种质冷冻保存平台依托于中国水产科学研究院黄海水产研究所，主要包括精子库、胚胎细胞库、基因库，是我国重要的水产养殖动物精子、细胞保藏及基因资源利用的专业平台，致力于我国水产养殖动物的种质收集、保藏以及基因功能挖掘。
在“十五”、“十一五”“十二五”期间，在国家863计划、国家自然科学基金的资助下，种质冷冻保存平台已完成多种海淡水鱼类及其它水产动物的精子的冷冻保存研究，建立了这些水生动物精子的超低温冷冻保存技术，共收集42种海淡水鱼类的精子，保存鱼类精子8000份以上；并且将冷冻精子应用到鱼类选择育种、杂交育种和性别控制等研究领域，促进了我国海水鱼类遗传育种和苗种繁育的发展。在胚胎冷冻保存方面，平台研究团队在国际上首次建立了海水鱼类胚胎玻璃化冷冻技术，突破了海水鱼类胚胎冷冻保存技术难关，多次获得冷冻复活的胚胎和鱼苗。目前开展了牙鲆、鲈鱼、大菱鲆、真鲷、星斑川鲽、七带石斑鱼和云纹石斑鱼等名贵水生动物胚胎的冷冻保存...
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半滑舌鳎富含亮氨酸的G蛋白偶联受体基因_LGR8_的分子克隆和表达分析
作 者： 吴娅红 陈松林 刘肖峰 董忠典 张宁 于洋 修旺姗 摘 要： 富含亮氨酸的G蛋白偶联受体(leucine rich repeat containing G protein coupled receptor 8, LGR8)在哺乳类动物雄性精巢下降过程中起重要作用。本研究在前期完成的半滑舌鳎(Cynoglossus semilaevis)全基因组测序与转录组测序的基础上，通过RACE 方法克隆得到半滑舌鳎LGR8 基因(GenBank 登录号：KP749830)，该基因全长2 768 bp，开放阅读框(open reading frame, ORF)为2 205 bp，编码734 个氨基酸，5'-非翻译区(untranslated regions, UTR)为272 bp，3'-UTR为296 bp。氨基酸序列中含有10 个LRRs 区域、1个LDL-A区域和1 个7tm-1 结构域。实时定量PCR(Real-time quantitative PCR, qRT-PCR)结果显示，在3龄性成熟鱼体中LGR8 主要在雄鱼脑和性腺中表达，且显著高于其他组织(P＜0.05)； -
半滑舌鳎3D-BAC池构建及性别连锁标记的物理定位
作 者： 张俊杰 张文婷 高峰涛 董忠典 张丽燕 刘琨 邵长伟 陈松林 摘 要： 半滑舌鳎(Cynoglossus semilaevis)属于雌配子异形染色体性别决定机,~(zw／zz)，其w染色体包含的大量重复序列阻碍了w染色体序列的精确组装。目前只有物理图谱可以有效地解决由重复序列所造成的组装难题。本研究在己有物理图谱和遗传图谱的基础上，利用3D(three．dimensiona1)-BAC(bacterial artificial chromosome)池构建和PCR筛选策略进行了性别连锁标记的物理定位。首先将所选44个384孔板的BAC克隆进行接种和预培养，再接种到4个96孔深孔板中。将每两个384孔微孔板的BAC 克隆分别汇集获得相应的板池、行池和列池，在提取DNA后，再将相应板池的部分DNA混合得到超级池。最终构建的3D—BAC池，共包括22个超级池和440个基池，覆盖半滑舌鳎基因组4．2倍，完成一个阳性超级池的基池筛选只需24个反应(20个BAC基池和4个对照)。然后选用159个半滑舌鳎性别连锁标记对这些克隆池进行两步PCR筛选。在对超级池的筛选中，有142个标记获得阳性扩增。对这些阳性超级池进行基池筛选后，最终有100个标记得到准确定位。 -
Molecular Method for Sex Identification of Half-Smooth Tongue Sole (Cynoglossus semilaevis) Using a Novel Sex-Linked Microsatellite Marker - See m
作 者： Xiaolin Liao, Genbo Xu and Song-Lin Chen 摘 要： Abstract: Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most important flatfish species for aquaculture in China. To produce a monosex population, we attempted to develop a marker-assisted sex control technique in this sexually size dimorphic fish. In this study, we identified a co-dominant sex-linked marker (i.e., CyseSLM) by screening genomic microsatellites and further developed a novel molecular method for sex identification in the tongue sole. CyseSLM has a sequence similarity of 73%–75% with stickleback, medaka, Fugu and Tetraodon. -
Differences in growth and sex reversion between normal and neomale stock in half-smooth tongue sole, Cynoglossus semilaevis
作 者： Qiaomu Hu Song-Lin Chen Fengtao Gao Yangzhen Li Shanshan Liu Feng Liu Jingfeng Yang Min Du 摘 要： The present study evaluated the difference in the sex ratio (the genotypic and phenotypic female ratios) and the growth parameters between normal male offspring and neomale offspring under tank culture conditions. The phenotypic female ratio of normal male offspring (20.46–63.79 %) in four families and three stocks was remarkably higher than that of neomale offspring (5.21–10.53 %) in three neomale families and three neomale stocks (p\0.01), while there was no significant difference between normal male offspring (30.41–70.60 %) and neomale offspring (29.7–70.83 %) in the genotypic female ratio (p[0.05). -
Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole (Cynoglossus semilaevis)
作 者： Qiaomu Hu, Ying Zhu, Yang Liu, Na Wang & Songlin Chen 摘 要： Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year ofage. In situ hybridization revealed that CS- wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. -
cloning and characterization of tesk1, a novel spermatogenesis-related gene, in the tongue sole (Cynoglossus semilaevis)
作 者： Liang Meng, Ying Zhu, Ning Zhang, Wanjun Liu, Yang Liu, Changwei Shao, Na Wang,Songlin Chen 摘 要： Protein kinases play a pivotal role in intracellular signal transduction systems, particularly those involved in the regulation of cell metabolism, proliferation and differentiation. They are divided into two broad categories based on their substrate specificity. Tyrosine kinases exhibit specificity toward the tyrosine residue, whereas serine/threonine kinases exhibit substrate specificity toward serine and threonine residues [1,2,3]. Testis-specific protein kinase 1 (Tesk1) is a member of the latter group and was first identified in the testis of rats and humans [4]. -
Cloning, expression and methylation analysis of piwil2 in half-smooth tongue sole (Cynoglossus semilaevis)
作 者： Liyan Zhang, Wanjun Liub, Changwei Shao, Ning Zhang, Hailong Li, Kun Liu, Zhongdian Dong, 摘 要： piwi is an important regulator gene in germ cell division during spermatogenesis. piwi homologous genes are involved in gametogenesis and germline specification, and knocking down these genes could affect germcellmeiotic progression. To understand the function of piwi-related genes in spermatogenesis, we cloned a Piwisubfamily member (piwil2 gene) from the gonad of Cynoglossus semilaevis. The full-length of piwil2 cDNA was 3314 bp, including a 3162 bp open reading frame (ORF), a 60 bp 5′-UTR, along with a 92 bp 3′-UTR, and encoded a predicted protein of 1053 amino acid residues. Phylogenetic analysis showed that the PIWIL2 putative protein belonged to the Argonaute protein family, and Piwi-subfamily, -
Molecular characterization and functional divergence of two Gadd45g homologs in sex determination in half-smooth tongue sole (Cynoglossus semilaevis)
作 者： Wan-Jun Liu , Li-Yan Zhang, Chang-Wei Shao, Na Wang, Kun Liu, Hai-Shen Wen , Ning Zhang ,Zhong-Dian Dong , Jun-Jie Zhang, Song-Lin Chen 摘 要： The growth arrest and DNA-damage-inducible protein 45 gamma (Gadd45g) is known to play a major role in embryonic development and sex determination. In this study, two Gadd45g genes were isolated from halfsmooth tongue sole (Cynoglossus semilaevis). Using chromosomal fluorescence in situ hybridization (FISH), Gadd45g1 and Gadd45g2 were located on the W and Z chromosomes, respectively. The full-length cDNA sequences of Gadd45g1 (1270 bp) and Gadd45g2 (1181 bp) were predicted to contain a 480-bp coding sequence that could encode a protein of 159 amino acids residues -
A Genome Scan for Quantitative Trait Loci Associated with Vibrio anguillarum Infection Resistance in Japanese Flounder (Paralichthys olivaceus) by Bulked Segregant Analysis
作 者： Lei Wang & Caixia Fan & Yang Liu & Yingping Zhang & Shoutang Liu & Deqiang Sun &Han Deng & Ying Xu & Yongsheng Tian & Xiaolin Liao & Mingshu Xie & Wenlong Li &Songlin Chen 摘 要： Japanese flounder (Paralichthys olivaceus) is an economically important fish in China, Japan, and Korea. In recent years, bacterial and viral diseases have seriously interfered with the development of Japanese flounder aquaculture. To prevent or at least diminish the devastating effects of these diseases, one useful method is to cultivate disease-resistant families or strains. -
Molecular characterization, sexually dimorphic expression, and functional analysis of 3'-untranslated region of vasa gene in half-smooth tongue sole (Cynoglossus semilaevis)
作 者： Jinqiang Huang, Songlin Chen, Yang Liu, Changwei Shao, Fan Lin, Na Wang, Qiaomu Hu 摘 要： Vasa is a highly conserved ATP-dependent RNA helicase expressed mainly in germ cells. The vasa gene plays a crucial role in the development of germ cell lineage and has become an excellent molecular marker in identifying germ cells in teleosts. However, little is known about the structure and function of the vasa gene in flatfish. In this study, the vasa gene (Csvasa) was isolated and characterized in half-smooth tongue sole (Cynoglossus semilaevis), an economically important flatfish in China. In the obtained 6425-bp genomic sequence, 23 exons and 22 introns were identified. The Csvasa gene encodes a 663-amino acid protein,