鲟鱼分子鉴定方法的整合应用

鲟形目物种是国家重点保护水生野生动物和CITES附录物种。其人工养殖种群数量众多, 种类丰富, 产品贸易量大, 但种类鉴定困难。本文在厘清当前鲟鱼商业类群的基础上, 通过分析现有种类鉴定方法, 整合了线粒体DNA遗传分析、SNP分析和微卫星DNA分析的鉴定方法, 探讨其鉴定国际贸易所涉鲟鱼的可行性。结果表明: 上述3种方法整合应用可在11种纯种鲟鱼及其正反杂交产生的杂交鲟范围内进行盲检。当前共有贸易鲟鱼36种, 其中杂交鲟14种, 杂交鲟的亲本共涉及9种鲟鱼。整合方法可准确鉴定小体鲟(Acipenser ruthenus)、达氏鳇(Huso dauricus)、施氏鲟(Acipenser schrenckii)、欧洲鳇(Huso huso)、闪光鲟(Acipenser stellatus)、高首鲟(A. transmontanus)两两杂交所产生的杂交鲟, 小体鲟为母本与纳氏鲟(Acipenser naccarii)或富氏鲟(A. fulvescens)或中华鲟(A. sinensis)产生的杂交鲟, 纯种的达氏鳇、高首鲟、富氏鲟和中华鲟, 但无法准确鉴定纯种的西伯利亚鲟(Acipenser baerii)和俄罗斯鲟(A. gueldenstaedti)以及父母本涉及此两种鲟鱼的杂交鲟。由于已开发的分子标记仍有限, 上述结果是对当前CITES贸易所涉鲟鱼鉴定的最大范围, 可以满足一些鲟鱼野生种群保护、贸易产品检测、种质资源管理等情景下的鉴定需求。     

养殖施氏鲟的性腺转录组特征分析

鲟是目前世界上最古老的软骨硬鳞鱼类之一, 雌雄个体之间无明显的第二性征。为了解人工养殖下鲟性腺发育的分子特征, 研究以人工养殖2龄施氏鲟(Acipenser schrenckii Brandt)为研究对象, 对其精巢与卵巢进行转录组测序分析。结果发现, 雌雄性腺中共有19690个差异表达基因转录本, 其中与性别分化相关基因包括转录因子Dmrt1、Sox9、Foxl2等和生长转化因子Amh、Bmp15、Gdf9等。另外, 通过差异表达基因KEGG代谢通路富集分析发现了4条与卵巢发育相关的通路, 分别为黄体酮介导的卵母细胞成熟、卵母细胞减数分裂、卵巢类固醇合成、促性腺激素释放激素信号通路。其中, 卵巢类固醇合成通路中18个差异表达基因的表达模式暗示了2龄施氏鲟限制卵巢雌激素的合成, 但精巢中雄激素的合成未受影响。研究结果为研究鲟性腺分化和发育机制以及今后在mRNA表达水平上鉴定鲟性别提供了基础。     

西伯利亚幼鲟对偶极子电场的避让行为研究

研究利用人工偶极子电场来模拟生物电场刺激, 对西伯利亚幼鲟的电感受能力进行了行为探究。结果显示西伯利亚幼鲟对本实验中的偶极子电场产生躲避行为, 其平均感受阈值在2月龄为(457.532.5) V/cm,3月龄为(29.52.5) V/cm, 7月龄为(101.0) V/cm。这表明西伯利亚鲟鱼的电感受敏感性随个体的发育而增强, 而这可能与电感受器官数量的增加有关(2月龄为2234470, 7月龄为5273523)。     

盐度对施氏鲟和西伯利亚鲟稚鱼的急性毒性

比较了盐度对施氏鲟(Acipenser schrenckii)和西伯利亚鲟(A. baerii)稚鱼的急性毒性效应.结果表明:施氏鲟稚鱼的96h盐度半致死浓度(LC50)为14.72,西伯利亚鲟为13.08;96h盐度LC50及盐度反应曲线显示,施氏鲟对盐度的耐受力大于西伯利亚鲟;在高盐度(≥14.70)下,2种鲟鱼依次表现出缓慢环游、狂躁环游、活动减弱、身体失衡和活动停止(死亡)等5种行为反应;西伯利亚鲟各行为反应出现的时间均早于施氏鲟,说明西伯利亚鲟对盐度的反应较施氏鲟敏感.     

5种养殖鲟鱼怀卵差异及周年繁殖

2007年11月至2009年11月通过对12批次(共计5 207尾个体)解剖观察,统计了5种性成熟鲟鱼[西伯利亚鲟(Acipenser baeri)、俄罗斯鲟(A.gueldenstaedti)、施氏鲟(A.schrencki)、黑龙江杂交鲟(Huso dauricus ♀×A.schrencki ♂)和欧洲杂交鲟(H...     

5种鲟鱼免疫球蛋白重链恒定区序列研究

为了探讨几种鲟鱼免疫球蛋白(IgM)所包含的信息与其亲缘和进化之间的关系, 分别对俄罗斯鲟(Acipenser. gueldenstaedtii)、小体鲟(A. ruthenus)、施氏鲟(A. schrenckii)、中华鲟(A. sinensis)和欧鳇(Huso huso)的IgM重链(IgH)恒定区进行了研究。采用RT-PCR的方法对IgH核酸序列进行了克隆, 通过软件获得了相应的IgH氨基酸序列。在分别对这5种鲟鱼免疫球蛋白重链恒定区4个区(CH1～CH4)进行研究后发现, 其CH4区氨基酸序列相似性最高。通过对CH4区序列氨基酸变异期望值(Kaa), 物种分化时间(T)及物种间系统进化树(Phylogenetic Tree)等参数的分析, 将克隆的5种鲟鱼IgH恒定区序列与已发表的西伯利亚鲟同源序列比对(源于NCBI序列)后发现: 西伯利亚鲟与俄罗斯鲟、施氏鲟与欧鳇、小体鲟各构成一个分支, 并与中华鲟相对。实验结果从体液免疫系统的演化这个角度, 反映了被研究的鲟鱼物种间的分类地位、地理分布及进化关系之间的联系。     

鲟鱼性别分化相关基因研究进展

鲟鱼是目前世界上最古老的鱼类之一,其肉质鲜美,营养丰富,鱼卵加工成的鱼子酱具有极高的经济价值。鲟鱼鱼子酱价格居高不下,因此养殖雌性鲟鱼比养殖雄鱼具有更高的经济前景。由于鲟鱼外形无法辨别雌雄,目前,通常借助超声波、内窥镜等仪器进行早期性别鉴定,但鉴别准确性无法保证。因此采用现代分子生物学技术探究鲟鱼性别决定及性别分化机制有助于早期鲟鱼性别鉴定技术的开发及实现鲟鱼全雌化苗种培育。总结了鱼类性别决定与性别分化相关基因的研究现状,综述了目前在鲟鱼上性别决定与性别分化相关基因的研究进展并预测了未来的研究方向及研究趋势。     

鲟鱼种质鉴定方法研究进展

鲟鱼是一类古老的大型经济鱼类,由于过度捕捞和人类对其生态环境的破坏及其鲟鱼自身具有的性成熟时间长、幼体成活率低等特点,使世界范围内的鲟鱼自然资源日趋枯竭。人工养殖鲟鱼已是鲟鱼制品的主要来源,但由于种质来源不清及大量杂交鲟的存在导致种质混乱情况比较严重,目前国内外均未有一套比较完善可行的鲟鱼种质鉴定体系。总结了目前进行鲟鱼种质鉴定的一些常用方法,分析了各个方法的优缺点,为确立一套稳定可靠的鲟鱼种质鉴定方法奠定基础。     

未产卵雌性黄鳝的性转变

为探讨产卵是否为雌性黄鳝(Monopterus albus Zuiew)性转变的必经过程, 研究分析了实验室内从受精卵或幼苗开始养殖至不同时间段的黄鳝性腺组织学状况, 采用性腺活检技术跟踪了34月龄雌性黄鳝性腺发育变化, 并以免疫组织化学方法探讨了黄鳝不同发育状态性腺中增殖细胞核抗原(PCNA)的分布。在养殖过程中, 实验黄鳝没有出现产卵现象或者繁殖行为。在5月龄黄鳝中, 间性占比13.3%, 雄性占比20.0%; 在12月龄(1龄)黄鳝中, 雄性占比17.6%; 34月龄(3龄)黄鳝中, 间性占比12.8%, 雄性占比8.5%。通过性腺活检技术对36条34月龄雌性黄鳝性腺发育变化进行了为期1个月的跟踪研究, 结果表明, 16.7%的雌性黄鳝发生了性转变, 性腺发育到间性阶段。黄鳝间性早期性腺生殖褶增厚, 部分细胞呈现明显PCNA免疫阳性, 包括间质细胞、精原细胞和初级精母细胞。上述结果提示, 产卵并非雌性黄鳝发生性转变的必经过程; 黄鳝性转变初期, 性腺结构变化包括生殖褶中间质细胞和精原细胞的发生和增殖。     

鲟鱼心外膜脓肿的病理学初步研究

作为新的养殖对象,我国各地先后开展了中华鲟(Acipenser sinensis Gray)、施氏鲟(A. schrenckii Brandt),以及引进的俄罗斯鲟(A. gueldenstaedti Brandt)、杂交鲟等鲟鱼人工养殖,由于养殖刚刚起步且养殖集约化程度较高等诸多因素,出现了多种新的非寄生性疾病,并造成大量死亡,经济损失极其严重,成为当前鲟鱼试养阶段的制约因素。1999年北京市一家鲟鱼养殖试验场鲟鱼患病,经检验和诊断,确诊为“鲟鱼心外膜脓肿”。本文报道了该病的病理解剖和组织病理学研究结果,为疾病的临床检验与诊断及有效控制提供科学依据。1 材料与方法1.1 …     

MRI在喉癌术前分期中的临床应用价值

目的:探讨MRI在喉癌术前诊断、分期中的临床应用价值。方法:对114例行电子喉镜检查并经病理学证实为喉癌的患者行术前MRI扫描,根据图像资料判断肿瘤侵及范围及判断有无淋巴结转移;同时进行术前分期、分型,并与术后病理分期、分型对照研究。结果:术前MRI T1期27例,其中25例经病理证实为T1期,2例为T2期,准确率为92.6%;术前MRI T2期39例,其中经病理证实35例为T2期,3例T1期,1例T3期,准确率为89.7%;术前MRI T3期29例,其中经病理证实25例为T3期,4例T2期,准确率为86.2%;术前MRI T4期17例,其中经病理证实15例为T4期,2例T3期,准确率为88.2%;MRI术前T分期总准确率为87.7%。N1期准确率为81.8%,N2期准确率为94.1%。结论:MRI图像能很好地显示喉癌肿块的侵及范围及淋巴结转移等,对喉癌的术前分期、分型及制定合理的手术方案具有指导意义。     

R法遗传参数估值置信区间的计算方法和重复估计次数的研究

探讨R法遗传参数估值置信区间的计算方法和重复估计次数(NORE)对参数估值的影响,利用4种模型通过模拟产生数据集。基础群中公、母畜数分别为200和2000头,BLUP育种值选择5个世代。利用多变量乘法迭代(MMI)法,结合先决条件的共扼梯度(PCG)法求解混合模型方程组估计方差组分。用经典方法、Box-Cox变换后的经典方法和自助法计算参数估值的均数、标准误和置信区间。结果表明,重复估计次数较多时,3种方法均可;重复估计次数较少时,建议使用自助法。简单模型下需要较少的重复估计,但对于复杂模型则需要较多的重复估计。随模型中随机效应数的增加,直接遗传力高估。随着PCG和MMI轮次的增大,参数估值表现出低估的趋势。     

壳聚糖载体的改性及其用于固定化漆酶的研究

利用有机酸改性壳聚糖,并用交联法制备酸化的壳聚糖载体,然后用改性壳聚糖载体固定漆酶。甲酸、乙酸改性壳聚糖的最适条件:壳聚糖与甲酸、乙酸的质量摩尔比(g/mol)分别为100∶1、100∶1.5,戊二醛的质量分数为1%,缓冲溶液的pH分别是4.4、5.0,反应时间为3h;壳聚糖与酒石酸、草酸的质量摩尔比(g/mol)分别为100∶0.5、100∶2,戊二醛的质量分数为2%,缓冲溶液的pH分别是3.6、4.2,反应时间为4.5h。不同有机酸改性的壳聚糖用于漆酶的固定,其酶活都有不同程度的提高,尤其用酒石酸改性的壳聚糖载体效果最好,其酶活提高了57%。     

The application of immunoperoxidase staining for the detection of causative fungi in tissue specimens of mycosis I

This study was performed in order to identify the fungi of four species (Aspergillus fumigatus, Fusarium anthophilum, Candida albicans, Cryptococcus neoformans) in formalin-fixed, paraffin-embedded tissue sections by the indirect method of immunoperoxidase staining. Mature albino rabbits were immunized by formalin-killed organisms. The antibodies were prepared by precipitation at a 50% saturation of ammonium sulfate and were checked for cross-reactivities by Ouchterlony's double immunodiffusion and precipitin test. The immunoperoxidase staining was applied to the paraffin-embedded tissue sections of infected mice, human autopsy and biopsy specimens. Although each fungus was stained clearly the cell wall, cross-reactivities appeared among them, however it was possible to identify four fungi by absorption and dilution of the antisera.     

藏绵羊子宫内膜炎主要致病菌多重PCR检测方法的建立与评价

本研究旨在建立一种多重PCR方法检测青海藏绵羊子宫内膜炎主要的病原菌。首先,提取5种标准菌株基因组,筛选出特异性引物;然后以标准菌株的基因组为模板,建立多重PCR方法。用无菌棉拭子涂抹藏绵羊子宫,置于LB培养液中培养并编号,48 h后提取样品基因组。运用单一PCR法对600份样品基因组进行检测,记录阳性样品;再挑取单一PCR法检测的阳性样品进行多重PCR检测,再次记录阳性样品,通过计算两种检测方法的符合率验证多重PCR方法;随机挑出30份阳性样品,进行病原菌分离鉴定菌种种类。单一PCR检测的样品中,无乳链球菌感染比例占47.33%,大肠杆菌占34.83%,金黄色葡萄球菌占6.5%,未检出沙门氏菌和化脓隐秘杆菌;多重PCR检测的阳性样品中,无乳链球菌感染比例占45.50%,大肠杆菌占33.50%,金黄色葡萄球菌占6.5%;两种检测结果相比较,多重PCR检测出的符合率均高于95%;分离鉴定的病原菌与两种PCR方法检测出的菌种结果基本一致。成功建立了多重PCR方法并检测出引起青海藏绵羊子宫内膜炎的主要病原菌为无乳链球菌、大肠杆菌和金黄色葡萄球菌。     

Purification and Properties of Proteases from a Marine-psychrophilic Bacterium

Protease which was found in the culture fluid of Pseudomonas sp. No. 548 was fractionated into four components with protease activity by a two step chromatography using DEAE-cellulose. Each protease was further purified by gel filtration on Sephadex G-100 and/or G-75. The protease of Ia was obtained in crystalline form and was shown to be homogeneous by analysis with electrophoresis, while the other three enzymes were also highly purified. The enzymatic properties of the proteases were investigated. All of the four enzymes were inactivated by ethylene diamine tetraacetate. Proteases Ia, Ib, and IIb were inactivated by diisopropylfluorophosphate. The optimum activity of protease Ia was shown to be at pH 10.0, and that of the other enzymes were at pH 7.0 to 8.0. The proteases of Ia, Ib, and IIb were stabilized by calcium ion. The effect of temperature, pH, and metal ions on the activity of the enzyme were also investigated.     

Quantitative analysis of oligosaccharide structure of glycoproteins

A sensitive and quantitative method for the structural analysis of oligosaccharide was established for the glycoform analysis of glycoproteins. In this study,N-linked oligosaccharides of human IgG and bovine transferrin were analyzed for the evaluation of the method. Carbohydrate moiety of glycoprotein was released by hydrazinolysis and purified by paper chromatography. The oligosaccharides were labeled with a fluorescent dye, 2-aminobenzamide, for the enhancement of detection sensitivity. Sialylated (acidic) oligosaccharides were separated from neutral oligosaccharide by employing a strong anion-exchange column (MonoO) followed by the treatment with sialidase. Enzymatically desialyated fractions and neutral fractions of oligosaccharides were applied to normal-phase HPLC to resolve the peaks according to glucose unit (GU). The structure of separated molecules was further determined by sequential digestion with exoglycosidases. As a result, disialylated biantennary complextype oligo saccharide was found to be a major sugar chain in bovine transferrin (63%). In human IgG, core fucosylated asialobiantennary complex oligosaccharides were dominant. These results coincided well with reported results.     

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo.

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.     

Studies of the regulation of purine nucleotide catabolism.

Ehrlich ascites tumor cells containing radioactive ATP were incubated in vitro with a range of concentrations of 2-deoxyglucose in order to produce different rates of ATP catabolism. Concentrations of all radioactive products of ATP catabolism were measured, and apparent rates of adenylate deaminase and inosinate dehydrogenase and of adenylate and inosinate dephosphorylation were calculated. It was concluded that these processes were reggulated primarily by the rate of formation of substrate, and to a lesser extent in some cases, by substrate concentration. No evidence was obtained for regulation of these processes by the concentration of ATP. The deoxyglucose-induced catabolism of radioactive GTP was also studied. When ATP catabolism was induced by incubation with 2,4-dinitrophenol, time courses of accumulation of purine nucleoside monophosphates and rates of alternative pathways of their metabolism were quite different than when deoxyglucose was used.