種質(zhì)冷凍保存平臺(tái)依托于中國(guó)水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所，主要包括精子庫、胚胎細(xì)胞庫、基因庫，是我國(guó)重要的水產(chǎn)養(yǎng)殖動(dòng)物精子、細(xì)胞保藏及基因資源利用的專業(yè)平臺(tái)，致力于我國(guó)水產(chǎn)養(yǎng)殖動(dòng)物的種質(zhì)收集、保藏以及基因功能挖掘。
在“十五”、“十一五”“十二五”期間，在國(guó)家863計(jì)劃、國(guó)家自然科學(xué)基金的資助下，種質(zhì)冷凍保存平臺(tái)已完成多種海淡水魚類及其它水產(chǎn)動(dòng)物的精子的冷凍保存研究，建立了這些水生動(dòng)物精子的超低溫冷凍保存技術(shù)，共收集42種海淡水魚類的精子，保存魚類精子8000份以上；并且將冷凍精子應(yīng)用到魚類選擇育種、雜交育種和性別控制等研究領(lǐng)域，促進(jìn)了我國(guó)海水魚類遺傳育種和苗種繁育的發(fā)展。在胚胎冷凍保存方面，平臺(tái)研究團(tuán)隊(duì)在國(guó)際上首次建立了海水魚類胚胎玻璃化冷凍技術(shù)，突破了海水魚類胚胎冷凍保存技術(shù)難關(guān)，多次獲得冷凍復(fù)活的胚胎和魚苗。目前開展了牙鲆、鱸魚、大菱鲆、真鯛、星斑川鰈、七帶石斑魚和云紋石斑魚等名貴水生動(dòng)物胚胎的冷凍保存...
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半滑舌鰨富含亮氨酸的G蛋白偶聯(lián)受體基因_LGR8_的分子克隆和表達(dá)分析
作 者： 吳婭紅 陳松林 劉肖峰 董忠典 張寧 于洋 修旺姍 摘 要： 富含亮氨酸的G蛋白偶聯(lián)受體(leucine rich repeat containing G protein coupled receptor 8, LGR8)在哺乳類動(dòng)物雄性精巢下降過程中起重要作用。本研究在前期完成的半滑舌鰨(Cynoglossus semilaevis)全基因組測(cè)序與轉(zhuǎn)錄組測(cè)序的基礎(chǔ)上，通過RACE 方法克隆得到半滑舌鰨LGR8 基因(GenBank 登錄號(hào)：KP749830)，該基因全長(zhǎng)2 768 bp，開放閱讀框(open reading frame, ORF)為2 205 bp，編碼734 個(gè)氨基酸，5'-非翻譯區(qū)(untranslated regions, UTR)為272 bp，3'-UTR為296 bp。氨基酸序列中含有10 個(gè)LRRs 區(qū)域、1個(gè)LDL-A區(qū)域和1 個(gè)7tm-1 結(jié)構(gòu)域。實(shí)時(shí)定量PCR(Real-time quantitative PCR, qRT-PCR)結(jié)果顯示，在3齡性成熟魚體中LGR8 主要在雄魚腦和性腺中表達(dá)，且顯著高于其他組織(P＜0.05)； -
半滑舌鰨3D-BAC池構(gòu)建及性別連鎖標(biāo)記的物理定位
作 者： 張俊杰 張文婷 高峰濤 董忠典 張麗燕 劉琨 邵長(zhǎng)偉 陳松林 摘 要： 半滑舌鰨(Cynoglossus semilaevis)屬于雌配子異形染色體性別決定機(jī),~(zw／zz)，其w染色體包含的大量重復(fù)序列阻礙了w染色體序列的精確組裝。目前只有物理圖譜可以有效地解決由重復(fù)序列所造成的組裝難題。本研究在己有物理圖譜和遺傳圖譜的基礎(chǔ)上，利用3D(three．dimensiona1)-BAC(bacterial artificial chromosome)池構(gòu)建和PCR篩選策略進(jìn)行了性別連鎖標(biāo)記的物理定位。首先將所選44個(gè)384孔板的BAC克隆進(jìn)行接種和預(yù)培養(yǎng)，再接種到4個(gè)96孔深孔板中。將每?jī)蓚€(gè)384孔微孔板的BAC 克隆分別匯集獲得相應(yīng)的板池、行池和列池，在提取DNA后，再將相應(yīng)板池的部分DNA混合得到超級(jí)池。最終構(gòu)建的3D—BAC池，共包括22個(gè)超級(jí)池和440個(gè)基池，覆蓋半滑舌鰨基因組4．2倍，完成一個(gè)陽性超級(jí)池的基池篩選只需24個(gè)反應(yīng)(20個(gè)BAC基池和4個(gè)對(duì)照)。然后選用159個(gè)半滑舌鰨性別連鎖標(biāo)記對(duì)這些克隆池進(jìn)行兩步PCR篩選。在對(duì)超級(jí)池的篩選中，有142個(gè)標(biāo)記獲得陽性擴(kuò)增。對(duì)這些陽性超級(jí)池進(jìn)行基池篩選后，最終有100個(gè)標(biāo)記得到準(zhǔn)確定位。 -
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,