GENETIC VARIATION AMONG SEA BASS POPULATIONS

采用水平淀粉凝胶电泳技术对花鲈群体的遗传结构进行了研究,共检测了中国沿海花鲈146 尾和40 尾日本 东京湾花鲈。其群体的多态位点比例为012667 —015333 ,观测杂合度和预期杂合度分别为010211 —010515 和 010398 —010797。中日花鲈在LDH 3 , GPI21 3 , GPI22 3 基因位点上的等位基因接近完全置换。中国花鲈各群体之 间的根井遗传距离为010004 —010011 ,平均值约为010080 ;而中日花鲈间的根井遗传距离为011870 —011954 ,平均值 为011926。以上结果表明中国花鲈群体间遗传变异很小,中日花鲈间遗传变异远大于中国花鲈群体间的遗传距 离。

Study on Mitochondrial DNA Cytochrome b Gene of Chinese sea bass, Lateolabrax sp.

参考鳗鲡等鱼类线粒体 DNA序列进行了中国花鲈线粒体 DNA细胞色素 b基因片断的引物设计、PCR扩增及其序列测定。得到中国花鲈的碱基序列为 4 10 bp,其 A、T、G、C含量分别为 10 1bp(2 4 .6 3% )、112 bp(2 7.32 % )、72 bp(17.56 % )、12 5bp(30 .4 9% ) ,与鳗鲡等其他鱼类相同基因片断序列碱基含量相似。

Differential population structuring and demographic history of two closely related fish species, Japanese sea bass (Lateolabrax japonicus) and spotted sea bass (Lateolabrax maculatus) in Northwestern Pacific

The Quaternary cold periods in the Northwestern Pacific are thought to have heavily influenced the amount and distribution of intraspecific genetic variation in marine fishes. To estimate the demographic history and genetic structure of Lateolabrax macula

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

日本花鲈细胞色素b基因部分序列的研究

首次报道日本花鲈线粒体DNA细胞色素b基因片段的PCR扩增及其序列测 定。得到410bp的碱基序列，其A、T、G、C含量分别为99bp(24．15哟、113bp(27．56旧、 72bp(17．56嗡、126bp(30．73哟，与其他鱼类相同基因片段碱基序列含量相似。

Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus)

Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

SNAP-23蛋白在鲈鱼巨噬细胞白细胞介素1β分泌过程中的功能

SNARE蛋白家族是所有真核细胞胞吐及分泌作用中的关键因子，由其介导的运输囊泡膜与靶膜的锚靠、融合为胞内蛋白的运出提供了一条重要途径。体外试验表明，Syntaxin6-Syntaxin7-Vti1b，SNAP-23-Syntaxin4等SNARE核心蛋白之间精确的相互作用是哺乳动物巨噬细胞肿瘤坏死因子α (TNF-α)运输和分泌的必备条件，在机体非特异性免疫应答反应过程中起重要作用。 本研究受上述启示，旨在揭示SNARE蛋白在海洋鱼类免疫细胞内重要细胞因子白细胞介素1β (IL-1β)的分泌过程中的作用。参照Percoll密度梯度离心技术，从鲈鱼头肾组织分离纯化巨噬细胞进行稳定培养；利用RT-PCR方法克隆出鲈鱼t-SNARE蛋白SNAP-23和Syntaxin3的部分cDNA序列，再结合先前克隆的VAMP2和已知的鲈鱼IL-1β，TNF-α和IL-8的基因序列，设计特异性引物。利用Real-time PCR技术在mRNA水平上精确测定鲈鱼巨噬细胞中上述6种基因在革兰氏阴性菌脂多糖(LPS)分子刺激下的表达变化，发现SNAP-23基因与三种细胞因子基因的表达正相关；通过免疫印迹检测SNAP-23蛋白表达变化，利用酶联免疫吸附试验(ELISA)检测IL-1β的分泌水平，在蛋白水平上验证了SNAP-23表达与IL-1β分泌的正相关性；利用5`RACE和3`RACE技术克隆出鲈鱼SNAP-23全长基因，结合定点突变策略和靶向PCR克隆手段，构建鲈鱼SNAP-23野生型融合质粒pEGFP-SNAP-23wt，Cys缺失突变融合质粒pEGFP-SNAP-23ΔCys和模拟E型肉毒神经毒素(BoNT/E)切割突变融合质粒pEGFP-SNAP-23ΔBoNT/E，以及鲈鱼IL-1β野生型融合表达质粒IL-1β-pEGFP和IL-1β-pEYFP。所有融合蛋白均在鲈鱼巨噬细胞内成功表达，结合ELISA实验结果发现，SNAP-23野生型的表达对IL-1β的分泌有促进作用，而Cys缺失突变体的表达则抑制IL-1β向胞外分泌。首次证实了鱼类巨噬细胞内SNAP-23蛋白在IL-1β分泌过程中的重要作用。此外通过与GFP共表达，定位了IL-1β分子在巨噬细胞内的分布，发现新合成的IL-1β分子很可能像TNFα一样经“内质网－胞质－伪足－胞外” 的分泌路径运出胞外。

评价水产动物生长性能的生理生化指标和分子生物学指标

以奥利亚罗非鱼(Oreochromis aureus)为实验对象，设计了3种不同的摄食类型，分别是鲜活饵料组、饥饿3周后饱食投喂组和人工饲料组。鲜活饵料组投喂冰冻赤子爱胜蚓，利用蚯蚓体内丰富的营养成分和活性物质，以期获得奥利亚罗非鱼良好的生长状况；饥饿后饱食组是指饥饿3周后，以人工饲料饱食投喂2周，用于研究饥饿与补偿生长获得快速生长时血液理化指标的变化情况；人工饲料组作为对照组。纯淡水条件下养殖，水温25±2℃。测定了奥利亚罗非鱼在3种摄食类型饲喂下某些血液生理生化指标变化的情况，并将指标变化情况与增重率做相关性分析，试图找出能够反映奥利亚罗非鱼生长性能的血液生理生化指标。 研究结果表明，奥利亚罗非鱼在饥饿3周后获得了补偿生长，补偿生长时的增重率和特定生长率显著高于人工饲料组(P<0.05)，高于鲜活饵料组，但差别不显著；相关性分析研究表明血清总蛋白、胆固醇、四碘甲状腺原氨酸（T4）与增重率极显著相关(P<0.01)，血红蛋白显著相关(P<0.05)，红细胞、白细胞、碱性磷酸酶高度相关(相关系数为0.580、0.551和0.557)，因此，建议血清总蛋白、胆固醇和血红蛋白可作为能够反映罗非鱼生长性能的新指标。 根据序列设计引物，PCR反应条件：变性温度：95 ℃，3 min；退火温度：57℃，20 sec；延伸温度：72℃，5 min，共36个循环，从牙鲆、黑鲪和鲈鱼中克隆出胰岛素样生长因子（IGF-Ⅰ）部分序列，首次证实了IGF-Ⅰ在3种海水鱼中的存在。 利用蛋氨酸与ZnSO4•7H2O，在pH 5.5、80℃下，反应1小时，采用蛋氨酸与硫酸锌2：1的配料比，合成出了产物蛋氨酸螯合锌，蛋氨酸螯合锌外观白色，粉状，室温下微溶于水，不溶于乙醇，并用原子吸收光谱法测定其含锌量为15%，螯合率为88.2%。

Development of the East Asian monsoon: Mineralogical and sedimentologic records in the northern South China Sea since 20 Ma

We here reconstruct the past change of the East Asian monsoon since 20 Ma using samples from Ocean Drilling Program (ODP) Site 1146 in the northern South China Sea based On a multi-proxy approach including a monomineralic quartz isolation procedure, identification of clay minerals by X-ray Diffraction (XRD) and grain-size analysis of isolated terrigenous materials. Terrigenous supply to ODP Site 1146 was dominated by changes in the strength of multiple sources and transport processes. Grain-size data modeled by an end-member modeling algorithm indicate that eolian dust from the and Asian inland and fluvial input have contributed on average 20% and 80% of total terrigenous material to ODP Site 1146, respectively. Specifically, about 40-53% of the total (quartz+feldspar) and only 6-11% of the total clay is related to eolian supply at the study site. Detailed analysis of the sedimentary environment, and clay minerals combined with previous studies shows that smectite originates mainly from Luzon, kaolinite from the Pearl River and illite and chlorite from the Pearl River, Taiwan and/or the Yangtze River. The proportion and mass accumulation rate (MAR) of the coarsest end-member EM1 (interpreted as eolian dust), ratios of (illite+chlorite)/smectite, (quartz+feldspar)% and mean grain-size of terrigenous materials at ODP Site 1146 were adopted as proxies for East Asian monsoon evolution. The consistent variation of these independent proxies since 20 Ma shows three profound shifts in the intensity of East Asian winter monsoon relative to summer monsoon, as well as aridity of the Asian continent, occurred at similar to 15 Ma, similar to 8 Ma and the youngest at about 3 Ma. In comparison, the summer monsoon intensified contemporaneously with the winter monsoon at 3 Ma. The phased uplift of the Himalaya-Tibetan plateau may have played a significant role in strengthening the Asian monsoon at similar to 15 Ma, 8 Ma and 3 Ma. (C) 2007 Elsevier B.V. All rights reserved.

Grain-size records at ODP site 1146 from the northern South China sea: Implications on the east Asian monsoon evolution since 20 Ma

273 samples from Ocean Drilling Program (ODP) Site 1146 in the northern South China Sea (SCS) were analyzed for grain-size distributions using grain-size class vs. standard deviation method and end-member modeling algorithm (EMMA) in order to investigate the evolution of the East Asian mon-soon since about 20 Ma. 10-19 mu m/1.3-2.4 mu m, the ratio of two grain-size populations with the highest variability through time was used to indicate East Asian winter monsoon intensity relative to summer monsoon. The mass accumulation rate of the coarsest end member EM1 (eolian), resulting from EMMA, can be used as a proxy of winter monsoon strength and Asian inland aridity, and the ratio of EM1/(EM2+EM3) as a proxy of winter monsoon intensity relative to summer monsoon. The combined proxies show that a profound enhancement of East Asian winter monsoon strength and winter monsoon intensity relative to summer monsoon occurred at about 8 Ma, and it is possible that the summer monsoon simultaneously intensified with winter monsoon at 3 Ma. Our results are well consistent with the previous studies in loess, eolian deposion in the Pacifc, radiolarians and planktonic foraminifera in the SCS. The phased uplift of the Himalaya-Tibetan Plateau may have played a significant role in strengthening the Asian monsoon at 8 Ma and 3 Ma.

Evolution and variability of the East Asian summer monsoon during the Pliocene: Evidence from clay mineral records of the South China Sea

The Late Pliocene is thought to be characterized by the simultaneous intensification of both the East Asian winter monsoon (EAWM) and East Asian summer monsoon (EASM). However, the evolution of the EASM during the Pliocene remains still controversial and only little is known about the dynamics of the EASM during the Pliocene on orbital time scales. Here we use clay mineral assemblages in sediments from Ocean Drilling Program (ODP) Site 1143 in the southern South China Sea (SCS) to obtain proxy records of past changes in the EASM climate during the Pliocene. Provenance analysis suggests that illite, chlorite and kaolinite originated mainly from the Mekong River drainage area. Smectite was derived mainly from the Indonesian islands. The kaolinite/illite ratio and the chemical index of alteration (CIA) of siliciclastic sediments allowed us to reconstruct the history of chemical weathering and physical erosion of the Mekong River drainage area and thus, the evolution of,the EASM during the Pliocene. Our clay minerals proxy data suggests a stronger EASM during the Early Pliocene than during the Late Pliocene. We propose that the long-term evolution of the EASM has been driven by global cooling rather than the uplift of the Tibetan Plateau. Spectral analysis of kaolinite/ illite ratio displays a set of strong periodicities at 100 ka, 30 ka, 28 ka, 25 ka, and 22 ka. with no clear obliquityrelated signal. Our study suggests that the Pliocene EASM intensity on orbital time scales is not only controlled by the Northern Hemisphere summer insolation, but also strongly influenced by equatorial Pacific ENSO-like ocean atmosphere dynamics. (C) 2010 Elsevier B.V. All rights reserved.

Records of the East Asian winter monsoon from the mud area on the inner shelf of the East China Sea since the mid-Holocene

AMS(14)C dating and analysis of grain size, major elements and clay minerals were applied to Core MZ01 from the mud area on the inner shelf of the East China Sea. Based on the environmentally sensitive grain size, clay mineral and major element assemblages, the history of the East Asia winter monsoon since the mid-Holocene could be reconstructed. These three proxies, mean grain size (>9.71 mu m), chemical index of alteration (CIA) and ratio of smectite to kaolinite in particular, show similar fluctuation patterns. Furthermore, 10 extreme values corresponding to the contemporary cooling events could be recognized since the mid-Holocene; these extreme values are likely to have been caused by the strengthening of the East Asia winter monsoon. The cooling events correlated well with the results of the delta O-18 curves of the Dunde ice core and GISP2, which therefore revealed a regional response to global climate change. Four stages of the East Asia winter monsoon were identified, i.e. 8300-6300 a BP, strong and unstable; 6300-3800 a BP, strong but stable; 3800-1400 a BP, weak and unstable; after 1400 a BP, weak but stable.

Development of the East Asian summer monsoon: Evidence from the sediment record in the South China Sea since 8.5 Ma

128 samples from Ocean Drilling Program (ODP) Site 1143 in the southern South China Sea were analyzed for grain size, clay minerals, biogenic opal content and quartz in order to reconstruct changes in East Asian monsoon climate since 8.5 Ma. An abrupt change of terrigenous mass accumulation rate (MAR), clay mineral assemblage, median grain size and biogenic opal MAR about 5.2 Ma suggests that between 8.5-5.2 Ma the source of terrigenous sediment was mainly in the region of surface uplift and basaltic volcanism in southern Vietnam. A simple model of East Asian summer monsoon evolution was based on the clay/feldspar ratio, kaolinite/chlorite ratio and biogenic opal MAR. The summer monsoon has two periods of maximum strength at 8.5-7.6 Ma and 7.1-6.2 Ma. Subsequently, there was a relatively stable period at 6.2-3.5 Ma, continued intensification about 3.5-2.5 Ma, and gradually weakening after 2.5 Ma. Since I Ma the monsoon has intensified, with remarkable high-frequency and amplitude variability. Simultaneous increase in sedimentation rates at ODP Sites 1143, 1146 and 1148, as well as in MAR of terrigenous materials, quartz, feldspar and clay minerals at ODP Site 1143 at 3.5-2.5 Ma, may be the erosional response to both global climatic deterioration and the strengthening of the East Asian summer monsoon after about 3-4 Ma. (c) 2006 Elsevier B.V. All rights reserved.

Coherence between solar activity and the East Asian winter monsoon variability in the past 8000 years from Yangtze River-derived mud in the East China Sea

AMS(14)C dating and grain-size analysis for Core PC-6, located in the middle of a mud area on the inner shelf of the East China Sea (ECS), were used to rebuild the Holocene history of the East Asian winter monsoon (EAWM). The 7.5-m core recorded the history of environmental changes during the postglacial transgression. The core's mud section (the upper 450 cm) has been formed mainly by suspended sediment delivered from the Yangtze River mouth by the ECS Winter Coastal Current (ECSWCC) since 7.6 kyr BP. Using a mathematical method called "grain size vs. standard deviatioW', we can divide the Core PC-6's grain-size distribution into two populations at about 28 mu m. The fine population (< 28 mu m) is considered to be transported by the ECSWCC as suspended loads. Content of the fine population changes little and represents a stable sedimentary environment in accord with the present situation. Thus, variation of mean grain-size from the fine population would reflect the strength of ECSWCC, which is mainly controlled by the East Asian winter monsoon. Abrupt increasing mean grain size in the mud section is inferred to be transported by sudden strengthened ECSWCC, which was caused by the strengthened EAWM. Thus, the high resolution mean grain-size variation might serve as a proxy for reconstruction of the EAWM. A good correlation between sunspot change and the mean grain-size of suspended fine population suggests that one of the primary controls on centennial- to decadal-scale changes of the EAWM in the past 8 ka is the variations of sun irradiance, i.e., the EAWM will increase in intensity when the number of sunspots decreases. Spectral analyses of the mean grain-size time series of Core PC-6 show statistically significant periodicities centering on 2463, 1368, 128, 106, 100, 88-91, 7678, and 70-72 years. The EAWM and the East Asian summer monsoon (EASM) agree with each other well on these cycles, and the East Asian Monsoon (EAM) and the Indian Monsoon also share in concurrent cycles in Holocene, which are in accord with the changes of the sun irradiance. (c) 2005 Elsevier B.V. All rights reserved.