物配体(RANKL)对大鼠血管平滑肌细胞体外钙化的调节作用,以及转鲑鱼降钙素基因酵母对RANKL调节作用的影响.体外培养大鼠血管平滑肌细胞,建立体外血管平滑肌细胞钙化模型,在培养介质中分别加入RANKL及转鲑鱼降钙素基因酵母蛋白上清液,观察细胞钙沉积、细胞碱性磷酸酶活性及骨钙素的变化情况.结果表明,RANKL促进钙化培养的大鼠血管平滑肌细胞的钙沉积、细胞碱性磷酸酶活性及骨钙素的表达,而转鲑鱼降钙素基因酵母能够抑制RANKL的促钙化作用.RANKL可能通过增加细胞碱性磷酸酶活性及骨钙素的表达从而促进血管平滑肌细胞向成骨细胞转化;转鲑鱼降钙素基因酵母可能通过OPG/RANKL/RANK途径抑制血管平滑肌细胞钙化.     

3种鲍16S rRNA基因片段序列的初步研究

本文对引自日本的盘鲍 (H aliotisdiscusdiscus)、皱纹盘鲍 (H.discushannai)和大鲍 (H.gi-gantiea) 3个自然群体的线粒体 DNA 1 6S r RNA基因片段进行了扩增和测序 ,分析了 52 8bp的碱基序列 ,结果显示 :3种鲍的基因序列中 A+T含量为 56.74%～ 57.1 2 %。 3个自然群体间碱基片段序列差异不显著 ,盘鲍与皱纹盘鲍、大鲍彼此均仅有 1处核苷酸检测到变异 ,皆为碱基转换 ,同源性为99.81 % ;皱纹盘鲍与大鲍有 2处碱基转换 ,同源性为 99.61 %。 1 6Sr RNA基因在鲍属内表现出很高的保守性。     

Phaeocystis globosa与Phaeocystis antarctica叶绿体psbA基因的比较

测定了2株球形棕囊藻Phaeocystis globosa P1、P2的psbA基因序列。发现得到的2个序列完全相同。以P2序列对比分析了P．globosa和P．antarctica的psbA基因在DNA序列、氮基酸序列和RNA二级结构上的差异性，发现2种棕囊藻psbA基因DNA序列和氨基酸序列非常保守，无插入／缺失，其核苷酸和氨基酸变异率分别为1．88％和1．13％。与核基因核苷酸的碱基替换不同，psbA基因核苷酸的碱基替换主要发生在密码子的第1位上。且不引起氨基酸的变化，引起氨基酸变化的碱基替换都发生在密码子的第2位和第3位上。在RNA二级结构上两序列的1～4茎环结构完全相同，表现出明显的棕囊藻属的特异性，其它结构区域差异较大。种间差异表现明显。由于psbA基因DNA序列和氨基酸序列非常保守，可能不适宜棕囊藻属的系统发育分析。但其RNA二级结构可能对于棕囊藻的分子分类有一定的参考价值。     

扇贝(Chlamys farreri)多肽通过EGFR抑制受紫外线B(UVB)诱导的HaCaT细胞凋亡

利用Hoechst 33258荧光染色法检测紫外线B(UVB)辐射诱导HaCaT细胞凋亡率。结果表明,扇贝多肽(Polypeptide from Chlamys farreri,PCF)可以剂量依赖性抑制UVB诱导的HaCaT细胞凋亡;表皮生长因子受体(EGFR)抑制剂AG1478能明显抑制UVB诱导的HaCaT细胞凋亡。采用3’-RACE法构建EGFR的cDNA片段,克隆测序检测突变位点。结果表明,UVB照射后EGFR发生碱基突变A→G,A→G,T→C,G→A,G→A;预加入5.69mmol/L的PCF,产生部分抗突变作用,1,2,3突变位点处未发生碱基的突变。     

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。     

海豚链球菌C5a肽酶功能活性区域表达及其对斑点叉尾(Ictalurus punctatus)的免疫保护作用

本研究对海豚链球菌DGX07株C5a肽酶基因序列进行了克隆和生物信息学分析, 表达了其功能活性区域, 并以斑点叉尾(Ictalurus punctatus)作为受试动物, 对该蛋白进行了免疫保护作用研究。结果显示, scpⅠ基因编码的氨基酸序列具有2个Peptidase_S8_S53超家族的保守结构域, 1个PA超家族的保守结构域和1个Fn3-like超家族的保守结构域, 推导其活性位点共有7个, 位于前501个氨基酸; 根据二级结构预测、疏水性分析、表面可及性、柔性分析和抗原表位分析, 推断该氨基酸序列的B细胞表位区域主要集中在第67—1098个氨基酸之间。因此, 本研究选取去掉信号肽的N段的前635个氨基酸(180—2085bp)进行表达。使用表达的重组蛋白pSCPⅠ免疫斑点叉尾, 抗体效价结果显示: 免疫后第四周抗体效价最高, 第五周后抗体效价开始下降, 相对免疫保护率为43.75%。以上结果表明重组pSCPⅠ蛋白具有较好的免疫保护作用, 能够作为海豚链球菌亚单位疫苗的候选之一。     

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。     

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.     

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。     

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.     

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.     

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.     

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.