微卫星DNA标记对乌苏里江哲罗鱼遗传多样性的分析

从网上查询获得鳟微卫星标记30对，并对乌苏里江哲罗鱼的基因组进行遗传多样性分析，结果筛选出10对具多态性的微卫星标记。并用其中6对微卫星引物对17尾乌苏里江哲罗鱼进行基因组DNA扫描检测。用2％的琼脂糖凝胶电泳检测微卫星标记的PCR扩增产物，并对扩增结果进行了统计分析，计算了6个基因座的等位基因频率、杂合度等。结果表明，乌苏里江哲罗鱼等位基因频率为0．0455～0．7857，多态信息含量(PIC)为0．2801～0．6351，杂合度(H)为0．3368～0．6563。统计结果初步表明乌苏里江哲罗鱼的遗传多样性程度处于中等偏下水平。同时，结合资源量下降的事实，建议在保护遗传多样性的前提下对其采取人工繁殖和放流的方法增加其种群数量。     

虹鳟、山女鳟及其杂交子代(虹鳟♀×山女鳟♂)的微卫星分析

利用虹鳟(♀)和山女鳟(♂)进行种间杂交,获得了90.00%的受精率,80.52%的发眼率,90.68%的孵化率和30.68%的鱼苗成活率.运用13个微卫星分子标记对杂交亲本与杂交子代进行了分子遗传机制的研究,结果表明:(1) 在13个微卫星位点中,3个位点只在虹鳟中得到扩增产物,6个位点扩增出虹鳟和山女鳟清晰的差异条带,另外4个位点在双亲中没有扩增出显著差异条带;(2) 双亲遗传分化显著,虹鳟和山女鳟存在杂交现象,虹鳟和山女鳟杂交子代的遗传符合孟德尔遗传规律,属两性融合生殖,是真正意义上的杂交种. (3) 杂交后代与虹鳟和山女鳟的遗传相似性系数分别为0.461 7和0.596 5,遗传距离分别为0.772 9和0.516 8, 表明杂交F1与两亲本的遗传差异不是对等的, 而是偏向父本一方,UPGMA系统树也同样证明了这一点.     

虹鳟、山女鳟及其杂交子代（虹鳟♀×山女鳟♂）的微卫星分析

摘 要：利用虹鳟（♀）和山女鳟（♂）进行种间杂交,获得了90.00%的受精率,80.52%的发眼率,90.68%的孵化率和30.68%的鱼苗成活率。运用13个微卫星分子标记对杂交亲本与杂交子代进行了分子遗传机制的研究,结果表明：(1) 在13个微卫星位点中,3个位点只在虹鳟中得到扩增产物,6个位点扩增出虹鳟和山女鳟清晰的差异条带,另外4个位点在双亲中没有扩增出显著差异条带;(2) 双亲遗传分化显著,虹鳟和山女鳟存在杂交现象,虹鳟和山女鳟杂交子代的遗传符合孟德尔遗传规律,属两性融合生殖,是真正意义上的杂交种。 (3) 杂交后代与虹鳟和山女鳟的遗传相似性系数分别为0.4617和0.5965,遗传距离分别为0.7729和0.5168, 表明杂交F1与两亲本的遗传差异不是对等的, 而是偏向父本一方,UPGMA系统树也同样证明了这一点。本研究结果将为虹鳟、山女鳟两种鱼的杂交育种工作的进一步开展提供依据。     

微卫星标记在坛紫菜丝状体品系DNA指纹构建中的应用

用从条斑紫菜EST数据库中筛选合成的微卫星引物对8个坛紫菜丝状体品系进行微卫星DNA指纹扩增。5个微卫星引物共扩增出32条带,其中3对引物所扩增出的5个条带(AU192094—127、AU187410—335、AU187410—190、AU194267—203和AU194267—328)被用来构建8个坛紫菜丝状体品系的DNA指纹。在这个图谱中,每个丝状体品系都有独一的指纹模式,彼此很容易被区分开。所获得的DNA指纹图谱,可用来进行孟德尔分离研究,及为坛紫菜纯系鉴定提供分子基础。     

哲罗鱼胰岛素样生长因子-II的原核表达与活性分析

在鱼类胚胎发育过程中,胰岛素样生长因子-II(insulin-like growth factors-II,IGF-II)起着关键的作用。本研究根据Gen Bank收录的鲑鳟鱼IGF-II基因序列设计引物,以哲罗鱼(Hucho taimen)肝RNA提取物为模板,利用RT-PCR方法扩增出哲罗鱼IGF-II基因开放阅读框。将目的基因IGF-II与原核表达载体p SUMO连接构建出重组表达载体p SUMO-IGF。将该重组质粒转化到大肠杆菌Rosetta中进行目的蛋白的诱导表达。SDS-PAGE电泳分析显示,约在40 k D处含有清晰的条带,与预期结果相符;目的蛋白以包涵体形式存在。对包涵体进行变性/复性后获得较纯的目的蛋白,利用ELISA和MTT方法对目的蛋白进行免疫学活性及生物学活性分析。ELISA结果显示该目的蛋白能够与商品化的抗鲑鳟鱼IGF-II的抗体发生特异性反应,并且呈现抗原浓度依赖性,该结果说明本研究获得了具有良好免疫原性的IGF-II蛋白;MTT方法测定IGF-II蛋白对鲤(Cyprinus carpio)上皮细胞(epitheliaoma papulosum cyprini,EPC)和虹鳟(Oncorhynchus mykiss)性腺细胞(rainbow trout gonad,RTG-2)的增殖效果来鉴定IGF-II蛋白的生物学活性,结果显示所表达的哲罗鱼IGF-II蛋白能够有效的刺激EPC细胞和RTG-2细胞增殖。该结果表明利用原核表达系统获得的哲罗鱼IGF-II蛋白具有良好的生物学活性。本研究为哲罗鱼的生长模式和生长繁殖的研究奠定了基础。     

施氏鲟、西伯利亚鲟、达氏鳇及其杂交种种质鉴定方法的建立及其应用

以施氏鲟（Huso dauricus）为研究材料,分别从线粒体基因组及核基因组两个层面进行物种分子鉴定方法研究。在线粒体基因组层面,对3种鲟及未知鲟种类共计119个样品的D-Loop区进行测序,通过同源序列比对,构建NJ进化树、计算群体间遗传距离,以鉴定其中30尾未知种类。在核基因组层面,利用15对微卫星标记扩增3种鲟DNA,筛选出特异性标记Ls19和SX226。Ls19在施氏鲟中扩增出特异条带126 bp、130 bp,在西伯利亚鲟中扩增出特异条带139 bp、143 bp,在达氏鳇中扩增出特异条带124 bp、127 bp;SX226在施氏鲟中扩增出特异条带185 bp,在西伯利亚鲟中扩增出特异条带260 bp、273 bp、283 bp,在达氏鳇中扩增出特异条带180 bp、182 bp。通过特异条带对未知鲟进行鉴定,结果显示：30尾未知鲟种类中,有西伯利亚鲟17尾,施氏鲟1尾,达氏鳇1尾,达氏鳇×施氏鲟2尾,施氏鲟×达氏鳇1尾,施氏鲟×西伯利亚鲟8尾。结果表明,特异性微卫星引物Ls19和SX226可以应用于施氏鲟、西伯利亚鲟、达氏鳇的纯种及杂交种分子水平种质鉴定。     

牙鲆基因组 (CAG)n微卫星 DNA 特征分析

通过磁珠富集法筛选牙鲆(Paralichthys olivaceus)的微卫星分子标记,采用限制性内切酶Sau 3A Ⅰ对牙鲆完整基因组DNA进行酶切;通过蔗糖溶液梯度离心,收集400～900 bp大小的片段,连接Brown接头,构建牙鲆基因组文库.用生物素标记的微卫星探针(CAG)15,对基因组文库进行杂交,利用磁珠富集含有微卫星的DNA单链序列,并对其进行PCR扩增;将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星序列文库.利用大量质粒检测法进行二次筛选,成功地从牙鲆基因组中分离出含有CAG重复的微卫星序列,测序其中的3000个单菌落,获得2805个(占93.5%)含有微卫星序列的克隆,其中含有微卫星座位3120个,完美型1808个,占57.97%;非完美型226个,占7.25%;混合型1085个,占34.78%.从中选出186个微卫星序列设计120对引物并合成,经过筛选,74对引物可扩增清晰条带,其中68对呈多态性.     

湖鲟微卫星引物在三种鲟鱼及杂交子代的通用性研究

利用12对湖鲟(Acipenser fulvescens Rafinesque)微卫星引物对小体鲟(Acipenser ruthenus Linnaeus)、施氏鲟(Acipenser schrenckii Brandt)、西伯利亚鲟(Acipenserbaeri Brandt)及三种杂交鲟进行种间的通用性研究.结果表明:12对湖鲟微卫星引物在六种鲟鱼中有较高的通用性,有6对引物(50%)在6种鲟鱼中扩增出PCR产物,但仅有4对在6种鲟鱼中得到清晰可辨且有多态性的条带,可以直接用来分析几种鲟鱼的遗传多样性和进行杂交鲟的辅助育种工作.其中,位点LS-68在三种纯种鲟鱼中的等位基因大小范围上有明显差异,可作为三种纯种鲟鱼的分子标记位点;位点AfuG112在除施氏鲟外的其他鲟鱼中都得到特异扩增产物,可以用于检测施氏鲟群体的纯度(施氏鲟无带,怎么能用来检测纯度),人工辅助育种的品种选择;位点AfuG122仅在三种杂交鲟中得到清晰可辨的条带,可以作为区分杂交种与纯种的分子标记位点.     

大西洋鳕微卫星引物对太平洋鳕的适用性

利用Genbank中大西洋鳕的7对微卫星引物,对太平洋鳕基因组DNA进行PCR扩增。这7对引物均可较稳定地扩增出条带,它们分别是Gmo2、Gmo3、Gmo8、Gmo19、Gmo35、Gmo36、Gmo37,并且扩增出的7个位点均表现出多态性,共获得69个等位基因,每个位点的等位基因数目为4～17,大小为111～227bp。在7个位点上2种鱼的等位基因的大小存在着不同程度的差异。其中Gmo37在大西洋鳕和太平洋鳕中扩增出的条带的长度差异最大,基本无重合部分,分别为220～290bp和144～226bp,本研究结果以期对2种鳕科鱼类遗传结构的进一步研究提供帮助。     

鲮微卫星引物的鉴定及其适用性检验

利用4种鲤科鱼类的14对微卫星引物对西江流域一批野生鲮进行PCR扩增。将各扩增条带进行克隆测序,发现引物MFW1、MFW2、MFW15、MFW17、Cc7、Cc11、Bgon22扩增出的产物含有微卫星重复序列。进一步对鲮的微卫星位点MFW1、MFW2、Cc7重新设计引物,并将其分别命名为Cm1、Cm2、Cm3,新引物对鲮的扩增特异性增强。采用引物Cm1、Cm2、Cm3、Cc11、MFW15、MFW17、Bgon22对西江流域一批野生鲮进行引物适用性检验。结果表明,除引物MFW15、Cc11无多态性外,其余5对引物(Cm1、Cm2、Cm3、MFW17、Bgon22)在取样群体中扩增图谱带型丰富,随引物不同,各标记在群体中检测到的等位基因数为2到16个。各微卫星座位的期望杂合度(He)及观察杂合度(Ho)范围分别为0~0.9038和0~1,平均分别为0.6881(0.1819SD)和0.7772(0.1931SD)。座位连锁分析显示Cm1与Bgon22之间存在显著性水平连锁关系(P<0.05),其余各座位之间未检测到明显的连锁关系(P>0.05)。研究群体的遗传多样性指数平均为0.6823,多态性水平相对较高。以上结果表明,筛选获得的7个微卫星座位适于对鲮进行遗传多样性分析。     

2003年天津及周边地区南美白对虾病毒性疾病的检测

采用随机取样临床观察和实验室PCR检验技术相结合的方法检测天津及周边地区养殖的南美白对虾类白斑杆状病毒WSBV(或WSSV)[1]和桃拉病毒TSV的感染情况。检测WSBV215例,检出率为31 2%,4月份幼体、仔虾阶段,检出率最高为80 95%,其次为7月份仔虾和成虾养殖期,检出率为58 33%。检测TSV67例,检出率为0%。目前天津地区南美白对虾养殖中流行的主要病毒病为由WSBV感染引发的白斑综合症。因此,加强苗种检疫,切断病毒传播途径是非常必要的。     

常见水产品中生物胺的调查及分析

为研究不同水产品中生物胺的产生情况,利用高效液相色谱-柱后衍生-荧光检测技术对蓝点马鲛、黄鳍金枪鱼、银鲑、牙鲆、中国明对虾、鹰爪虾、中华绒螯蟹、杂色蛤、杂色鲍中的7种生物胺含量进行调查与分析。试验结果表明,鲜活水产品中几乎不含生物胺,或仅含少量的精胺、亚精胺;腐败水产品中检出大量的生物胺,其中蓝点马鲛、黄鳍金枪鱼、银鲑、牙鲆中的组胺含量分别为422.57、640.00、309.63、151.86mg/kg,而虾、蟹、贝类等无脊椎动物几乎不产生组胺。不同水产品在腐败过程中产生的生物胺种类、含量、比例不同。生物胺总量可作为水产品腐败变质程度的指标。     

熟制与贮藏对凡纳滨对虾挥发性成分的影响

为研究熟制与贮藏对凡纳滨对虾挥发性成分的影响,采用电子鼻及固相微萃取—气相色谱—质谱联用技术分析虾肉的挥发性成分,并采用相对气味活度值和感官评定法评价虾肉风味的变化。结果显示,熟制与贮藏对虾肉中的挥发性成分影响显著。凡纳滨对虾生虾、熟虾、熟虾冷却、熟虾冷却过夜及二次熟制虾分别被检出50、68、63、49和41种挥发性成分。二次熟制后,虾肉中的挥发性成分有所减少,感官上虾的特征鲜香气也略微减弱;一次熟制对虾肉的风味影响不大。生虾肉中检测出的挥发性成分如壬醛和癸醛等对风味有显著性影响;熟虾肉中检测出壬醛、癸醛和十四醛等;二次熟制的虾肉中检测出对风味影响较大的挥发性成分主要是十八醛、二丁基羟基甲苯等。熟虾经过夜(4°C)放置后,其中检测出的挥发性成分种类和总量均减少,但总量差异不显著。研究表明,熟制可显著增加挥发性风味成分种类和总量,二次熟制与一次熟制相比,凡纳滨对虾的关键风味物质种类和含量显著降低;熟虾冷却后4°C贮藏12 h风味差异不显著。     

白斑症病毒在日本对虾体内的感染增殖

用投喂患白斑症病毒病的虾组织人工感染日本对虾稚虾,每日取样,整虾冰冻切片,单克隆抗体的荧光抗体方法,原位观察病毒在虾体内的感染增殖,结果表明：感染后三天内,在感染虾的各组织器官内均未观察到明显的病毒感染的阳性细胞,每四天首先在鳃丝腔内的小量血细胞观察到病毒感染;第五天除血细胞外同时在血窦,鳃上皮组织,皮下组织内观察到,第六天进而在心脏,胃上皮组织内观察到：第七天进一步又在淋巴器官,中肠内观察到,八     

Herpesvirus cyprini: a search for viral genome in infected fish by infected fish by in situ hybridization

Abstract. Herpesvirus cyprini (CHV) genome was traced in carp, Cyprinus carpio L., after acute infection by the method of in situ hybridization with biotinylated probes. The viral genome was detected in several tissues including cranial nerve ganglia. Subcutaneous tissue and spinal nerves. However, at this stage, viral antigens were not detected and the virus was not isolated. The viral genome was also detected in the same fish tissues when papillomas were present which contained viral antigens and even infective virus particles. After papilloma regression, the viral genome was still detected in these tissues. It is suggested that CHV becomes latently established in cranial nerve ganglia, subcutaneous tissue and spinal nerves, and is associated with the induction and recurrence of papillomas.     

黄鳝质量安全风险分析

黄鳝(Monopterus albus)养殖产业近年发展较快,已在中国18个省、自治区、直辖市进行养殖,连续10年产量超过30万t。黄鳝养殖方式主要为池塘网箱养殖,其特殊的生态养殖方式对水环境污染小,影响黄鳝产品质量安全的原因主要为养殖环境及投入品。养殖环境中的重金属、农药残留等通过食物链及其他途径影响黄鳝产品质量。在人工养殖环境下,黄鳝体内重金属未超标,而野生环境下的黄鳝,尤其是黄鳝苗,其体内重金属超标风险较高。通过对黄鳝体内农药残留进行调查,主要检测了敌敌畏、氟虫腈、五氯酚钠和林丹4种农药,从检测结果看,黄鳝体内这4种农药的残留情况总体良好,敌敌畏和氟虫腈都未检出,林丹和五氯酚钠仅在个别样品有检出;对黄鳝配合饲料的检测未发现重金属超标,未检测到雌二醇、己烯雌酚和甲基睾酮等性激素;研究发现,作为动物饲料源的水蚯蚓和蚯蚓可能是黄鳝体内砷的重要来源,螺等鲜活饵料可能是黄鳝其他重金属的重要来源;渔用药物中,防治寄生虫药物阿苯达唑在投喂养殖期间的黄鳝体内有一定比例检出并超标,呋喃类药物、氯霉素,氟苯尼考、环丙沙星、恩诺沙星和磺胺类等有部分检出,但含量未超标。通过近10年的抽样调查,没有发现违禁药物与激素在黄鳝体内超标,近10年黄鳝质量安全是稳定的。     

Characterization of rainbow trout, Oncorhynchus mykiss (Walbaum), serum creatine kinase isoenzymes and isoforms by means of electrophoresis and isoelectric focusing

Serum samples were obtained from rainbow trout, Oncorhynchus mykiss (Walbaum), from a commercial fish farm. Creatine kinase (CK; EC 2.7.3.2) isoenzymes and sub-bands (isoforms) were studied using agarose gel electrophoresis and polyacrylamide gel isoelectric focusing. Creatine kinase isoenzymes and isoforms (sub-bands) were found to display disparate electrophoretic mobility and isoelectric points, respectively, when compared with CK patterns of human origin. The CK-BB isoenzyme, rarely detected in human serum, was detected in all trout samples assayed. Up to eight CKMM isoforms were detected by the polyacrylamide gel IEF method described.     

Analysis of germ cell proliferation, apoptosis, and androgenesis in the Lake Van fish (Chalcalburnus tarichi) during testicular development

In the present study, the testis histology, gonadosomatic index (GSI), germ cell proliferation and apoptosis, and the plasma 11-ketotestosterone (11-KT) and testosterone (T) levels of male Chalcalburnus tarichi were analyzed. According to the histological examinations of the specimens that were caught between February 2009 and January 2010, three testicular stages were determined. Those stages were as follows: (1) recrudescence or prespawning (July–April), (2) spawning (May–June), and (3) postspawning (July). It was observed that the GSI increased gradually, starting from the recrudescence stage, and it reached peak values at the spawning stage, while the lowest values were in the postspawning. Germ cell proliferation in the testis was detected using a proliferating cell nuclear antigen (PCNA), and germ cell apoptosis was detected by transferase dUTP nick end labeling staining. The germ cell PCNA and apoptosis index values were calculated. It was indicated that germ cell proliferation was observed in all of the testicular stages. The highest germ cell PCNA index (PI) levels were detected in July, August, and September, which then dropped in October and stabilized between February and April. The lowest PI values were detected in the spawning stage (May–June). Germ cell apoptosis was observed in all of the months, and the highest apoptotic index values were detected in August, September, October, May, and June. Plasma 11-KT and T levels were at their highest levels in May and June, and it was detected as stabile in the other months. There was a correlation between GSI, PI, and plasma androgen levels. In conclusion, the present data illustrate testicular development stages for C. tarichi and show changes in the level of GSI and sex steroid biosynthesis through spermatogenesis.     

Movements of immature hatchery-reared Mekong giant catfish <Emphasis Type="Italic">Pangasianodon gigas</Emphasis> released in the Mekong River,measured using acoustic telemetry

Twenty-eight immature hatchery-reared Mekong giant catfish Pangasianodon gigas tagged with acoustic transmitters were released in the Mekong River, Thailand from 2002 to 2004. Twenty-four and four fish were tagged with normal transmitters and pressure-sensitive transmitters, respectively. Five to seven automated monitoring receivers were used for monitoring the post-release movements. The tagged catfish could be detected for up to 97 days, the first detection taking place at the release point, where the fish remained for several days. Sixteen tagged fish (57%) were not detected at any later point. These fish may have passed along the opposite (Laos) side of the river without notice because the width of the river was larger than the detection range of the transmitter. The remaining 12 tagged fish (43%) could be detected by the receivers installed, excluding the release point receiver. Of these 12 tagged fish, six showed long-distance (30–80 km) upstream movements and one long-distance (50 km) downstream movement. These seven fish (25%) were detected only during the daytime, suggesting that the Mekong giant catfish is diurnal.     

Fish mycobacteriosis: morphopathological and immunocytochemical aspects

Abstract. A morphopathological study of fish tuberculosis was carried out using histological and immunocytochemical techniques. The clinical signs observed were emaciation, lack of movement coordination and body deformation. Macroscopically, small, light-coloured, visceral nodules were detected. Immunocytochemical techniques were found to be effective in detecting infections where conventional staining methods failed to reveal, or detected only small numbers of bacilli.