雄性半滑舌鳎雄激素受体基因的克隆与表达分析

利用RT-PCR方法从半滑舌鳎(Cynoglossus semilaevis)中克隆了雄激素受体(Androgen receptor,AR)基因cDNA的部分序列(434 bp),经BLAST比对与狼鲈(Dicentrarchus labrax)、花溪(Kryptolebias marmoratus)、三斑海猪鱼(Halichoeres trimaculatus)和三刺鱼(Gasterosteus aculeatus)的雄激素受体同源性分别为84%,82%,82%和81%.组织表达分析表明,AR基因在半滑舌鳎的性腺、肝、胃、脾、肾、头肾、肠、鳃、心、脑和肌肉这11种组织中均有表达,表达量有所差异.肾中表达量最丰富,鳃中最少.根据其他硬骨鱼类表达模式,推测半滑舌鳎可能只含有1种AR.     

半滑舌鳎（Cynoglossus semilaevis）促性腺激素受体在雄性生殖周期中的表达

采用RT-PCR方法,研究了促性腺激素受体（FSHR和LHR）基因在雄性半滑舌鳎繁殖周期中的季节表达规律。结果表明,FSHR mRNA在雄鱼精巢、脑和脾中及LHR mRNA在雄鱼的精巢和脾中均呈季节规律性变化。3月份样品中,FSHR mRNA在精巢表达量最低,12月份,表达量最高;FSHR mRNA在脑中表达模式与精巢...     

半滑舌鳎稚鱼脂肪分解关键酶基因的克隆及饲料中脂肪水平对其表达的调控

克隆了半滑舌鳎脂肪分解关键酶基因即激素敏感脂肪酶(Hormone-sensitive lipase,HSL)和脂肪甘油三酯脂肪酶(Adipose triglyceride lipase,ATGL)cDNA部分序列,并探讨了饲料中脂肪水平对半滑舌鳎稚鱼HSL及ATGL基因表达的影响。克隆所得到的HSL和ATGL片段长度分别为589bp和581bp,序列和系统进化分析表明半滑舌鳎的HSL和ATGL与大部分鱼类具有较高的同源性。采用不同水平的鱼油配制成5种不同脂肪水平(6.68%、9.84%、13.47%、17.89%和21.88%干物质)的等氮饲料,饲养35日龄半滑舌鳎稚鱼30d,养殖过程中每天饱食投喂5次,养殖实验结束后采用实时荧光定量PCR分别测定各处理稚鱼内脏团的HSL及ATGL mRNA相对表达量。结果表明,高脂饲料组(21.88%)显著促进了半滑舌鳎稚鱼内脏团HSL基因的表达(P0.05),但饲料脂肪水平对于半滑舌鳎稚鱼内脏团的ATGL表达量未产生显著影响(P0.05)。     

半滑舌鳎(Cynoglossus semilaevis)GnRHR基因全长cDNA 的克隆与组织表达分析

采用同源克隆和末端快速扩增(RACE)方法, 克隆了半滑舌鳎中 GnRHR 全长 cDNA。通过基因全长以及推断的氨基酸序列分析, 得知该序列包含一个 7tm-1 保守结构域, 为 G 蛋白偶联受体家族成员。利用 PHYLIP 3.5c 邻位相连法以及 DNAstar 中的 CLUSTAL W 方法对相应的氨基酸序列进行了聚类分析和序列相似度分析。结果表明, 半滑舌鳎 GnRHR 鳉 与鲈鱼、琥珀鱼、虹鳟以及青 等鱼类中的 GnRHR聚为一支, 亲缘关系较近, 且相似度较高, 分别为 90.1%、 89.7%、 79.0%以及 78.3%, 而与高等哺乳动物人、小鼠相似性仅分别为 18.8%和 16.2%, 亲缘关系较远。应用半定量 RT-PCR 技术分析其组织表达, 发现 GnRHR 广泛表达于各个组织, 但表达量差异较大, 在性腺、脑和肾中表达量较高, 其它组织较弱。     

半滑舌鳎的一种细胞凋亡抑制因子的克隆、鉴定与分析

克隆半滑舌鳎(Cynoglossus semilaevis)的1种细胞凋亡抑制因子survivin基因,并对其序列及表达模式进行分析.结果表明,该基因cDNA 序列全长704 bp(GenBank登录号为EU499314),包括86 bp5'非翻译区,444 bp开放阅读框以及含 poly(A)信号的174 bp的3'非翻译区,编码147个氨基酸.氨基酸序列同源性分析表明,半滑舌鳎survivin氨基酸序列与斑马鱼、非洲爪蟾、鸡、小鼠和人survivin氨基酸序列的同源性分别达到了63.3%,49.0%,43.7%,43.6%和41.5%.RT-PCR分析表明,survivin基因在半滑舌鳎成体雌鱼卵巢中大量表达,在雌鱼的肾脏中微量表达,在其它雌鱼的组织中无表达,在雄性的任何组织中均无表达.该基因在受精卵、多细胞和囊胚3个胚胎发育的早期阶段,大量表达,而随着胚胎发育的进程,survivin基因的表达逐渐减少,在孵化前尾芽期胚胎中只有微量表达,在孵化后5 d的仔鱼中,几乎检测不到survivin基因的表达.表明该基因可能参与卵子形成和胚胎的早期发育.     

促激素及其受体在雄性半滑舌鳎脑垂体和精巢中的定位

应用兔抗血清对抗鱼类和哺乳类促激素及其受体的多克隆抗体对雄性半滑舌鳎脑垂体和精巢进行了免疫组织化学定位.其结果显示GnRHR、LHR和GtH免疫活性定位在不同发育时期的腺垂体和精巢中.GtH免疫阳性细胞的数量和免疫强度随着精巢发育成熟而明显增强.同时还发现睾酮（T）和雄激素受体（AR）在半滑舌鳎精巢中的定位有别于其他鱼类,尤其是雄激素受体（AR）免疫活性在精原细胞中的数量很少.依据这个特点文中还讨论了其可能与精巢发育的关系,同时证明了半滑舌鳎精巢发育像其他硬骨鱼类一样受激素的调控.     

半滑舌鳎des基因的表达及其启动子功能分析

利用NCBI上已经发表的半滑舌鳎(Cynoglossus semilaevis)基因组及转录组序列,发现了半滑舌鳎两个有结蛋白(Desmin)基因(des和des-like),且des-like具有两个剪切型des-like_X1、des-like_X2。系统进化树及基因结构分析发现,半滑舌鳎的des基因和des-like基因分别与硬骨鱼的des基因和des-like基因聚为一支,物种间des和des-like氨基酸序列相似性分别在90%和85%以上。保守性与染色体共线性分析发现,半滑舌鳎的des基因和哺乳动物的des基因具有相同的起源,而其des-like基因是通过硬骨鱼的第三次全基因组复制获得的。qRT-PCR的检测分析发现,des基因在肠和心脏中表达量较高,在肌肉中只是痕量表达;而des-like基因则在心脏和肌肉中表达最高,在肠中痕量表达,说明des与des-like已经出现了功能的分化而且可能存在功能上的互补效应。有趣的是,des-like_X1只在心脏中表达,本研究推测des-like_X1对于半滑舌鳎的心脏发育具有重要作用。des基因呈现母源性表达,而des-like在胚胎发育早期表达量较低,从神经胚后表达量开始升高,此时是肌节与心脏发育的时期,说明des-like可能参与胚胎发育时期的肌节与心肌发育。此外,通过PCR克隆了半滑舌鳎结蛋白基因不同长度的启动子片段,并对其进行启动活性分析。结果表明,半滑舌鳎des基因ATG上游长度为667 bp的片段是其核心启动子,为进一步研究des基因的功能奠定了基础。     

性类固醇激素及其受体在半滑舌鳎性腺分化发育过程中的表达与生理功能

首次用17β-雌二醇(E_2)及其受体(ERα)、睾酮受体(AR)的多克隆抗体在半滑舌鳎(Cynoglossus semilaevisG(u|¨)nther)性腺分化发育过程中进行免疫细胞化学定位研究。结果表明:在半滑舌鳎原始性腺中已有E_2、ERα和AR的分布和表达;卵原细胞、初级卵母细胞、精原细胞、初级精母细胞、次级精母细胞对E_2及ERα、AR抗体均呈现不同程度的阳性反应,在精细胞和精子中对E_2和ERα呈免疫阴性反应,而AR则一直保持较强表达,揭示了半滑舌鳎性腺中类固醇激素可能通过旁分泌调控机制刺激卵子和精子发育和成熟。性类固醇激素及其受体免疫阳性物定位在生殖细胞的胞质、胞膜和核膜等广泛部位并有表达,在性腺分化和不同发育时期的表达强度有所不同,表明其在性腺分化发育过程各阶段所起生理功能不同。这些结果为证明性类固醇激素及受体参与调节半滑舌鳎性腺分化发育提供了重要形态学新证据。     

10 个半滑舌鳎家系 MHC IIB 基因多态性初步研究

为检测10个半滑舌鳎(Cynoglossus semilaevis)家系MHCⅡB(Cyse-DAB)基因的多态性水平、MHCⅡ类B位点数目以及平衡选择的作用,作者利用多聚酶链式反应(PCR)和直接测序的方法对10个半滑舌鳎家系MHCⅡB基因位点遗传变异和平衡选择进行了研究。用特异性引物和PCR扩增的半滑舌鳎MHCⅡB基因片段大约397 bp,包含一部分第一外显子,全部第一内含子和全部第二外显子。10个半滑舌鳎家系中,每家系选取5个体,每个体5个克隆序列分析发现60个不同序列,代表60个等位基因,其中有28个是新发现的,已提交到Gen Bank。同源分析表明60个等位基因相似性为89.36%。共50个个体中,有6个存在5个不同等位基因,表明在半滑舌鳎至少存在3个座位。有9个家系的MHCⅡB序列多肽结合区(PBR)的非同义替换(dN)显著高于同义替换(dS)。     

半滑舌鳎骨形态发生蛋白2基因的克隆、表达分析及启动子活性分析

骨形态发生蛋白2（BMP2）在脊椎动物的骨形成、尤其是诱导成骨方面具有至关重要的作用。本研究克隆了半滑舌鳎（Cynoglossussemilaevis）BMP2基因的cDNA序列，分析其在半滑舌鳎成鱼的组织表达差异及其在早期发育阶段的定位和表达变化趋势，并检测了其启动子活性。研究结果表明，半滑舌鳎BMP2基因cDNA全长为2048bp，编码422个氨基酸。BMP2mRNA在半滑舌鳎成鱼的14个组织中具有广泛的分布，并在脊髓中表达量最高。此外，BMP2基因在早期胚胎发育阶段（原肠期）具有极高水平的表达，其表达量约为其他发育阶段表达量的10倍。整体原位杂交检测结果表明，BMP2mRNA在1日龄仔鱼主要在冠状幼鳍、心和肝表达，在脊索有微量表达；随后，3日龄仔鱼的BMP2mRNA主要表达于脊索。通过对BMP2基因5''上游调控区片段进行启动子活性分析，发现了-179～+109片段可能包含启动子活性增强原件，生物信息学预测该片段包含的锌指转录因子等可能参与了BMP2的转录调控。     

Application of a two-way nested model to the seamount problem

A two-way nested model has been constructed and applied to the idealized ocean where a uniform mean flow impinges on the isolated Gaussian-shaped seamount and produces two eddies (cold and warm) in the depths. The performance of the nested model has been evaluated subjectively and objectively. Both subjective and objective analyses confirm the traditional view that the nested model can well capture the performance of isolated eddies. Objective analysis, however, reveals some quantitatively important features of a two-way nested model. One is penetration of improved features into the coarse domain and another is the deterioration of mean flow field inside the nested area, neither of which is clear from subjective analysis. With successful application of two-way nested model to the seamount problem, we expect that such a nested model will also be applicable to other oceanic phenomena, particularly to some coastal problems whose time scale is short and where the topographic effects are dominant.     

Staggered nested designs to identify hierarchical scales of variability

Staggered nested sampling designs are virtually unknown in biology, but are widely used in manufacturing because they are highly efficient. I used a staggered nested design to address variation in the abundance of an intertidal clam Austrovenus stutchburyi (Gray) at four spatial scales. The scales were addressed by sampling cells containing five samples spaced at 50, 5, 0.5, and 0.05 m from one another. Estimates of variation from staggered nested designs can be made via ANOVA and principal component methods. ANOVA and principal component methods estimated roughly equal variability among those four spatial scales of abundance, and at all but the smallest scales variances were estimated very imprecisely. Staggered nested designs are highly efficient, applicable to any nested design, and deserve to be investigated more widely.     

Application of finite volume coastal ocean model to hindcasting the wind-induced sea-level variation in Fukuoka bay

The wind-induced sea-level variations at Hakata tidal station in winter are reproduced realistically using a one-way nested model. This nested model is constructed with a structured finite-difference Princeton Ocean Model (POM) for the Tsushima-Korea Straits, and an unstructured Finite Volume Coastal Ocean Model (FVCOM) for Fukuoka Bay divided into triangular-cell grids. The correlation coefficient and root-mean-square error between observed and modeled results are 0.742 and 1.88 [cm], respectively. Moreover, the results show that the nested model with FVCOM is more accurate than the model in which FVCOM is replaced with a high-resolution POM for Fukuoka Bay. This indicates that the nested model constructed with structured and unstructured models works effectively in hindcasting the wind-induced sea-level variations.     

应用巢式逆转录聚合酶链反应检测鱼类病毒性出血性败血症病毒(VHSV)

参照鱼类病毒性出血性败血症病毒序列,根据PCR引物设计的原则,设计巢式PCR引物,采用巢式逆转录聚合酶链反应(RT-PCR)方法,对如何快速检测鱼类病毒性出血性败血症病的方法进行了较为系统的研究,并对RT-PCR的反应条件进行了优化.结果表明,巢式RT-PCR扩增获得279bp的特异性片断,阴性对照无扩增条带.巢式RT-PCR扩增出的特异性片段经测序分析,结果证实与报道的序列完全一致.该方法最低可检测出0.1pg的鱼类病毒性出血性败血症病毒RNA.初步建立了VHSV的巢式RT-PCR检测方法,该方法灵敏、特异,可为VHSV的检测提供一个快速、有效的手段.     

Numerical analysis of effects of tidal variations on storm surges and waves

This study examines the effects of tides on surges, wave setups and waves, in terms of tidal amplitudes and phases, by using a coupled numerical model of Surge, WAve and Tide (called as SuWAT). The SuWAT model, composed of depth integrated nonlinear shallow water equations and Simulating WAves Nearshore (SWAN) model, is able to simultaneously run with an arbitrary number of nested domains by using the Message Passing Interface. The results for an idealized case indicate that surge and wave setup are increased in the phase of low water and decreased in the high water phase; on the other hand, waves change in a reverse manner. Such changes are enhanced by large tidal variations. The conventional method (e.g., surge plus tide independently) has the possibility of overestimation for the total water level. The hindcast results for Typhoon Ewiniar in 2006 show that the run with tides is more accurate 10% than that without tides in coastal areas of Korea. The nested scheme improves the accuracy up to 40% for the prediction of water levels in the simulations. It is shown that the present coupled model, SuWAT, is capable of predicting both water levels and waves under storm events with reasonable accuracy against the observations.     

套式PCR检测斑节对虾白斑症病毒(WSSV)

解剖患白斑症斑节对虾的鳃组织 ,制备成不同稀释倍数的模板 ,对其进行白斑症病毒 (WSSV)的 PCR扩增 ,结果显示所建立的套式 PCR的灵敏度大约为一步 PCR的 10 4 倍 ;对感染病毒后不同时期的斑节对虾进行 PCR检测 ,发现感染早期经一步 PCR检测为 WSSV阴性的样品 ,套式 PCR的检测结果为阳性 ;对发病虾塘中的几种甲壳类动物进行 PCR检测 ,发现经一步 PCR检测为阴性的长臂虾、秉氏厚蟹和褶痕相手蟹等宿主 ,套式 PCR的检测结果为阳性。表明所建立的 WSSV套式 PCR检测法较普通的一步 PCR有更大的应用价值和意义     

天津地区养殖鲤鱼(Cyprinus carpio)鲤浮肿病毒(CEV)PCR检测与人工感染试验

对2015—2016年天津地区鲤鱼养殖场采集的发生鲤浮肿病毒(CEV)/锦鲤睡眠病(KSD)的鲤鱼样品,进行套式PCR检测和人工浸泡感染试验,并测定养成期鲤鱼感染率。实验观察研究发现,所采集样品均表现为体色发黑,头部上方颅骨软组织周围皱缩,眼球凹陷,鳃粘脏,黏液较多,常滞游于浅水区水面。套式PCR检测结果显示全部10个样品均出现特异性目的条带,为CEV阳性。目的产物测序后经NCBI BLAST,与已报道的CEV相关序列相似性介于85%—99%;系统发育树分析显示样品编号JY1856与JY1650位于同一分支,与已知CEV病毒株亲缘关系较近,而其余8个样品位于同一分支,与已知CEV病毒株亲缘关系较远。通过人工浸泡感染健康鲤鱼,半数致死时间为55d,累计死亡率为71.1%±3.2%,并从试验感染鱼组织中检测到CEV病毒DNA。养成期鲤鱼CEV携带率为77.5%。以上结果表明,天津地区养殖鲤鱼中存在CEV感染,揭示了我国鲤鱼养殖产业可能面临新的疫情,需引起养殖业的高度重视。     

The nested structure of Arctic thermohaline intrusions

Arctic thermohaline intrusions have a “nested” temperature/salinity structure characterized by the lining up of widely separated profiles along a series of well-defined lines in the T–S plane. The nesting pattern is coherent across much of the Arctic Basin (>2000 km), and roughly 90% of the water column between 150 and 350 m depth is found to lie along these nesting lines. We propose the nested structure results from a type of slanted convection occurring within the thick, salt-finger stratified layers in the intrusions. The convection cells are reminiscent of those investigated in laboratory experiments done by Thorpe, Hutt, and Soulsby, and it is estimated that the cells have horizontal dimensions ranging from 50 to 100 km at depths close to the Atlantic Water temperature maximum. Simple theoretical ideas suggest the convective cells may appear when the intrusions reach a critical amplitude, driving them toward a nested configuration. Our analysis provides a new estimate of the effective lateral diffusivity due to the intrusions (≃50 m² s⁻¹), as well as an estimate of the vertical diffusivity near the core of the Atlantic Water layer (≃0.8×10⁻⁵ m² s⁻¹).     

Catching a wave? A case study on incorporating storm protection benefits into Habitat Equivalency Analysis

Since Hurricane Sandy, there has been heightened attention to increasing the resilience of coastal communities to extreme events, including storm protection provided by coastal ecosystems. Storm protection benefits (SPB) are the ability of ecosystems, including wetlands, reefs, and beaches/dunes, to attenuate waves and storm surge. SPB are a topic of growing interest in the scientific and policy spheres, including discussions of how to incorporate SPB into existing policies. As an engine for restoration and a leading mechanism for the evaluation of ecosystem services, Natural Resource Damage Assessment (NRDA) provides a platform for better internalizing this and other services into decision-making particularly using Habitat Equivalency Analysis (HEA). HEA does not explicitly account for impacts to ecosystem services that flow from one habitat to an adjacent one. This study examines a hypothetical case study of an oil spill that impacts a marsh with resulting impacts on SPB to the adjacent upland forest. To more fully assess these impacts, a “nested HEA” was developed which accounts for cross-habitat ecosystem service flows. The nested HEA captures the impacts of the marsh loss on the forest due to wave and saltwater intrusion that would not be captured by a traditional HEA. By adapting the HEA approach with a nested HEA, NRDA could quantify direct ecosystem services losses as well as additional cross-service flows between habitats. However, additional data are needed in order to perform a nested HEA, and in the case of SPB, location-specific data likely will be needed to appropriately specify the model.     

Ecosystem-based marine management in European regional seas calls for nested governance structures and coordination—A policy brief

Marine governance in European seas is at a crossroad aiming towards implementation of eco-system based marine management (EBMM) through integration of different EU policies or directives to protect the environment, while at the same time expected to facilitate growth and employment in support of the blue economy. This article shows that the governance landscape at the regional sea level is very complex, fragmented and faced with several dilemmas. It examines the present governance structures in the four European seas (Baltic, Black, and Mediterranean Seas and North East Atlantic Ocean). It is argued that the implementation of EBMM at the regional sea level is characterized by a highly fragmented European governance system where there is lack of coordination between relevant DGs within the European Commission, between EU, International organisations, Regional Sea Conventions and the Member States and between sectoral governance arrangements that should provide sectoral management measures that support EBMM. The article develops suggestions for a nested governance system in which institutions, policies, laws and sectors are nested into a tiered, internally consistent and mutually re-enforcing planning and decision-making system. Developing institutional interaction and soft modes of governance between the EU, the Regional Sea Conventions, Member States and the governance arrangements of the different marine sectors will be crucial in evolving towards such a nested governance system for EBMM. Moreover, there is no one size fits all approach in implementing EBMM, which means that for each European Sea a context-dependent nested governance system should be developed.