大黄鱼(Larimichthys crocea)I型干扰素基因的特征与表达分析

采用RT-PCR和RACE-PCR技术克隆了大黄鱼(Larimichthys crocea)Ⅰ型干扰素基因全长cDNA序列及其基因组序列,并利用荧光定量PCR技术研究了该基因在大黄鱼不同组织中的表达谱,以及副溶血弧菌(Vibrio parahaemolyticus)、LPS、polyI:C刺激后大黄鱼脾脏、肝脏和头肾组织中该基因在转录水平的表达变化。结果表明,大黄鱼Ⅰ型IFN基因全长cDNA为878bp,开放阅读框558bp,编码185个氨基酸。推测的大黄鱼Ⅰ型IFN氨基酸序列N端含有20个氨基酸的信号肽,其后包含一个保守的IFabd结构域。大黄鱼Ⅰ型IFN基因包含5个外显子,4个内含子,在大多数组织和细胞中都有表达,其中在血细胞中表达量最高,肌肉中表达量最低。PolyI:C刺激可诱导大黄鱼Ⅰ型干扰素基因在脾脏、头肾和肝脏中的表达量显著上调;LPS刺激可诱导大黄鱼Ⅰ型干扰素基因在脾脏和肝脏中的表达量显著上调;灭活的副溶血弧菌可诱导大黄鱼Ⅰ型干扰素基因在头肾和肝脏中的表达量显著上调。     

鳜鱼(Siniperca chuatsi)脂肪酸去饱和酶和延长酶基因全长cDNA序列的克隆与分析

利用RT-PCR和RACE方法克隆得到鳜鱼肝脏中控制高不饱和脂肪酸合成的脂肪酸去饱和酶(fatty acid desaturase,FAD)和脂肪酸延长酶(fatty acid elongase,ELO)基因的全长cDNA序列。FAD基因的全长cDNA序列1882bp,编码445个氨基酸,该蛋白序列含有FAD全部的特征结构区,包括3个组氨酸簇、2个跨膜区和1个细胞色素b5结构域,与其它鱼类FAD氨基酸序列的同源性为66.5%-90.1%,系统树分析显示其与欧洲鲈鱼和金头鲷等海水鱼类的亲缘关系最近。获得的ELO基因全长cDNA序列1401bp,编码294个氨基酸,该蛋白序列含有单一的氧化还原中心组氨酸簇、内质网停留信号和多个跨膜区等ELO特征结构,与其它几种鱼类ELO氨基酸序列的同源性为71.8%-86.7%,系统进化分析显示其与南方蓝鳍金枪鱼和金头鲷的亲缘关系较近。鳜鱼FAD和ELO基因全长cDNA序列的获得可为进一步研究其HUFA合成能力及调控机理奠定基础。     

大黄鱼(Larimichthys crocea)IGF-Ⅰ基因的克隆及序列分析

采用RT-PCR和RACE技术克隆了大黄鱼胰岛素样生长因子-Ⅰ(IGF-Ⅰ)cDNA的全序列,序列全长2027bp,由57bp的5’非翻译区、1409bp的3’非翻译区和561bp的开放阅读框组成。序列分析表明,大黄鱼IGF-Ⅰ编码区包括信号肽、成熟肽(B、C、A、D)和E等6个区域的186个氨基酸,形成成熟肽时,信号肽和E区被切除,成熟肽分子量7.5kDa,等电点(pI)为7.76,成熟肽的B区和A区有6个保守的半胱氨酸残基,形成3对二硫键,起到了稳定IGF-Ⅰ三维结构的作用。与其它物种的IGF-Ⅰ比对后发现A、B区域比较保守,C、D区域保守性较差;E区域的分析结果表明,大黄鱼IGF-ⅠcDNA序列为Ea-4亚型。核苷酸同源性分析发现,大黄鱼IGF-ⅠcDNA与美国红鱼同源性最高,为99.47%;与斜带石斑鱼、金头鲷和褐牙鲆的同源性依次降低,分别为97.68%、97.50%和95.90%。     

大黄鱼(Larimichthys crocea)Tristetraprolin基因的克隆与功能分析

Tristetraprolin (TTP)是广泛存在于真核生物的RNA结合蛋白。TTP通过促进mRNA降解或抑制翻译在转录后水平抑制炎症因子表达,是炎症性疾病的潜在治疗靶点。采用RACE技术获取了大黄鱼(Larimichthys crocea) TTP (命名为LcTTP) cDNA的全长序列。LcTTP全长cDNA 1 508 bp,包括77 bp的5′-非编码区(5’-UTR)、183 bp的3’-UTR和1 248 bp的开放阅读框(ORF),编码418个氨基酸残基。推导的LcTTP理论分子量44 897.78 Da,预测等电点pI 8.37;哺乳动物TTP的重要功能结构域:N-端核输出序列(NES)、中央串联锌指结构域(TZF)、C-端NOT1-结合结构域(NOT1-BD)也在LcTTP中保守存在;系统发育分析显示LcTTP和其他脊椎动物TTP聚为一支,并与哺乳动物ZFP36家族其他成员ZFP36L1、ZFP36L2和ZFP36L3的进化支分离。组织表达特异性分析显示检测的9个组织均表达LcTTP mRNA,但不同组织间表达水平差异大,其中肌肉组织表达水平最高。大黄鱼头肾细胞系...     

泥蚶(Tegillarca granosa) cDNA 文库的构建及铁结合蛋白基因(Ferritin)序列分析

取正常泥蚶肌肉组织获得总RNA,纯化mRNA后,利用含SfiⅠ酶切位点的CDSⅢ引物逆转录合成cDNA第一链,通过LD—PCR合成cDNA双链,经胶回收除去短片段,与pDNR—LIB载体进行连接,电转化到大肠杆菌DH10B中,构建形成原始文库,进行滴度测试和重组率鉴定。结果表明原始文库的滴度为3．17×10^5cfu／ml,重组率为86．3％,插入片段多在500--2500bp间。随机挑选458个克隆测序,经分析获得94种已知基因及87种未知基因。其中包括铁结合蛋白（Ferritin）的全长cDNA序列。该基因序列全长895bp,5’-端非编码区为163bp,3’-非编码区为213bp,开放阅读框为519bp,编码172个氨基酸。推测其蛋白分子量和等电点分别为20kDa和4．89。氨基酸序列与皱纹盘鲍、太平洋牡蛎、血红扇头蜱、文昌鱼、中华蟾蜍、非洲爪蟾、小家鼠以及人等的同源性有62％-82％。比对结果表明,铁结合蛋白基因在动物进化中高度保守。     

大黄鱼(Larimichthys crocea)白细胞介素10(IL-10)基因克隆及溶藻弧菌(Vibrio alginolyticus)侵染后表达变化分析

白细胞介素10(interleukin10,IL-10)是鱼类先天性免疫因子之一,在炎症反应和免疫调节中有着重要作用。本研究获得大黄鱼(Larimichthys crocea)IL-10基因cDNA序列,由899个核苷酸组成,含1个555bp的开放阅读框、编码184个氨基酸,预测编码蛋白分子量为21.24kDa、N端有22个氨基酸组成的信号肽序列。氨基酸序列多重比对表明大黄鱼IL-10具有IL-10家族特征性序列和构成2对二硫键的4个保守性半胱氨酸,与欧洲鲈(Dicentrarchuslabrax)IL-10序列相似性最高(78.5%);系统进化树分析显示大黄鱼IL-10和其他鱼类IL-10形成一个大簇,与欧洲鲈进化关系最近。荧光定量PCR(quantitative real-time PCR, qRT-PCR)检测结果显示健康大黄鱼IL-10基因在鳃和肠中高表达,肝和头肾中低表达;溶藻弧菌(Vibrio alginolyticus)侵染后大黄鱼肠、脑、脾、肝和头肾中IL-10基因表达量均显著上调(P0.05),其中头肾和肝中上调幅度最大(分别为菌侵染前的10.06倍和8.79倍)。综上,大黄鱼IL-10基因表达与病原菌感染密切相关,为深入研究大黄鱼IL-10的生物学功能及免疫机制提供了基础资料。     

大黄鱼(Pseudosciaena crocea)细胞色素P450 CYP4F7基因的克隆及表达分析

CYP4F7是细胞色素P450超基因家族中CYP4F亚家族中的同工酶之一,而细胞色素P450在通过电子传递参与生物体代谢和转化内源和外源化合物方面起着重要的作用。以本实验室构建的大黄鱼消减杂交cDNA文库中623bp的CYP4F7片段设计引物,以大黄鱼的总RNA为模板,利用RACE-PCR的方法,克隆鉴定出了大黄鱼细胞色素P450 CYP4F7基因。结果表明:基因全长1934bp,5'端非编码区59bp,3'端非编码区264bp,开放阅读框1611bp,编码536个氨基酸。序列分析表明大黄鱼的CYP4F7氨基酸序列与舌齿鲈的相似性为91%。利用RT-PCR方法研究CYP4F7在大黄鱼各组织中的表达特征,结果表明CYP4F7在肝脏、脾脏中表达量最高,在心脏、肾脏、头肾、鳃丝中表达量次之。另外,CYP4F7在注射了细菌脂多糖的大黄鱼各组织中的表达量均低于正常的大黄鱼,认为细菌脂多糖可能是CYP4F7表达的抑制剂,说明CYP4F7可能与鱼类免疫反应有一定相关性。     

大黄鱼(Pseudosciaena crocea)Cathepsin L基因cDNA片段的克隆与稳定性研究

采用RACE-PCR和RT-PCR方法获得大黄鱼组织蛋白酶L(Cathepsin L)基因的cDNA中间片段,片段长度为773bp。BLAST分析显示,大黄鱼Cathepsin L基因cDNA片段与鱼的同源性最高达到95%,与斑马鱼同源性为80%。实时定量分析了大黄鱼Cathepsin L基因在宰杀当天和冷藏期间的mRNA水平,结果表明,大黄鱼0℃条件下冷藏0、5、10、15、20d肌肉中mRNA都具有相对稳定性,但是含量水平有差异并且是呈现下降趋势,宰杀当天的大黄鱼肌肉中Cathepsin L基因mRNA含量水平最高,与冷藏5、10、15、20d的Cathepsin L基因mRNA水平差异性极显著(P<0.01),冷藏5d的Cathepsin L基因mRNA水平与15、20d的水平也达到了极显著水平(P<0.01)。同时结果表明,在不同储藏时间Cathepsin L基因的mRNA含量水平与肌肉TPA和pH等也表现了高度的相关性,表明Cathepsin L基因的mRNA含量可以作为评价大黄鱼冷藏期间肉品质指标的可行性和准确性。     

大黄鱼肌肉组织cDNA全长文库的构建及EST分析

以大黄鱼肌肉组织为材料,利用Creator Smart cDNA Library Construction Kit构建了大黄鱼肌肉组织的全长cDNA文库.文库质量分析表明：cDNA文库容量不低于1.68×106cfu/mm3,重组率达96%,插入片断的平均长度大于1 000bp.随机挑取512个cDNA克隆进行5’端测序,其中430条ESTs的长度大于100bp;并初步拼接得到了230个单基因簇（unigene）,其中包括58个重叠群（contigs）,172个单拷贝ESTs（singletons）,冗余度为46.51%.经检索,35个ESTs在BLASTx上无明显的同源性（E值≤1.00×10-10）,为新基因.195个ESTs与已报道的基因有较高的同源性;其中与能量相关基因的ESTs丰度最高,占总数的20.00%.蛋白质合成、细胞骨架和信号传导次之,比例分别为13.48%、12.17%、10.00%,其中包括高迁移率族蛋白B1、瘦素受体、β1-热激蛋白等.这些EST数据为进一步筛选和克隆大黄鱼肌肉特异性表达基因提供了平台.     

鲫鱼(Carassius auratus)slc7A8基因分子特征及表达分析

采用RT-PCR和RACE技术克隆鲫鱼slc7A8基因的全长cDNA序列,对基因序列用生物软件对基因进行生物信息分析,用Real-time PCR方法检测基因在组织中的mRNA表达丰度。结果表明,鲫鱼slc7A8cDNA全长为2709bp,包含195bp的5′UCR序列,921bp的3′UCR序列,1593bp开放阅读框,编码530个氨基酸序列;鲫鱼与斑马鱼基因同源性和编码氨基酸同源性分别为88.9%和95.5%,而与其他动物分别在70.1%-74.8%和80.6%-82.1%之间;预测编码蛋白的分子量为57719.84,等电点为4.8,对其结构预测显示该蛋白具有与哺乳动物十分相似的12个螺旋跨膜结构,跨膜区氨基酸高度保守,不同动物间的同源性都在92.2%以上;Real-time PCR检测鲫鱼组织中的mRNA表达丰度高低顺序为前肠、中肠、脑、后肠、肌肉、肾、鳃、心、肝脏。     

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。     

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.     

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.     

A Tidal Flow Model for the Gulf of Kachchh, India

A vertically integrated model has been used to study the tidal circulation and currents in the Gulf of Kachchh along the west coast of India. The model is fully nonlinear and uses a semiexplicit finite difference scheme to solve the basic hydrodynamic equations on a staggered grid. The model is forced by prescribing the tides along the open boundary of the model domain. The flow is simulated both with and without the presence of the proposed tidal barrage across the Hansthal Creek in the Gulf of Kachchh. The results show a considerable change in the behavior of the tidal flow in the presence of the barrage.     

利用中国GF-3卫星SAR和HY-2A卫星微波散射计的首次台风联合定量观测

<正>The typhoon,as a mature tropical cyclone that develops in the western part of the North Pacific Ocean with high wind speed and heavy rainfall,is one of the most lethal and costly of natural disasters for the densely populated countries of East Asia.It can be easily detected by space-borne sensors operated at microwave,visible or infrared bands(Liu et al.,2014).Synthetic Aperture Radar(SAR)is     

离子交换—原子吸收光谱法测定海水中痕量铜、铅、锌、镉、铁、锰

本文介绍了用国产D401型螯合树脂分离富集海水中铜、铅、锌、镉、铁、锰等痕量元素,并用原子吸收光谱仪测定其含量的方法。讨论了各元素的分离条件选择及干扰元素的影响,并与溶剂萃取法的结果作了比较。各元素检测的定量下限为:铜0.5μg/L、铅0.1μg/L、锌1.0μg/L、镉0.01μg/L、铁2.0μg/L、锰2.0μg/L。方法精密度在4—8％之间,回收率为90—102％。     

Biogeochemistry of Mangrove Soil Organic Matter: a Comparison BetweenRhizophoraandAvicenniaSoils in South-eastern Brazil

Soil core samples fromRhizophora mangleL. andAvicennia schauerianaStapf & Leech forests from south-eastern Brazil were analysed for their total organic matter content and their sugars, amino acid and amino sugars composition. Organic carbon and nitrogen contents were higher inAvicenniathan inRhizophorasoils. The contribution of sugars and amino acids to the total organic carbon pool was constant with depth inRhizophorasoils whereas inAvicenniasoils it increased. Spectral distribution of sugars and amino acids showed a dominance of Ca-affine monomers, particularly acidic amino acids, and the sugar arabinose. Biogeochemical indicators derived from ratios of individual sugar and amino acid monomers confirm previous studies which showed that organic matter in both soils is mainly of mangrove origin. The results further suggest accumulation of organic matter inRhizophorasoils and a continuing degradation of organic matter inAvicenniasoils. The latter may thus release more nutrients to adjacent ecosystems thanRhizophorasoils.     

A review of orange roughy Hoplostethus atlanticus fisheries,estimation methods,biology and stock structure

Orange roughy Hoplostethus atlanticus are unusual fish. They form dense aggregations that have fuelled lucrative fisheries at great depths (600–1 400 m), especially off Namibia, New Zealand and Australia. They are thought to be very long-lived (>100 years, maturity at 22 – 40 years), and to have exceptionally low natural mortality (M = 0.045–0.064 year^?1) and slow growth rates (K = 0.055–0.070 year^?1). In addition, they spawn large eggs and have low fecundity. These factors combine to make orange roughy highly susceptible to overfishing; most stocks are below 30% of pristine levels. Assessments are obtained from indices of catch rate and trawl, acoustic and egg surveys. Acoustic estimates are the most direct, but are confounded by the species' low target strength (?50 to ?53 dB)–attributable to the wax-filled swim bladder. Extracellular wax esters are stored in abundance and comprise mostly mono-unsaturated fatty acids, with low concentrations of the ω-3 fatty acid family. This unusual composition (resultant from the species' diet) ensures neutral buoyancy. Stock separation has been inferred mainly from biological studies, but genetic studies have also found differences among stocks within New Zealand and Australia. Deep-water habitat may be damaged by trawling operations and may take many years to recover, so in some quarters there is a call for a portion of suitable habitat to be set aside for preservation. Although Namibian orange roughy are shallower, smaller and younger than those in other stocks, the Namibian fishery sustained high catches for only a few years before quotas were reduced, from 12 000 to 1 875 tons. Three management lessons are suggested for developing orange roughy fisheries based on the Namibian experience: (1) imposition of catch limits during exploratory fishing; (2) starting the acoustic surveys earlier in the fishery, if possible; (3) greater reliance on trends in catch rate until a survey series has been established.