栉孔扇贝血细胞活性检测方法的筛选与应用

筛选栉孔扇贝(Chalmys farreri)血细胞活性的检测方法,研究苯并[a]芘(B[α]P)对栉孔扇贝血细胞活性的影响.结果表明96孔培养板中栉孔扇贝血细胞接种密度在(1～32)×10~4 cell/孔时,血细胞活性无显著性差异(P>0.05),且细胞存活率在97.42%～98.82%之间,台盼蓝排斥法、四甲基偶氮唑(MTT)、二甲氧唑黄(XTT)比色法、乳酸脱氢酶(LDH)活性测定法均可作为栉孔扇贝血细胞活性的测定方法,而当血细胞接种密度在(12～32)×10~4/孔时,中性红比色法亦可作为血细胞活性的检测方法;同时实验采用Eagle培养液进行血细胞原代培养在0～24 h内血细胞活性较好.采用中性红比色法检测B[α]P对栉孔扇贝血细胞活性具有显著抑制作用,且抑制程度与B[α]P作用浓度、时间呈正相关,其余方法均未检测出B[α]P对血细胞活性的影响.由此可见,B[α]P对栉孔扇贝血细胞活性具有免疫毒性作用,中性红比色法可以作为评价B[α]P对栉孔扇贝血细胞免疫活性的敏感指标.     

4种养殖扇贝的群体遗传多样性及特异性AFLP标记研究

应用AFLP技术对我国4种主要养殖扇贝进行了遗传分析.虾夷扇贝、海湾扇贝、华贵栉孔扇贝、栉孔扇贝4种养殖扇贝群体中,种内遗传相似度依次分别为0.841 5,0.786 3,0.719 0和0.673 1,香侬氏指数分别为43.52,58.87,80.16和92.83,栉孔扇贝和华贵栉孔扇贝的遗传多样性水平明显高于海湾扇贝和虾夷扇贝;AFLP标记共享位点分布表明,在4种扇贝中栉孔扇贝与华贵栉孔扇贝之间的共享位点最多,同时4种扇贝的种间遗传相似度也以栉孔扇贝和华贵栉孔扇贝之间为最高(0.769 5),海湾扇贝和虾夷扇贝之间为最低(0.662 6),揭示所研究的几种扇贝中栉孔扇贝和华贵栉孔扇贝的遗传关系最近(遗传距离为0.262 1);找到了21个种内特异性AFLP标记和种间共享标记,可用于种质鉴定和分子辅助分类.     

专家为扇贝养殖持续发展开出处方

自1997年以来,我国北方沿海栉孔扇贝相继发生大面积死亡事件。据了解,仅山东省经济损失就近40亿元,尤其是视栉孔扇贝为衣食父母的长岛县,1997年和1998年两年直接经济损失近10亿元,严重影响了长岛县人民的切身利益。栉孔扇贝的大量死亡,也对整个山东省沿海扇贝养殖业可持续发展提出了新的课题。在     

氨海水与5-羟色胺对栉孔扇贝解剖卵的体外促熟作用

研究氨海水与5-羟色胺(5-HT)对栉孔扇贝解剖获得卵母细胞的促熟作用。通过体外浸泡的方法处理栉孔扇贝卵母细胞使其成熟,并完成人工受精。结果表明一定浓度的氨海水可显著促使栉孔扇贝卵母细胞生发泡破裂,但经过氨处理的卵母细胞受精率却不高;5-羟色胺不能诱导栉孔扇贝卵母细胞生发泡破裂,但可有效地使已经发生生发泡破裂的卵母细胞进一步成熟。先用氨海水处理然后用5-羟色胺处理可以得到最佳诱导效果,受精率可达(40.02±2.73)%。     

栉孔扇贝、虾夷扇贝及其杂交子代线粒体COI和Cytb基因遗传多样性分析

为了解栉孔扇贝(Chlamys farreri)、虾夷扇贝(Patinopecten yessoensis)及其杂交子代(F1)的种群遗传多样性和遗传结构,对3个群体的线粒体COI和Cytb基因的部分序列进行了扩增和分析。经比对分别获得781bp和725bp核苷酸片段,74个样本共检测到47个单倍型;F1群体的单倍型数、单倍型多样性指数、核苷酸多样性及平均核苷酸差异数都是最高的,而双亲的较低;F1和栉孔扇贝间的遗传距离最小,其次为栉孔扇贝和虾夷扇贝群体之间,F1和虾夷扇贝群体之间的遗传距离最大;F1和栉孔扇贝之间的遗传分化系数较小而两者间的基因流比较大,F1和栉孔扇贝与虾夷扇贝之间的遗传分化系数较大而基因流较小,说明栉孔扇贝和虾夷扇贝群体群体很早就发生了遗传分化;采用UPGMA法和简化的中介网络法构建的系统树表明,3个群体的所有个体被分为2个族群,栉孔扇贝和F1交叉聚为一类,虾夷扇贝独自聚为一类,2个分支间没有交叉;使用特异性引物分别对3个群体进行PCR扩增检验,结果栉孔扇贝的特异引物能在杂交子代中扩增,而虾夷扇贝的特异引物不能在子代中扩增出条带,说明栉孔扇贝和虾夷扇贝杂交,其子代的线粒体遗传模式为严格的母系遗传。本研究结果表明杂种优势的形成与线粒体遗传多样性的变化有关。     

栉孔扇贝养殖病害原因及其防治措施

栉孔扇贝筏式养殖在山东省长岛已有21年的历史。1983～1993年连续出现扇贝大量死亡的现象,累计2万多亩受灾,造成经济损失达3亿多元。因此,迅速查明栉孔扇贝死亡的原因便成为扇贝养殖业的重要课题。 1 栉孔扇贝死亡原因 1.高温期进行分苗,使贝苗受到高温刺激,是引起扇贝大量死亡的重要原因之一。 2.由于栉孔扇贝在分苗养成初期挂的水层偏浅,而在牡蛎繁殖高峰期内扇贝放养的水层又没有下调到深水层来避开牡蛎苗的附着水     

栉孔扇贝三倍体与二倍体的生长比较

近几年在我国栉孔扇贝养殖业中出现了单产降低、品质下降、大面积死亡问题 ,其主要原因是缺少生长快、品质优、抗逆能力强的养殖新品种。对于养殖贝类来说 ,性成熟往往导致生长抑制、抗逆性下降和肉质下降。目前广泛开展的多倍体贝类研究的主要目的是产生不育的三倍体 ,充分利用三倍体的不育性和生长优势 ,为海水养殖业健康、稳定、持续地发展提供技术支持。通过对栉孔扇贝三倍体和二倍体的生长比较 ,证实栉孔扇贝三倍体具养殖实用价值。１材料与方法１．１材料栉孔扇贝Ｃｈｌａｍｙｓ(Ａｚｕｍａｐｅｃｔｅｎ) ｆａｒｒｅｒｉ取自青岛太平…     

栉孔扇贝(Chlamys farreri)与华贵栉孔扇贝(C.nobilis)远缘杂交子代的胚胎发生及幼虫生长发育的初步研究

采用单一自由度独立比较的方法,研究了两个栉-华杂交组合HCH(栉孔扇贝长岛群体×华贵栉孔扇贝)和HJH(栉孔扇贝胶南群体×华贵栉孔扇贝)以及栉孔扇贝种内交配组合ZZ的胚胎发生及幼虫生长发育过程。结果表明,栉孔扇贝和华贵栉孔扇贝的种间杂交F1在生长和发育的两个环节上是不一致的,在涉及器官及其功能形成的发育环节上,杂交表现负效应;而在涉及形态变化和增大的生长环节上,杂交表现正效应,存在杂种优势现象。两个杂交组合中,HJH综合效果较好,授精22h平均孵化率达到95.8%,与ZZ基本接近;30日龄平均变态率达61.5%,明显好于组合HCH的42.0%;8日龄幼虫平均大小达143.4μm×123.2μm,表现出更强的生长优势。这些结果为全面评价栉孔扇贝与华贵栉孔扇贝杂交体系应用价值和选配优良杂交组合提供了理论依据。     

栉孔扇贝体内寄生的病毒的分离纯化及其形态学观察

栉孔扇贝(Chlamys farreri)是我国传统海水养殖贝种,养殖范围主要分布于黄海、渤海沿岸的山东、河北、辽宁等地,但是,近年来因病害发生,造成了巨大的经济损失.扇贝大规模死亡流行病学调查研究已进行多年,流行病学调查、病理学、病原感染实验及病原分离纯化等研究初步认为类立克次体是扇贝(包括栉孔扇贝和海湾扇贝Argopecten irradians)的病原生物[1～4].在用泛影葡胺密度梯度离心纯化栉孔扇贝类立克次体时发现一种具囊膜的病毒粒子,本文首次报道栉孔扇贝体内该球形病毒纯化的形态学特征.     

栉孔扇贝热休克蛋白70（HSP70）基因的BAC-FISH 定位

利用BAC-FISH技术,将包含HSP70基因的BAC克隆定位到栉孔扇贝的一对同源染色体的长臂上.HSP70基因的染色体定位将对深入研究该基因的结构及功能并将其应用于生产实践提供基础支持.同时,本研究是首次对栉孔扇贝低拷贝基因进行染色体定位的实践,其结果将为栉孔扇贝的染色体的深入研究以及染色体鉴别等工作提供必要的参考.     

Cadmium speciation and transport in the blood of the bivalve Mytilus edulis.

Cadmium (Cd) speciation in the blood plasma of Mytilus edulis was investigated using the metal speciation model MINTEQA2. In the presence of inorganic ions alone, Cd-chloro complexes dominated the speciation (97% of total Cd), with 3% as Cd2+. Inclusion of a novel Cd-binding histidine-rich glycoprotein (HRG) purified from mussel blood plasma decreased the contribution of chloro-complexes to 11.9%, with 86.8% of the Cd bound to the HRG and 1.3% present as Cd2+. Cd transfer from the blood plasma to the kidneys in vivo was studied by injecting 109Cd (both with and without additional chelation) into mussels. Oxine and EDTA complexed a significant amount of blood-borne Cd (23.7% Cd Oxine; 57.1% CdEDTA). In the presence of each chelator, plasma retained significantly more Cd, although there was no significant difference in Cd uptake by tissues (kidney, gill-mantle, and remaining viscera).     

灰树花糖蛋白中总糖含量的测定

探讨糖蛋白中总糖含量测定的简捷方法,以2种灰树花糖蛋白(GF6A,GF6B)为原料,分别以硫酸-苯酚法和次亚碘酸钠法对其中的总糖含量进行了比较分析。结果显示,应用前者方法测定多糖含量分别为85.45%和38.46%,采用后者方法测定结果分别为87.03%,36.51%,2种方法测定的结果相近,其中以硫酸-苯酚法因不需对样品进行预水解,方法简便、可靠,更适合于糖蛋白中总糖含量测定。     

PHA体外诱导草鱼白细胞产生γ-干扰素的研究

于1997年10月－1999年3月，采用半数细胞病变抑制等方法，进行植物凝集素（Phytoagglutinin,PHA)体外诱导草鱼白细胞产生γ－干扰素的研究，结果表明，在PHA诱导的草鱼白细胞培养液中出现了一种抗病毒因子，经理化和生物学性质鉴定证明，该因子是一种干扰素活性物质。但它不同于病毒诱导的α／β－干扰素，主要表现为：对56℃、pH＝2和0．1％SDS敏感；其活性不能被病毒诱导的干扰素抗体所中和；其诱生剂为PHA，来源于白细胞；在诱导条件下，佛波酯（PMA）和白细胞介素－2（IL－2）能显著地促进其诱生，这些特性与人类和高等脊椎动物中报道的γ－干扰素相符，表明是一种γ性质的干扰素。     

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.     

Effects of hexachlorobenzene and pentachlorophenol on cellular and humoral immune parameters in Glycera dibranchiata

The effects of exposure to two model marine pollutants, hexachlorobenzene (HCB) and pentachlorophenol (PCP) on components of the defense system of a polychaete, Glycera dibranchiata, were determined. Although both pollutants were markedly concentrated in the tissues, the levels reached in these studies produced no mortality or morbidity. Certain parameters (in vitro phagocytosis by amebocytes, total and differential coelomocyte counts, hemolysin, hemagglutinin and bacterial agglutinin activity) were not affected by our experimental procedures. However, the activity of a bactericidal glycoprotein was consistently induced in PCP- or HCB-exposed Glycera. We suggest that the release of this factor from coelomocytes is increased by a mechanism similar to that of macrophage activation, a characteristic of the leukocytes of higher animals. Exposure to PCP caused an apparent reduction in the numbers of amebocytes capable of forming rosettes with formaldehyde-treated rabbit erythrocytes. This was interpreted as an impairment in the ability of the cells to recognize foreign material, a process required for the normal functioning of these potentially protective blood phagocytes.     

鱼类甲状腺轴对胚胎发育、仔鱼变态及性别分化的调控作用研究进展

鱼类甲状腺轴由下丘脑、垂体、甲状腺组成,下丘脑通过分泌促甲状腺激素释放激素、促肾上腺皮质激素释放激素调节垂体促甲状腺激素的合成和分泌,促甲状腺激素促进甲状腺滤泡合成并释放甲状腺激素(主要为甲状腺素—T4),T4运输至肝脏等外周组织中,经过脱碘转化形成生物活性更强的三碘甲腺原氨酸(T3),T3主要通过同核受体结合发挥生物学功能。甲状腺激素对鱼类胚胎发育、仔鱼变态及性别分化等早期生长发育过程具有重要调控作用:甲状腺激素为鱼类胚胎发育期所必须,外加适量甲状腺激素能够提高胚胎或仔鱼存活率;仔鱼变态过程中,某些外部形态变化及相关组织分化依赖甲状腺激素的调控,牙鲆等仔鱼变态期体内甲状腺激素水平及其受体表达量会显著升高;性腺发育期,甲状腺激素对调控鱼类性别分化方向可能具有一定作用。     

Enhanced extracellular enzymatic peptide hydrolysis in the sea-surface microlayer

The accumulation of dissolved organic matter (DOM) at the air–sea interface is controlled by dynamic physical processes at the boundary between ocean and atmosphere. Much of the DOM concentrated in the surface microlayer is thought to be protein or glycoprotein. Enzymatic hydrolysis of these and other biopolymers is an important step in the microbial uptake of dissolved and particulate organic matter in many aquatic environments. We employed a sensitive fluorescence technique to investigate differences between extracellular enzymatic peptide hydrolysis in the sea surface microlayer and corresponding subsurface water from Stony Brook Harbor, NY. We separated the microlayer from its underlying water and thus measured hydrolysis potential rather than an in-situ process. Peptide turnover was always faster in the microlayer than in subsurface waters. This was confirmed by allowing a new surface film to form on subsurface water; hydrolysis was still faster in the new surface film. In a year-long study, we found the relative difference between turnover times in the surface film and subsurface waters to vary greatly with season. While rate constants of peptide hydrolysis were generally higher in both microlayer and bulk water samples in spring/summer than in fall/winter, the difference in activity between the two environments was greatest in winter. Enhanced hydrolysis in the sea surface microlayer is likely due to the greater concentrations of DOM in the microlayer. Seasonal changes in distribution of hydrolytic activity between surface film and subsurface water probably reflect seasonal variation in the mechanisms of DOM enrichment, which depend on water temperature, substance and energy fluxes across the water–air boundary, activity of aquatic organisms and other seasonal variables.     

Metallothionein-like proteins induced by cadmium stress in the scallop Mizuhopecten yessoensis

Organisms have evolved a cellular response called stress protein response that increases their tolerance in adverse environmental conditions. Well known stress proteins that bind essential and toxic metals are metallothionein (MT). The scallop Mizuhopecten yessoensis is the most interesting organism because it is able to accumulate toxic cadmium in its digestive gland. However, in the tissue of the digestive gland of Mizuhopecten yessoensis MT (metallothioneins) have not been found. Eastern scallops, Mizuhopecten yessoensis, were collected from two locations ?? one clean and one polluted site. The concentrations of cadmium (Cd), copper (Cu) and zinc (Zn) were measured in the digestive gland. There was a significant increase in Cd concentrations in this studied tissue. We found that in the presence of cadmium Mizuhopecten yessoensis can induce high molecular proteins. The results of experiments have shown that Cd-binding ligands have a number of properties similar to MT: acetone and temperature stability; the ability to bind some metals, including Cd, Cu and Zn. Protein chromatography (FPLC, Superosa 12) from the digestive gland of scallop M. yessoensis has shown that cadmium is associated with high molecular weight Cd-binding proteins (72 kDa and 43 kDa). The major cadmium-binding protein 72 kDa is glycoprotein. In experiments we have demonstrated that Cd-binding proteins can be induced when there is cadmium exposure. The results of this study strongly suggest that the far eastern scallop Mizuhopecten yessoensis has a unique and well-developed system for the detoxification of heavy metals and it allows for biochemical systems to be maintained in a relatively stable manner in the presence of heavy metals.     

Presence of carcinoembryonic antigen in hepatic neoplasms of Rivulus ocellatus marmoratus

Carcinoembryonic antigen (CEA), a glycoprotein, is found in the embryonic gut and liver, and is in malignant neoplasms of the intestine and liver of humans. CEA is used as a diagnostic marker for liver and gut neoplasms. The objective of this preliminary study was to determine the usefulness of CEA as a diagnostic marker in liver neoplasms of the mangrove rivulus, Rivulus ocellatus marmoratus, and to compare the results to those reported in mammals. A commercially-available polyclonal antibody kit, Autoprobe III (Fisher Scientific, Houston, Texas), which utilizes avidin-biotinperoxidase complex was used to stain CEA. Diethylnitrosamine-induced neoplasms and control livers from a previous study were used. These paraffin sections were stored at room temperature for at least six years. Both Bouin's-fixed and formalin-fixed tissues were used. A positive control (1-day-old mangrove rivulus larvae), and a negative control (omission of CEA antibody) were also used. CEA was found as red deposits in the gut mucosa, epidermis, and the brush boarder and tubular epithelium of the kidney of the 1-day-old-fish, control fish and the fish that had liver neoplasms. No CEA was found in the negative controls, the livers of postive controls and the normal adult livers. The liver tumors that had characteristics of biliary neoplasms stained positively for CEA, but the hepatocellular neoplasms were negative. The presence of CEA in the gut mucosa and the biliary neoplasms of fish is similar to the results of similar studies of human tissues.     

The inventory and chemical characterization of dissolved proteins in oceanic waters

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high resolution two-dimensional electrophoresis (2-DE) were applied to separate protein molecules in dissolved organic matter (DOM) from oceanic waters. Results were: (1) The 2-DE distinguished a total of 412 protein spots in 10 samples from five water columns over the Pacific, although fewer than 30 proteins were resolved as bands from the identical samples by SDS-PAGE. (2) Major and ubiquitous protein bands (34 and 39 kDa proteins) on the SDS-PAGE gel were resolved into horizontally spread arrays (trains) of spots on the 2-DE gels, indicating that these bands were a mixture of protein species that have the same molecular weight (MW) but different isoelectric points (pIs). (3) Proteins that exhibited such electrophoretic patterns on the 2-DE gels were glycosylated with variable linkages between the sugar and polypeptide chains. (4) N-terminal amino-acid sequencing demonstrated that individual spots within each train of spots had identical N-terminal amino-acid sequences.The N-terminal amino-acid sequences of the 39 and 34 kDa glycoprotein spots in samples collected at different sites were also identical. Protein isoforms with the same amino-acid sequence but different glycosylation profiles, termed glycoforms, were often observed on the 2-DE gel. Thirty-one and 24 spots on the 2-DE gels were glycoforms of two glycoproteins with MWs of 39 and 34 kDa, respectively; they were one protein species. The glycoforms of the 39 kDa protein were identified as a low molecular weight alkaline phosphatase (L-AP) of Pseudomonas aeruginosa PAO1 by a homology search through five amino-acid sequence databases. The present and earlier work indicates that all identified source organisms of dissolved proteins belong to the Pseudomonas group. We propose the hypothesis that proteins associated with membrane vesicles liberated from a minor member of the bacterioplankton assemblage, the marine Pseudomonas group, are one of the important sources of dissolved proteins in oceanic waters. This hypothesis may apply to the source pathway and survival not only of proteins and also to the universally occurring bacterial peptidoglycan and lipopolysaccharide components in DOM.