人工诱导大鳞副泥鳅雄核发育二倍体克隆鱼的产生

首次获得人工诱导雄核发育二倍体大鳞副泥鳅克隆鱼。方法是:用紫外线210mJ/cm^2辐射浸泡在人工合成卵巢液中的泥鳅卵,使其染色体遗传失活,再与大鳞副泥鳅精子“受精”。在室温26℃条件下“受精”后15min开始,每隔2min一组,将“受精卵”置于39℃温水中热休克处理2min,以阻止第一次有丝分裂的发生,结果表明:二倍诱导率距“受精”后15～19min和27～29min出现两个高峰,最高二倍诱导率为61.1％,经染色体和形态特征鉴定表明,鱼苗为大鳞副泥鳅雄核发育二倍体克隆鱼,其染色体数(2n=48)和外部形态均与父本相同。

虚拟克隆鱼实验

提出了构建一个虚拟克隆鱼实验系统,该系统采用虚拟现实技术来仿真鱼的克隆实验过程,可以通过实验快速的进一步观察实验结果。更方便快捷的使用户的在实验结果中得到结论。此虚拟克隆鱼实验还可以减少真实世界中对克隆鱼进行实验造成不必要的浪费,以及在实验过程中漫长的等待过程。为用户节约了大量的时间。还探讨了虚拟现实技术在人工生命中的应用以及该实验的开发和应用。

克隆技术及其在水产上的应用

为让大家进一步了解动物克隆技术，本文简要地介绍了克隆技术的基本概念和发展史。并着重介绍了我国鱼类克隆的研究进展。作者以鲤鲫移核鱼和草鱼肝细胞克隆鱼为例，分析了克隆技术在培育鱼类新品种上的特点和优势。同时阐述了克隆技术在水产育种和种质保存上的应用前景。

世间克隆技术第一人——纪念著名科学家童第周诞辰100周年

2000年,中、美、英等国科学家同时宣布,人类基因工程图工程全部完成。然而,早在20世纪60年代,一位中国科学家就将人类对生物进化和细胞遗传与变异研究推进到了世界前列;开创了人类按照需要而人工培育新物种的历史先河,被誉为世间克隆第一人。他就是原中国科学院副院长、第五届全国政协副主席、宁波鄞州籍的杰出科学家童第周。

Cloning and Expression of PKR Gene of Ctenopharyngodon idellus Induced by PolyI:C in vitro

[Objective] The study aimed at cloning PKR gene from Ctenopharyngodon idellus induced by PolyI：C in vitro,so as to provide foundation for study on the anti-virus genes of C.idellus.[Method] By referring to the PKR gene sequences of zebra fish（AJ852023.1） and Carassius auratus（AY293929.1） in Genbank,three pairs of degenerate primers were designed with Primer Premier 5.0 software;in vitro C.idellus kidney cells（CIK） were treated with 100 μg/ml of Poly I：C for 12,36 and 48 h,and then total RNA of the cells treated was extracted for amplifying the PKR gene by RT-PCR.[Result] The PKR gene was amplified from the cells treated with Poly I：C for 36 and 48 h,but not from the cells treated for 12 h;in addition,the expression level increased with the processing time.Part of the amplified sequence of C.idellus shared the homology of 100% and 81.48% with the sequences of carp and zebra fish separately.[Conclusion] Part of the PKR gene sequence was cloned successfully from C.idellus.Moreover,we have proved that PolyI：C induction is effective for PKR protein expression,which will provide reference for treating viral diseases of C.idellus.

Cloning and Sequence Analysis of Interferon γ-2β Full-length cDNA in Cyprinus carpio L.

[Objective] The research aimed to carry out the cloning, identification and sequence analysis of full-length cDNA of carp interferon γ-2β （IFNγ-2β）. [Method] The cDNA library of peripheral blood leucocytes which were separated from carp and stimulated with mitogen was screened by a probe labeled with DIG. The IFNγ- 2β EST sequence was picked out from the constructed cDNA library of peripheral blood leucocyte, and the full length of carp interferon γ-2β was cloned. In addition, the sequence analysis was carried out. [Result] Three positive clones were obtained. Sequence analysis indicated that the sequence had a 119 bp 5’-UTR and a 218 bp 3’-UTR, and the open reading frame （ORF）of this gene was 537 bp which putatively coded 178 amino acids and there were several instable motifs for mRNA （ATTTA） in the 3’-untranslated region. Its homology with IFN from GenBank was up to 97% . Analysis on protein sequence and structure showed that the predicted protein sequence was identified as an IFN family signature. [Conclusion] The research laid the foundation for further studying the expression manner, function characteristic and regulation mechanism of IFNγ-2β in vivo and the action mechanism in the inflammatory reaction, emergency reaction and immune response.

Cloning,Identification and Differential Expression Analysis of Full-length cDNA of Carp Interleukin-1β

[Objective] The research aimed to carry out the cloning,identification and differential expression analysis of carp interleukin-1β （IL-1β） cDNA. [Method] By using DD-RTPCR method,the differential expression cDNA fragments were gained. The cDNA library of carp peripheral blood leucocytes which was stimulated by the mitogen was screened,and the full length cDNA of carp IL-1β was cloned. Moreover,the sequence analysis and differential expression analysis were carried out. [Result] The positive clone which had a whole ORF that encoded 276 amino acids was obtained. The cluster analysis showed that the amino acid sequence of carp IL-1β and Japanese carp closely gathered as a branch,and the homoeology of amino acid sequence reached 95%. The clustering order was the carassius,zebra fish,pig,cattle,horse,human and mouse in turn. The differential expression analysis showed that the expression of IL-1β in the leucocytes significantly increased in the prior period （4 h） after the mitogen stimulated. But as the time went by （12 and 24 h）,it didn＇t increase in the same period. The total trend of expression amount presented the peak type. [Conclusion] The research laid the foundation for further studying the expression manner,function characteristic,regulation mechanism of IL-1β in vivo and its action mechanisms in the inflammatory reaction,emergency reaction and immune response.

广告技艺拾零

全世界每年有数以千亿美元的费用用以广告。以香港为例,今年的广告总经费约为60亿港元。可以说,目前没有不做广告的商人,也没有不依赖于广告进行商品销售的商业活动。“商战”离不开广告的攻心战,其中有一批精心制作的广告,它们的手法耐人寻味。

谋略

茫茫大海;烟波浩瀚,海底深处聚集着很多动物,他们要召开海洋大会。宽敞的会议厅里已座无虚席,他们一会儿交头接耳,一会儿窃窃私语……

报刊大看台

中央红军在长征中屡遭敌人围追堵截,倍受挫折,为什么徐海东的队伍却能突围出来?原来,这里有一段秘闻。 1934年春季,蒋介石在武汉召开军事会议,军长以上的几十人参加,决定集中兵力"围剿"鄂豫皖的徐海东部队。这个计划由一个参谋监印了有数的几份后,就将原稿销毁。总司令有一份,给参谋长,参谋长又给了秘书。而这个秘书却是我党地下工作者谢捕生的挚友,在谢的影响下,为我党传送情报。当这个"围剿"计划到了谢捕生的手里,他及时将情报送往上海,转至苏区。中央立即又派程子华通过秘密路线找到了李感,经地下交通员与徐海东接头。徐海东、程子华带领二十五军,按照情报了解敌人兵力配置,就从敌人的空隙中突围出去,成了长征中的一个奇迹。蒋介石得知后,气急败坏地喊:"怎么让他们跑掉了。"

Cloning and Characterization of Largemouth Bass(Micropterus salmoides) Myostatin Encoding Gene and Its Promoter

Myostatin or GDF-8, a member of the transforming growth factor-β （TGF-β） superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass （Micropterus salmoides）. The myostatin encoding gene consisted of three exons （488bp, 371 bp and 1779bp, respectively） and two introns （390bp and 855 bp, respectively）. The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 （MEF2）, serum response factor （SRF）, activator protein 1 （AP1）, etc., and muscle-specific Mt binding site （MTBF） were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This infunnation will be useful for studying the tran- scriptional regulation of myostatin in fish.

Molecular cloning and expression of a heat-shock cognate 70 (hsc70) gene from swordtail fish (Xiphophorus helleri)

Heat shock proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In the present study, a full-length cDNA, encoding the constitutively expressed 70-kDa heat shock cognate protein （Hsc70）, was isolated from swordtail fish （Xiphophorus helleri） and designated as XheHsc70. The Xhehsc70 cDNA was 2 104 bp long with an open reading frame of 1 941 bp, and it encoded a protein of 646 amino acids with a theoretical molecular weight of 70.77 kDa and an isoelectric point of 5.04. The deduced amino acid sequence shared 94.1%-98.6% identities with the Hsc70s from a number of other fish species. Tissue distribution results show that the Xhehsc70 mRNA was expressed in brain, heart, head kidney, kidney, spleen, liver, muscle, gill, and peripheral blood. After immunization with formalin-killed Vibrio alginolyticus cells there was a significant increase in the XhehscT0 mRNA transcriptional level in the head kidney of the vaccinated fish compared with in the control at 6, 12, 24, and 48 h as shown by quantitative real time RT-PCR. Based on an analysis of the amino acid sequence of XheHsc70, its phylogeny, and Xhehsc70 mRNA expression, XheHsc70 was identified as a member of the cytoplasmic Hsc70 （constitutive） subfamily of the Hsp70 family of heat shock proteins, suggesting that it may play a role in the immune response. The Xhehsc70 cDNA sequence reported in this study was submitted to GenBank under the accession number JF739182.

Genetic discrimination for three gynogenetic clones of silver carp Hypophthalmichthys molitrix, based on restriction endonuclease analysis of Nd5-Nd6 region of mitochondrial DNA

Three artificial gynogenetic clones of silver carp were produced for the analysis of restriction enzyme digestion patterns of ND5-ND6 region from mtDNA of the clones. It is revealed that all intraclonal individuals shared completely the same digestion patterns but among interclonal individuals did not. The three clones were mixed and cultured in a pond together for two years, and restriction endonuclease digestion patterns of ND5-ND6 were used as genetic markers to assess the growth performance of each clone.

Molecular cloning and mRNA expression analysis of myosin heavy chain(MyHC)from fast skeletal muscle of grass carp,Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

SERUM IMMUNOGLOBULIN OF THE MANDARIN FISH,SINIPERCA CHUATSI WITH DEVELOPMENT OF POLYCLONAL ANTIBODY

Serum immunoglobulin from the mandarin fish, or the so called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM of S. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains of S. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein (TCTP) Gene from Flatfish Turbot (Scophthalmus maximus)

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot （Scophthalmus maximus）, SmTCTP, was isolated with rapid amplification of cDNA Ends （RACE）. SmTCTP consisted of a 5＇ untranslated region （UTR） of 84 bp, a 3＇ UTR of 451 bp and an open reading frame （ORF） of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5＇UTR of SmTCTP started with a 5＇-terminal oligopyrimidine tract （5＇-TOP）, a typical feature for translationally controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known vertebrate TCTPs in a range of 58.8% to 64.1%. The length of fish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch （Lateolabraxjaponicus） is 170 aa in length, while that of zebrafish （Danio rerio） and rohu （Labeo rohita） is 171 aa in length. Northern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further investigation of the biological function of TCTP in fish.

cDNA cloning and expression of a collectin from red-spotted grouper(Epinephelus akaara)

Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin （EALecl） from Epinephelus akaara using RACE. The complete EALecl cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly（A） tail. The EALecl cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain （CRD）, indicating that EALecl is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALecl in 12 different tissues using RT-PCR. EALecl was expressed in all tissues, though at different levels. In addition, we inserted EALecl into an expression vector （pET-28a） for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALecl recombinant expression vector.

愿效老牛,为国捐躯

1996年7月5日,一只名叫“多莉”的小羊出生在英国爱丁堡市罗斯林研究所。它是世界上第一个用已经分化成熟的体细胞克隆出来的哺乳动物,被美国《科学》杂志评为1997年世界十大科技进步的第一项,也是当年最引人注目的国际新闻之一。但几乎没有人知道,在一位中国老人呕心沥血的努力下,我们早在1981年就创造了世界上第一条克隆鱼!