绿色荧光蛋白基因向花鲈胚胎的转移及其表达

通过显微注射的方法,将绿色荧光蛋白(Green fluorescent protein,GFP)基因注射入花鲈(Lateolabrax japonicus)单细胞期受精卵动物极细胞质内。初步研究了显微注射后的花鲈胚胎的存活状况。在荧光显微镜下观察到了GFP的表达,多数胚胎表现为嵌合性表达。利用PCR技术,从花鲈尾芽期胚胎DNA中扩增出了特异片段,大小为750bp,表明显微注射胚胎中整合了外源GFP基因。     

鱼类生殖细胞移植技术的发展及应用前景

鱼类生殖细胞移植技术是通过诱导不同物种之间生殖系嵌合体来实现。原始生殖细胞和精原细胞(或卵原细胞)是诱导生殖细胞系嵌合体的关键材料。在过去的十多年中,通过采用不同的供体生殖细胞和不同发育阶段(囊胚期胚胎、初孵仔鱼和成鱼)的鱼为受体,开发出多种鱼类生殖细胞移植技术。这些成果的取得,为生殖细胞移植技术应用于诸多水产养殖新兴领域打下了坚实基础。该技术可以应用于:(1)生殖细胞生物学和转基因鱼等基础研究;(2)遗传资源和濒危鱼种的保护;(3)鱼类性别选择育种;(4)有效提高放流鱼苗的遗传多样性。     

鱼类原始生殖细胞发育与生殖操作技术研究进展

原始生殖细胞是胚胎发育过程中最早建立的一群生殖干细胞，是有性生殖动物生殖发育的基础。鱼类原始生殖细胞特化遵循“先成论”的模式，早期胚胎发育过程中获得母源生殖质组分的细胞特化为原始生殖细胞，特化形成的原始生殖细胞需要维持其生殖干细胞命运，并经过长距离迁移最终到达性腺原基的位置。原始生殖细胞特化、迁移和命运维持过程受到多种基因和信号通路的综合调控。研究鱼类原始生殖细胞发育不仅有助于深入理解脊椎动物细胞特化、迁移和命运维持等基本生物学过程的调控机理，而且是开发新的养殖鱼类生殖控制和生殖干细胞移植技术的重要基础。本文概述了鱼类原始生殖细胞发育的基础理论以及生殖操作技术研究进展，并展望了未来的发展趋势，以期为开发重要养殖鱼类优良种质创制新技术提供参考。     

动物转基因技术的研究现状与应用

转基因动物就是指通过人工的方法将外源基因导人动物染色体基因组,使之稳定表达并能遗传给后代的一类动物。转基因技术的一般方法有原核期胚胎的显微注射法(Microjection)、逆转录病毒载体法、精子载体法、体细胞核移植技术、胚胎干细胞介导的基因转移,这几种方法各有其公优缺点,在动物转基因上均有不同的应用,目前在动物抗病育种、建立诊断和治疗人类疾病的动物模型、利用转基因动物生产药用蛋白质等方面应用越来越广泛。     

类基因组研究概述

本文概述了近些年鱼类基因组研究的进展状况，鱼类基因组研究起步比较晚，但目前在鱼类遗传连锁图谱的构建、功能基因的筛选与克隆，胚胎干细胞和基因打靶技术的研究上已经取得很好的进展。具体研究在淡、海水鱼类中都有所体现，并且对与鱼类密切相关的藻类的基因组也进行了研究。     

鱼类生殖细胞移植的研究进展及应用前景

生殖细胞移植是指将供体的生殖细胞移植到同种或异种受体体内,供体生殖细胞嵌合到受体性腺,经过增殖、分化并最终发育为功能性配子的过程。作为辅助生殖技术,它不仅为珍稀濒危动物的繁育和保护提供了新途径,同时也为生殖干细胞的功能研究提供了有效手段。鱼类生殖细胞移植研究首先在模式鱼类斑马鱼中开展,经过十多年的发展,取得了一系列突破性的进展:主要包括先后建立了以胚胎、仔鱼和成鱼为受体的生殖细胞移植体系,精原和卵原干细胞的发现拓宽了供体生殖细胞的选择,受体的选择与制备方法的完善。该技术在缩短鱼类性成熟周期、性控育种、珍稀濒危鱼类保护等方面具有巨大的应用前景,已成功在多种淡水和海水鱼类中开展了研究和应用。本文结合作者的研究实践和经验,系统地梳理和总结了鱼类生殖细胞移植的研究进展,指出了该技术实践应用的关键问题,并探讨了其应用前景。     

不同因子对花鲈胚胎干细胞增殖的影响

胚胎干细胞(ES细胞)是从动物早期发育胚胎中分离出来的具有发育全能性的一种未分化细胞系。维持花鲈胚胎干细胞(LJES1)的体外生长、增殖及未分化状态需要在培养基中添加一些生长因子。实验通过配制省略1种或多种因子的培养基PESM0～10，计数LJES1细胞在各培养基中增殖的数量，确定各种生长因子对LJES1细胞的增殖作用。同时，对LIF因子和bFGF因子的作用进行了重点的研究，发现LIF因子对早期的LJES1细胞增殖几乎没有作用，其主要作用是维持LJES1细胞的未分化状态，但对晚期LJES1细胞的增殖有一定的作用；bFGF因子对LJES1细胞有强烈的刺激增殖的作用；鲈鱼胚胎抽提液(PEE)以及鲈鱼血清(FS)也促进了LJES1细胞的增殖，2-巯基乙醇(2-ME)具有还原血清中的含硫化合物、防止过氧化物对LJES1细胞的损害及促进贴壁的作用，因而也促进了LJES1细胞的增殖。     

花鲈大规格鱼种淡化培育试验

花鲈(Lateolabrax japonicus)，又称鲈子、海鲈、七星鲈鱼，属鲈形目、鮨科、花鲈属，是典型的凶猛肉食性鱼类。其肉质细嫩，味鲜美，是名贵经济鱼类之一，也是目前主要出口创汇的商品鱼之一。花鲈较耐低温，适温范围0℃～33℃，生长水温14℃-30℃，最适生长温度24℃～27℃；花鲈属广盐性鱼类，在0～38‰的盐度范围内均能生长。经驯化后能摄食配合饲料。     

黄河口地区淡水池塘花鲈养殖试验

正花鲈又称鲈鱼、海鲈等,主要分布于我国、日本、朝鲜半岛沿海。花鲈因其肉质鲜美,颇受普通消费者欢迎,是我国海水养殖的传统种类和淡水养殖的优质鱼类。由于花鲈对水温、盐度适应性强,目前已成为水产养殖业备受关注的品种之一。东营市淡水池塘养殖的鱼类多为普通淡水鱼,经济效益较低。通过实施花鲈淡水池塘养殖技术研究,开展淡化技术、越冬技术及混养技术,形成本地优势鱼类养殖品种,笔者在2014-2015年开展了相关试验,现将养殖技术总结如下。     

花鲈水库网箱养殖技术

花鲈（L．japonicusC．etv）又称鲈鱼、板鲈、七星鲈，属鲈形目，科，鲈属。花鲈广泛分布在我国沿海，是一种生活在近岸浅海中、下层，富有较高经济价值的广温、广盐性名贵鱼类。它适合在各种水体进行各种形式的养殖，具有生长速度快，肉质坚实，洁白细嫩，味道鲜美，营养丰富等特点，因而具有广阔的市场前景。江西省景德镇市于１９９９年开始在乐平民兵水库开展网箱养殖花鲈商品鱼，经过几年的探索已基本掌握花鲈水库网箱养殖技术。一、养殖场地选择花鲈属温水性鱼类，其生存水温为２℃～３４．５℃，最适水温为１５℃～２５℃，１０℃以…     

Embryonic stem cells in fish: current status and perspectives

Totipotent embryonic stem (ES) cells represent a bridge that links in vitro and in vivo manipulations of animal genomes and have enormous potential for genetic engineering of livestock. We have recently established feeder cell-free conditions for culturing cells of midblastula embryos (MBE) of the medaka (Oryzias latipes) and obtained several stable cell lines that show all features of mouse ES cells in vitro. One of these lines, MES1, has been demonstrated to retain a diploid karyotype and can be induced to differentiate into various cell types in vitro. Upon microinjection into albino host blastulae, MES1 cells are able to form pigmented chimeras. Genotype-specific PCR analysis revealed that 90% of host blastulae transplanted with MES1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. Transplantation of genetically labelled MES1 cells revealed a wide contribution to numerous organs derived from all three germ layers and differentiation into various types of functional cells. These ES properties of MES1 line was not abolished by stable gene transfer and long-term selection. Thus MES1 cells may represent a first promising cellular vehicle for the production of genetically modified fish. The genetic background has been found to have a profound effect on the efficacy of ES cell derivation and of chimera formation.     

Germ cell transplantation as a potential biotechnological approach to fish reproduction

Although the use of germ cell transplantation has been relatively well established in mammals, the technique has only been adapted for use in fish after entering the 2000s. During the last decade, several different approaches have been developed for germ cell transplantation in fish using recipients of various ages and life stages, such as blastula-stage embryos, newly hatched larvae and sexually mature specimens. As germ cells can develop into live organisms through maturation and fertilization processes, germ cell transplantation in fish has opened up new avenues of research in reproductive biotechnology and aquaculture. For instance, the use of xenotransplantation in fish has lead to advances in the conservation of endangered species and the production of commercially valuable fish using surrogated recipients. Further, this could also facilitate the engineering of transgenic fish. However, as is the case with mammals, knowledge regarding the basic biology and physiology of germline stem cells in fish remains incomplete, imposing a considerable limitation on the application of germ cell transplantation in fish. Furthering our understanding of germline stem cells would contribute significantly to advances regarding germ cell transplantation in fish.     

Biotechnology applied to fish reproduction: tools for conservation

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.     

受精卵不同发育时期导入外源基因时整合效率影响的研究

从1985年以来,世界各国先后应用不同外源基因导入虹鳟鱼、鲤鱼、鲶鱼等有经济价值的养殖鱼类。但受精卵发育到何时注基因源射外获得高的成活率(孵化率)和整合率一直是研究人员所关心的问题。法国的Chourout等在1986年就做了虹鳟鱼受精卵不同发育时期注射外源基因与孵化率关系实验。美国的Thoms.T.then等人也做了鲤鱼等受精卵在不同发育时期注射外源基因对其成活率、整合率影响的研究。由于外源基因对受体鱼的整合机理比较复杂,不仅这方面的研究不多,而且各实验室之间的研究结果也很不一致,因此至今对某种鱼类的受精印发育到何时是注射外源基因的最佳时期还没有形成一致的意见。本研究以鲤鱼受精卵为材料,通过显微注射技术,在受精卵的不同发育时期把鲤鱼MT启动因子基因,大麻哈鱼CH基因的融合基因导入受精卵中,按孵出的鱼苗数分别计算成活率,再通过斑点杂交和Southern Blot杂交检查整合情况,计算整合率,以探索外源基因导入鲤鱼受精卵的最佳时期。     

大菱鲆受精卵显微注射技术的建立

本研究将100~300 pg含有肌肉特异表达启动子和绿色荧光蛋白(Green fluorescent protein,GFP)基因的重组质粒(smyd1:gfp)显微注射到大菱鲆(Scophthalmus maximus)受精卵动物极细胞中,通过细心培育,成功孵化出鱼苗约120尾。统计分析显示,显微注射后,大菱鲆胚胎存活率为4.8%。利用荧光显微镜观察大菱鲆胚胎及仔鱼,只在注射smyd1:gfp质粒的胚胎及仔鱼的肌肉中发现有绿色荧光。通过进一步PCR扩增检测,在注射的大菱鲆胚胎及仔鱼DNA中扩增出了GFP特异片段,大小约为340 bp。研究表明,本研究成功建立了大菱鲆显微注射技术,可为大菱鲆基因功能研究和遗传育种奠定基础。     

Development of Microsatellite Markers in Sea Perch,Lateolabrax Japonicus,from Codominant Amplified Fragment Length Polymorphism Bands

A fast and cost‐effective protocol to develop candidate microsatellite markers from sea perch, Lateolabrax japonicus, was described here. Ten suites of codominant bands that contained seven microsatellites were discovered in this marine fish, in which no microsatellite development was reported previously. All the seven microsatellites were found to be polymorphic among tested 20 individuals of sea perch. Five of the above seven loci conformed to Hardy–Weinberg equilibrium (HWE) as determined by using the Markov‐Chain method implemented, the other two loci significantly deviated from HWE, both of them showed a large heterozygote excess (H_E). Out of 21 possible pair‐wise comparisons among the seven loci applied to sea perch, none showed significant linkage disequilibrium (LD) (P > 0.008). Five additional fish species assessed for cross‐species amplification revealed that some of the loci appear to be applicable in close‐related species.     

人工鱼礁生态诱集技术的机理及研究进展

人工鱼礁是用于营造海洋牧场、调控和优化海洋生态环境、增殖渔业资源的人工设施。人工鱼礁所形成的环境可以作为鱼类良好的栖息、庇护和产卵场所,有利于鱼类的生存和繁殖,因此对鱼类具有良好的诱集效应。本文从多个角度论述了人工鱼礁生态诱集技术的机理以及鱼类行为和生态诱集技术的研究进展,探讨了目前人工鱼礁生态诱集技术研究所面临的问题和主要发展方向,旨在为我国沿海人工鱼礁建设提供一定的理论基础。