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半滑舌鰨富含亮氨酸的G蛋白偶聯受體基因_LGR8_的分子克隆和表達分析
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蛋白偶聯受體(leucine rich repeat containing G protein coupled receptor 8, LGR8)在哺乳類動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)全基因組測序與轉錄組測序的基礎上，通過RACE 方法克隆得到半滑舌鰨LGR8 基因(GenBank 登錄號：KP749830)，該基因全長2 768 bp，開放閱讀框(open reading frame, ORF)為2 205 bp，編碼734 個氨基酸，5'-非翻譯區(untranslated regions, UTR)為272 bp，3'-UTR為296 bp。氨基酸序列中含有10 個LRRs 區域、1個LDL-A區域和1 個7tm-1 結構域。實時定量PCR(Real-time quantitative PCR, qRT-PCR)結果顯示，在3齡性成熟魚體中LGR8 主要在雄魚腦和性腺中表達，且顯著高于其他組織(P＜0.05)； -
半滑舌鰨3D-BAC池構建及性別連鎖標記的物理定位
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,~(zw／zz)，其w染色體包含的大量重復序列阻礙了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上，利用3D(three．dimensiona1)-BAC(bacterial artificial chromosome)池構建和PCR篩選策略進行了性別連鎖標記的物理定位。首先將所選44個384孔板的BAC克隆進行接種和預培養，再接種到4個96孔深孔板中。將每兩個384孔微孔板的BAC 克隆分別匯集獲得相應的板池、行池和列池，在提取DNA后，再將相應板池的部分DNA混合得到超級池。最終構建的3D—BAC池，共包括22個超級池和440個基池，覆蓋半滑舌鰨基因組4．2倍，完成一個陽性超級池的基池篩選只需24個反應(20個BAC基池和4個對照)。然后選用159個半滑舌鰨性別連鎖標記對這些克隆池進行兩步PCR篩選。在對超級池的篩選中，有142個標記獲得陽性擴增。對這些陽性超級池進行基池篩選后，最終有100個標記得到準確定位。 -
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, -
Epigenetic modification and inheritance in sexual reversal of fish
作 者： Changwei Shao, Qiye Li, Songlin Chen, Pei Zhang, Jinmin Lian, Qiaomu Hu, Bing Sun, Lijun Jin, Shanshan Liu, Zongji Wang, Hongmei Zhao, Zonghui Jin, Zhuo Liang, Yangzhen Li, Qiumei Zheng, Yong Zhang, Jun Wang, and Guojie Zhang 摘 要： Environmental sex determination (ESD) occurs in divergent, phylogenetically unrelated taxa, and in some species, cooccurs with genetic sex determination (GSD) mechanisms. Although epigenetic regulation in response to environmental effects has long been proposed to be associated with ESD, a systemic analysis on epigenetic regulation of ESD is still lacking. Using half-smooth tongue sole (Cynoglossus semilaevis) as a model—a marine fish that has both ZW chromosomal GSD and temperature-dependent ESD—we investigated the role of DNA methylation in transition from GSD to ESD. Comparative analysis of the gonadal DNA methylomes of pseudomale, female, and normal male fish revealed that genes in the sexdetermination pathways are the major targets of substantial methylation modification during sexual reversal. -
2014.3.20 A first generation BAC-based physical map of the half-smooth tongue sole (Cynoglossus semilaevis) genome
作 者： Junjie Zhang, Changwei Shao, Liyan Zhang, Kun Liu, Fengtao Gao, Zhongdian Dong, Peng Xu 摘 要： Half-smooth tongue sole (Cynoglossus semilaevis Günther) is a marine flatfish that belongs to the family Cynoglossidae in the order Pleuronectiformes, and is widely distributed in Chinese coastal water [1,2]. Because of its rarity and delicacy, half-smooth tongue sole has been exploited as a commercially important cultured marine fish, especially in the Shandong Peninsula [3]. Because female grows 2–3 times faster than male, the development of all-female stocks of this fish would be of significant benefit for aquaculture and this fish could be an ideal model for the study on sex-determination mechanisms in teleosts [4]. Genetic studies, especially on the sex-determining mechanism of this species, have been carried out in the last decade. The chromosomal sexdetermining mechanism in half-smooth tongue sole was determined to be female heterogametic with the ZW -
whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle
作 者： Songlin Chen, Guojie Zhang, Changwei Shao, Quanfei Huang, Geng Liu, Pei Zhang,Wentao Song, Na An, Domitille Chalopin, Jean-Nicolas Volff, Yunhan Hong, Qiye Li, Zhenxia Sha,Heling Zhou, Mingshu Xie, Qiulin Yu, Yang Liu, Hui Xiang, Na Wang, Kui Wu, Songlin Chen, Guojie Zhang, hangwei Shao, Quanfei Huang, Geng Liu, Pei Zhang,Wentao Song, Na An, Domitille Chalopin, Jean-Nicolas Volff, Yunhan Hong, Qiye Li, Zhenxia Sha,Heling Zhou, Mingshu Xie, Qiulin Yu, Yang Liu, Hui Xiang, Na Wang, Kui Wu, Changgeng Yang,Qian Zhou, Xiaolin Liao, Linfeng Yang, Qiaomu Hu, Jilin Zhang, Liang Meng, Lijun Jin, Yongsheng Tian,Jinmin Lian, Jingfeng Yang, Guidong Miao, Shanshan Liu, Zhuo Liang, Fang Yan, Yangzhen Li, Bin Sun,Hong Zhang, Jing Zhang, Ying Zhu, Min Du, Yongwei Zhao, Manfred Schartl, Qisheng Tang & Jun Wang 摘 要： The existence of males and females, which are often strikingly different in morphology, reproductive strategies and behavior, is one of the most widespread phenomena in biology. However, the genetic mechanisms that generate this ubiquitous pattern are surprisingly diverse and do not follow a phylogenetic pattern. Sex-determination echanisms can differ between even closely related species and arise frequently and independently. Fish provide a paradigmatic example, -
Screening and analysis of PoAkirin1 and two related genes in response to immunological stimulants in the Japanese flounder (Paralichthys olivaceus)
作 者： Chang-Geng Yang, Xian-Li Wang, Bo Zhang, Bing Sun, Shan-Shan Liu and Song-Lin Chen 摘 要： Biological processes are primarily performed and controlled by proteins. Therefore, clarifying the biological functions of proteins and their biological response mechanisms at the cellular level has become the main objective of proteomics research. Protein-protein interactions play a crucial role in various biological functions, including the formation of polymer structure, cell signal transduction, gene regulation, and metabolic pathways. In the post-genome sequencing era -
Construction of a microsatellite-based genetic linkage map for half-smooth tongue sole Cynoglossus semilaevis
作 者： Wentao SONG, Guidong MIAO, Yongwei ZHAO, Yuze NIU, Renyi PANG, 摘 要： The half-smooth tongue sole Cynoglossus semilaevis is an important cultured marine fish and a promising model fish for the study of sex determination. Sex-specific genetic linkage maps of half-smooth tongue sole were developed with 567 markers (565 microsatellite markers and two SCAR markers). The parents and F1 progeny (92 individuals) were used as segregating populations. The female map was composed of 480 markers in 21 linkage groups, covering a total of 1388.1 cM, with an average interval 3.06 cM between markers. The male map consisted of 417 markers in 21 linkage groups, spanning 1480.9 cM, with an average interval of 3.75 cM. The female and male maps had 474 and 416 unique positions, respectively. The genome length of half-smooth tongue sole was estimated to be 1522.9 cM for females and 1649.1cM for males. Based on estimations of map length, the female and male maps covered 91.1% and 89.8% of the genome, respectively. Furthermore, two female-specific SCAR markers... -
Molecular cloning and multifunctional characterization of GRIM-19 (gene associated with retinoid-interferon-induced mortality 19) homologue from turbot (Scophthalmus maximus)
作 者： Na Wang，Xianli Wang, Changgeng Yang, Xiaojie Zhao, Yuxi Zhang, Tianzi Wang，Songlin Chen a 摘 要： GRIM-19 (gene associated with retinoid-interferon-induced mortality 19), a novel cell death regulatory gene, plays important roles in cell apoptosis, embryogenesis, mitochondrial respiratory chain and immune response. To date, little information is known about fish GRIM-19 characteristics except orange-spotted grouper (Epinephelus coioides). Here a new GRIM-19 gene is identified and characterized from turbot (Scophthalmus maximus), an economic marine fish in China and Europe. Briefly, turbot GRIM- 19 is a 595-bp gene encoding a 144 amino acids protein -
Characterization of the cyp19a1a gene froma BAC sequence in half-smooth tongue sole (Cynoglossus semilaevis) and
作 者： SHAO Changwei, LIUGeng, LIU Shanshan, LIUChanglin, CHENSonglin 摘 要： The cyp19a1a gene encodes an aromatase that plays a key role in sex differentiation of the gonad. The first bacterial artificial chromosome (BAC) sequence of half-smooth tongue sole (Cynoglossus semilaevis) containing the intact cyp19a1a gene was reported and the conservation and synteny of the cyp19a1a gene among teleosts were analyzed in the study. The BAC is 107 367 bp in size, with an overall guanine-cytosine (GC) content of 43.44%, and contains 4.38% transposable elements. Nine genes were predicted, including seven functional genes and two hypothetical genes. The cyp19a1a gene of all tested teleosts has nine exons and eight introns, and potential binding sites flanking the transcriptional start site are conserved. The expression pattern among teleosts is also similar during ovarian differentiation. Synteny analysis revealed a conserved gene cluster PKH4B-SL9A5-FHOD3-CEBPG-CEBPA among teleosts. These findings suggest that, among teleosts, cyp19a1a genes not... -
Molecular cloning, subcelluar location and expression profile of signal transducer and activator of transcription 2 (STAT2) from turbot
作 者： Na Wang, Xian-Li Wang, Chang-Geng Yang, Song-Lin Chen 摘 要： Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signalling pathway. To date, little STAT2 homologue is available in fish except Atlantic salmon and goldfish. In this paper, STAT2 was firstly cloned and characterized from turbot, a marine flatfish with high economic value. Briefly, turbot STAT2 cDNA is 3206 bp in length encoding a predicted protein of 793 amino acids. The phylogenetic tree shows that turbot STAT2 protein shared the closest relationship with Atlantic salmon. Analysis of subcellular distribution indicates that STAT2 is mainly present in the cytoplasm of TK cells -
cloning, genomic structure and expression analysis of ubc9 in the course of development in the half-smooth tongue sole
作 者： Qiaomu Hu, Songlin Chen 摘 要： The small ubiquitin-like modifier (SUMO) pathway is an essential biological process in eukaryote, and Ubc9 is an important E2 conjugating enzyme (UBE2) for SUMO pathway and plays a critical role in cellular differentiation, development and sex modification in various species. However, the relationship between Ubc9 and sex modification and development in fish remains elusive. To elucidate the impact of Ubc9 on sex modification and development, the full length of the cDNA and genomic sequence was cloned from half-smooth tongue sole, Cynoglossus semilaevis. Real-time quantitative RT-PCR demonstrated that ubc9 was ubiquitously expressed in different tissues, and the expression levels varied in the different stages of embryonic and gonadal development. In addition, the expression level was significantly higher in the temperature-treated females than the normal females and males. Moreover, the PET-32-Ubc9 plasmid was constructed... -
Iron-metabolic function and potential antibacterial role of Hepcidin and its correlated genes (Ferroportin 1 and Transferrin Receptor) in Turbot (Scophthalmus maximus)
作 者： Chang-Geng Yang, Shan-Shan Liu, Bing Sun, Xian-Li Wang, Na Wang, Song-Lin Chen 摘 要： Antimicrobial peptide plays an important role in fish immunity. The small molecular antimicrobial peptide Hepcidin in turbot was studied and reported in this paper. The Ferroportin 1 (FPN1) and Transferrin Receptor (TFR) genes, which are related to Hepcidin, were cloned in turbot. The characteristics of Hepcidin and its related genes were studied, including an analysis of the expression patterns and cloning of the Hepcidin promoter, the relationship between Hepcidin and NF-kB and the regulation of iron-metabolism. The results showed that the promoter of SmHepcidin contains the binding sites of NFkB, and NF-kB may directly or indirectly receive feedback signals from SmHepcidin. In the liver, spleen and kidney -
Polymorphism and Balancing Selection of MHC Class II DAB Gene in 7 Selective Flounder (Paralichthys olivaceus ) Families
作 者： Min Du, Song-lin Chen, You Liang, LeiWang,Feng-tao Gao, Yang Liu, and Xiao-Lin Liao 摘 要： In order to determine the genetic variation of the MHC class IIB exon2 allele in the offspring, 700 fry from seven families of Japanese flounder challenged with V. anguillarum were studied, and different mortality rates were found in those families. Five to ten surviving and dead fry from each of the seven families were selected to study the MHC class II B exon2 gene with PCR and a direct sequencing method. One hundred and sixteen different exon2 sequences were found and 116 different alleles were identified, while a minimum of four loci were revealed in the MHC class II B exon2 gene. The ratio (dN/dS) of nonsynonymous substitution (dN) to synonymous substitutions (dS) in the peptide-binding region (PBR) of the MHC class IIB gene was 6.234, which indicated that balancing selection is acting on the MHC class IIB genes. The MHC IIB alleles were thus being passed on to their progeny. Some alleles were significantlymore frequent in surviving than dead individuals... -
Construction of a High-Density Microsatellite Genetic Linkage Map and Mapping of Sexual and Growth-Related Traits in Half-Smooth Tongue Sole (Cynoglossus semilaevis)
作 者： Wentao Song, Yangzhen Li, Yongwei Zhao, Yang Liu, Yuze Niu, Renyi Pang, Guidong Miao, Xiaolin Liao, Changwei Shao, Fengtao Gao, Songlin Chen 摘 要： High-density genetic linkage maps of half-smooth tongue sole were developed with 1007 microsatellite markers, two SCAR markers and an F1 family containing 94. The female map was composed of 828 markers in 21 linkage groups, covering a total of 1447.3 cM, with an average interval 1.83 cM between markers. The male map consisted of 794 markers in 21 linkage groups, spanning 1497.5 cM, with an average interval of 1.96 cM. The female and male maps had 812 and 785 unique positions, respectively. The genome length of half-smooth tongue sole was estimated to be 1527.7 cM for the females and 1582.1 cM for the males. Based on estimations of the map lengths, the female and male maps covered 94.74 and 94.65% of the genome, respectively -
牙鲆遺傳連鎖圖譜的構建
作 者： 龐仁誼, 宋文濤, 高峰濤, 廖小林, 趙永偉, 牛余澤, 王磊, 趙文，田永勝, 陳松林 摘 要： 利用基因組測序得到的大量微衛星序列, 以681383B 為父本、6812E36 為母本雜交獲得的F1 為作圖群體, 構建了牙鲆(Paralichthys olivaceus)微衛星標記(SSR)遺傳連鎖圖譜。雌雄圖譜共定位SSR 標記529 個, 其中雄性連鎖圖譜包括418 個標記, 分布在24 個連鎖群上, 總長度1 418.1 cM, 標記平均間隔3.62 cM, 圖譜覆蓋率為88.7%; 雌性連鎖圖譜包括437 個標記, 分布在24 個連鎖群上, 總長度1 298.1 cM, 標記平均間隔為3.16 cM, 圖譜覆蓋率為89.1%。牙鲆中密度遺傳圖譜的構建為QTL 分析以及分子標記輔助育種進一步奠定基礎, 并可以有效推動牙鲆的遺傳改良工作, 推動牙鲆養殖業的可持續發展。