用团头鲂精子诱导金鱼雌核发育研究

本文用紫外灭活的团头鲂(Megalobrama amblycephala)精子激活金鱼 (Carassius auratus Goldfish)卵子,用0－4℃冷水冷休克处理卵子使其染色体加倍,得到成活的雌核发育金鱼。使用与金鱼不同亚科的团头鲂精子做为激活源能极大提高雌核发育后代的鉴定效率,只需依据外形特征、染色体数目和性腺发育程度,就能容易地将雌核发育金鱼和与团头鲂杂交后代区分开。雌核发育金鱼有两种体色不同的后代,但都为双尾,体形似金鱼,染色体数目为2n=100,全雌,性腺发育正常;而杂交后代为单尾,体形似鲫鱼,染色体数目为3n=124,性腺发育滞后。本实验为证明金鱼的性别决定方式为XX/XY型提供了细胞遗传学证据。得到两种体色皆不同于母本体色的后代,体色不同可能是基因座位纯化导致后代性状分化,也可能是异精效应导致。     

人工诱导白鲢（hypophthalmichthys molitrix）雌核发育及性转

为了获得白鲢自交品系,以解决该鱼种退化问题,本文用失活鲤鱼精子诱导白鲢雌核发育,再用17α－甲基睾丸激素促使其发生性别转换。经紫外线照射过的锂鱼精子,遗传物质遭到破坏。用这种失活的鲤鱼精子与白链卵“受精”,精子不参与卵发育,但能激发卵的发育,得到单倍体“受精卵”。这种单倍体“受精卵”自身不能成活,要在41℃条件下热处理3分钟,当单倍体复制成二倍体以后才能继续发育。当雌核发育的鱼长到10cm时,将含有17α－甲基睾丸激素的Silastic管插入其腹腔中。激素通过管壁逐渐向外释放,影响鱼体性发育。待实验鱼性腺与对照白鲢一样都发育至第Ⅱ期时,可以观察到：实验鱼性腺发生了退化 现象（Fig.1)。制备血清,做等电聚焦电泳可以观察到：实验鱼的同功酶谱与白鲢鱼的相同,而不同于鲤鱼（Fig.2)。排除鲤鱼精子参与受精的可能。     

异源精子诱导的罗非鱼人工雌核发育

异源精子诱导的罗非鱼人工雌核发育潘锋,胡玫,王和海（中国水产科学研究院淡水渔业研究中心,无锡２１４０８１）ＡＲＴＩＦＩＣＩＡＬＩＮＤＵＣＥＤＧＹＮＯＧＥＮＥＳＩＳＢＹＡＬＬＯＧＬＮＯＵＳＳＰＥＲＭＩＮＴＩＬＡＰＩＡ（Ｏｒｅｏｃｈｒｏｍｉａｕｒｅｕｓ）...     

人工诱导南方鲇雌核发育的最适参数

用紫外线照射南方鲇(Silurus meridionalis)精子,用遗传失活的精子人工授精,采用热休克方法抑制南方鲇第二极体的排出。根据热休克起始时间、持续时间和休克温度三因子三水平设计正交试验,探索获得人工诱导南方鲇雌核发育的最理想条件。结果表明,紫外线照射精子15 min,热休克起始时间为受精后5 min,持续时间为1 min,休克温度为41℃,对人工诱导南方鲇雌核发育最有利。该实验为进一步分析人工繁殖南方鲇的雌化机制以及南方鲇性别决定的分子机制奠定了基础。     

人工诱导泥鳅雌核发育及人工转性的研究

泥鳅(Misgurnus anguillicaudatus)肉味鲜美,营养价值高,是市场上畅销的特种水产品之一。但泥鳅生活周期短,一定程度上限制了群体生长。       

大黄鱼人工诱导雌核发育后代的微卫星标记分析

通过冷休克法诱导2组大黄鱼雌核发育,并采用6对微卫星标记引物 （C7、C13、C27、C44、D19、F27）对雌核发育家系G1、G2中24尾鱼苗及双亲进行PCR扩增,并进行微卫星标记比较分析。结果表明：冷休克诱导雌核发育二倍体获得了35.3%的孵化率和45日龄9.9%的成活率;有4对引物可扩增出清晰的亲本差异条带,其中引物C44、C27扩增结果显示G1子代中均未出现雄鱼基因,全部为雌核发育产物,C27扩增结果显示G2子代中也未出现雄鱼基因,但D19扩增结果显示G2子代中有3尾出现雄鱼基因,属正常受精个体,两家系后代的基因纯合率分别达93.8%和72.9%,平均为83.3%。研究表明雌核发育是促进基因纯合的一个有效途径,微卫星标记技术是是鱼类雌核发育鉴定和遗传分析的有效方法。     

异源精子诱导稀有鮈鲫的人工雌核发育

用经过紫外线灭活的异源精子启动稀有鮈鲫卵子发育,再经过热休克分别抑制极体排放和第一次卵裂,得到了极体雌核发育和有丝分裂雌核发育的存活个体。通过正交试验确定诱导极体雌核发育的最佳参数为受精后2min、40℃休克处理2min;诱导有丝分裂雌核发育的最佳参数为受精后17min、40℃休克处理2min。雌核发育个体的形态学特征没有显示出受到父本影响的迹象。RAPD分析表明雌核发育个体扩增片段全部来自于母本,没有发现异源父本DNA成分进入稀有鮈鲫基因组的迹象。实验中还发现,鲤×稀有鮈鲫的正常杂交组合能以极低的几率产生出雌核发育的稀有鮈鲫。     

人工诱导泥鳅雌核发育的细胞学研究和诱导参数优化

为培育泥鳅(Misgurnus anguillicaudatus)全雌品种,研究通过细胞荧光染色后进行细胞学观察确定人工诱导泥鳅雌核发育的热休克起始时间,并进一步利用核型和流式细胞分析等方法对人工诱导雌核发育泥鳅进行鉴定。结果显示,二倍体泥鳅受到灭活的鲤(Cyprinus carpio)精子刺激后,第二极体与卵核分开的时间在人工授精后3—5min;卵子在人工授精后3.5min后再热休克2min的诱导孵化率达到10.26%。通过分析野生型泥鳅胚胎、人工诱导雌核发育泥鳅胚胎、泥鳅×鲤杂交胚胎和单倍体泥鳅胚胎的发育,发现部分雌核发育的胚胎可以正常发育,而杂交胚胎和单倍体胚胎会出现明显的发育障碍。核型和流式细胞检测分析表明利用本研究获得的热休克参数进行处理可得到二倍体雌核发育子代,二倍率达64.71%。研究从细胞学层面获得泥鳅雌核发育的诱导参数,可为利用人工诱导雌核发育开展全雌泥鳅育种提供指导。     

热休克诱导虹鳟二倍体雌核发育

本文采用温度休克的方法进行了虹鳟二倍体雌核发育试验。发眼率为１２．６６％,孵化率为０．２１％,获得雌核发育二倍体虹鳟鱼苗１７尾。试验结果表明,只要处理方法得当就可以获得二倍体雌核发育的虹鳟幼苗。     

人工诱导栉孔扇贝雌核发育后代的微卫星标记分析

用紫外线灭活栉孔扇贝精子和6-DMAP处理受精卵抑制第二极体,获得A、B、C 3组雌核发育栉孔扇贝。采用筛选获得的5对多态性微卫星引物对3个家系雌核发育组30个个体、双亲及对照组40个个体进行了遗传变异分析。结果显示：3个家系雌核发育组后代中均有部分个体出现父本基因,表明精子遗传物质失活不彻底,雌核发育组中存在正常受精个体;在具多态性的5个基因座位上均发生了基因-着丝点之间的重组,重组率在26.7%—55.0%之间,表明通过抑制第二极体排出获得的栉孔扇贝雌核发育后代在个体和群体水平上具有一定的基因杂合;3个家系后代的平均基因纯合率为59.1%,而其对照组家系为2.5%,雌核发育使基因的纯合率提高了56.6%。研究表明雌核发育是促进基因纯合的一个有效途径,微卫星标记技术是贝类雌核发育鉴定和遗传分析的有效方法。     

Induction of Gynogenesis in Japanese Crucian Carp (Carassius cuvieri)

Diploid gynogenesis was induced in Japanese crucian carp (Carassius cuvieri) eggs using UV-irradiated genetically inactive spermatozoa from mirror carp (Cyprinus carpio L.) or blunt snout bream (Megalobrama amblycephala), with or without cold shock. The optimal radiation dosage was 4200 mJ/cm² and 3600 mJ/cm² for mirror carp and blunt snout bream sperm, respectively. At this dosage and without cold shock, the yields were (32.4±3.3)% vs. (33.8±1.4)% gynogenetic haploids and (0.7±0.3)% vs. (0.5±0.3)% hybrid diploids, respectively. At the optimal UV dosage but with cold shock (2 min after fertilization, 0-4°C for 40 min), the hatching rates were (27.8±2.1)% and (29.4±3.3)%, respectively. From hatching to feeding, (15.7±3.4)% and (23.6±4.1)% normal gynogenetic diploids were recorded, respectively. Survival of normal gynogenetic diploids was 56% out of the hatched fry when using irradiated spermatozoa of mirror carp, which was lower than that (up to 80%) when using irradiated spermatozoa of blunt snout bream. This indicated that the sperm of blunt snout bream, with distant genetic relation to the maternal Japanese crucian carp, was more effective than that of mirror carp to induce diploid gynogenesis. The nature of the gynogenetic progeny was identified with external appearance, chromosome number and gonad structure. The presence of only females in gynogenetic progeny probably suggested XX genotype in the female Japanese crucian carp. The gynogenetic diploids have potential values such as faster growth and stronger disease resistance than the normal Japanese crucian carp. All gynogenetic progeny possessed 100 chromosomes whereas all J x B crosses were triploid with 124 chromosomes. The formation of the new triploid hybrids in J x B crosses may be useful in aquaculture.     

日本白鲫IGF-1基因全长cDNA克隆及组织表达分析

通过已获得的多个物种IGF-1基因的cDNA序列,设计合成简并引物,用日本白鲫的肝脏总RNA反转录获得的cDNA做模板,克隆获得了IGF-1中间序列.在此基础上,通过SMART RACE,获得了IGF-1全长cDNA序列.经过序列对比分析,所得到的序列是日本白鲫(Carassius cuvieri)IGF-1 cDNA全长,其中开放阅读框为486 bp,编码含161个氨基酸的蛋白质,该蛋白质含有保守的信号肽、B、C、A、D和E结构域和6个半胱氨酸残基.同时,系统进化分析表明,在进化过程中IGF-1基因在鱼类中保持着高度保守的进化特征.IGF-1在日本白鲫中广泛表达于多个组织,尤其在肝脏中表达量最高.在垂体、心脏、肾脏和肌肉中有较高的表达.而在脑、脾脏、精巢和卵巢中的表达量较低.     

彭泽鲫两个雌核发育克隆的染色体组型分析

采用PHA和秋水仙素体内注射法直接制作肾细胞染色体标本,对彭泽鲫种群内两个不同雌核发育克隆的亲本进行染色体数目及组型分析。结果表明,彭泽鲫种群内的两个不同克隆存在染色体数目及组型差异,其中克隆H包含6条超数染色体在内的染色体众数是156,150条基本染色体的组型公式为：42M 36SM 39ST 33T,NF=228;克隆L包含12条超数染色体在内的染色体众数是162,150条基本染色体的组型公式为：36M 45SM 33ST 36T,NF=231。两个不同雌核发育克隆的发现及其染色体的差异说明彭泽鲫种群内同样存在着类似银鲫种群内的遗传多样性。     

Genetic Diversity in Crucian Carp (Carassius auratus)

A survey of restriction fragment polymorphism in mitochondrial DNA of three subspecies of Carassius auratus throughout four provinces in China was undertaken using 17 restriction enzymes. Two carp, Cyprinus carpio rubbrofuscus and Cyprinus carpio carpio, were included as the outgroup. A total of 16 haplotypes was observed: 5 in tetraploids of C. auratus auratus; 8 in hexaploids of C. auratus auratus; and 2 in C. auratus gibelio and C. auratus cuvieri, respectively. The tetraploids and hexaploids share three common haplotypes as I, V, and VI. C. a. Cuvieri may have diverged first among the three subspecies. Interestingly, C. a. auratus and C. a. cuvieri did not form monophyletic clades, which indicated that the classification of Carassius auratus required further studies. The current hypothesis, that hexaploids originated from tetraploids by a polyploidy event, is less favorable, based on the distribution of haplotypes and the lower diversity in tetraploids than in hexaploids. Our data also indicate that divergence of hexaploids and tetraploids might be recent and mtDNA polymorphism existed before the divergence. Meanwhile, genetic isolation exists between the hexaploids and the tetraploids.     

彭泽鲫雌核发育的细胞学研究

通过对彭泽鲫♀×彭泽鲫♂、彭泽鲫♀×尖鳍鲤♂“杂交”后代的细胞学观察发现 ,同源与异源精子在彭泽鲫卵中的发育情况基本一致。精子入卵后一般呈固缩状态 ,不解凝 ,不形成雄性原核 ,但都能看到未解凝精核与雌性原核接触 (未融合 )的现象。刚产出的成熟卵子没有看到极体 ,但受精 1 0分钟有极体排出 ,是卵子发育排出的惟一极体。根据观察结果 ,彭泽鲫在细胞学上表现出典型的雌核发育行为特征 ,是以雌核发育方式繁殖的种群     

A Set of Polymorphic Trinucleotide and Tetranucleotide Microsatellite Markers for Silver Crucian Carp (Carassius auratus gibelio) and Cross-Amplification in Crucian Carp

Silver crucian carp (Carassius auratus gibelio Bloch), as a gynogenetic fish, is a promising model for the study of the evolutionary genetics of vertebrates. We have developed 59 polymorphic trinucleotide and tetranucleotide markers for the silver crucian carp through the biotin capture method and radioactive-labeling hybridization. The number of alleles ranged from 2 to 12 in the population, and the average proportion of heterozygotes (including tri- and diallelic) at polymorphic loci was 76.8%. In addition, these loci were successfully applied to a close relative, the crucian carp (Carassius auratus), by cross-amplification, as shown by the range of alleles (2–19), observed heterozygosity (0.1765–0.9706), expected heterozygosity (0.2392–0.9421), and polymorphism information content (0.2186–0.9236).     

市售鲫鱼肠道中沙门菌的调查研究

目的 为摸清市场中出售鲫鱼沙门菌的污染情况.方法 以徐州市某市场上出售的鲫鱼为材料,主要采用SS琼脂平板对120份肠道样品中的沙门菌进行初步鉴定和培养计数.结果 在120份鲫鱼肠道中共检出沙门菌32份,阳性率为26.7％ (32/120),菌群数量分布在(0.32 ～9.12)×103 CFU/g.结论 本研究对徐州地区市售鱼类沙门菌检疫及疾病防治等提供重要参考.     

雌核发育银鲫与两性生殖彩鲫卵壳蛋白组分的比较分析及其受精前后的变化

以雌核发育银鲫和两性生殖彩鲫的成熟卵为材料,分离卵壳,经处理得到卵壳可溶性蛋白组分。SDS－PAGE梯度凝胶电泳分析在雌核发育银鲫中揭示出3条较明显的差异蛋白带。同时,采用相同处理方法对受精前后卵壳蛋白组分进行比较分析后发现,这些差异蛋白带在受精后发生了变化,其带纹表现为减弱或消失,表明这些差异蛋白可能与受精过程相关。 Abstracts:Egg chorions were isolated from unfertilized and fertilized eggs of gynogenetic silver crucian carp and gonochoristic color crucian carp by homogenization and further purification techniques.Then,soluble proteins were extracted from the isolated egg chorions,and were analyzed by gradient SDS-PAGE.Three differential protein bands were revealed between the gynogenetic silver crucian carp and gonochoristic color crucian carp.Furthermore,these differential proteins were demonstrated to undergo obvious changes during fertilization.It was suggested that these differential proteins should be related to the special fertilization process in gynogenetic silver crucian carp.     

Genetic Diversity among Different Clones of the Gynogenetic Silver Crucian Carp, Carassius auratus gibelio, Revealed by Transferrin and Isozyme Markers

Genetic diversity among four clones (A, D, E, F) of gynogenetic silver crucian carp was studied using transferrin and isozymes in the blood as markers. Of the five proteins investigated, three (transferrin, esterase and superoxide dismutase) indicated polymorphism and eight polymorphic loci were detected. These loci were probably encoded by codominant alleles and their inheritance patterns were analyzed. Intraclonal homogeneity and interclonal heterogeneity were observed in these clones, which allowed us to infer the clonal nature and evolutionary relationship between them. Clonal diversity in this population of silver crucian carp in China was also compared with data reported from gynogenetic crucian carp in Germany.     

Induction of Tetraploid Gynogenesis in the European Sea Bass (Dicentrarchus labrax L.)

A preliminary study on tetraploid gynogenetic induction in the European sea bass was performed by pressure-blocking the second polar body release and the first cleavage in eggs fertilized with ultraviolet-irradiated sperm. Fertilization of eggs with genetically inactivated sperm produced only haploid development that terminated around hatching. Pressure treatments (8.500 psi for 2 min) applied at 6 and 65 min after fertilization (a.f.) produced variable levels (7–95%) of tetraploid larvae at hatching. A small proportion of mosaics (3.8n/4.2n) was also recorded.