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贾永义, 郑建波, 顾志敏, 陈立侨, 罗琛, 迟美丽, 刘士力, 蒋文枰, 程顺. 翘嘴鲌Sox9基因的克隆及CpG岛甲基化与基因表达的关系[J]. 水生生物学报, 2019, 43(3): 473-478. DOI: 10.7541/2019.058
引用本文: 贾永义, 郑建波, 顾志敏, 陈立侨, 罗琛, 迟美丽, 刘士力, 蒋文枰, 程顺. 翘嘴鲌Sox9基因的克隆及CpG岛甲基化与基因表达的关系[J]. 水生生物学报, 2019, 43(3): 473-478. DOI: 10.7541/2019.058
JIA Yong-Yi, ZHENG Jian-Bo, GU Zhi-Min, CHEN Li-Qiao, LUO Chen, CHI Mei-Li, LIU Shi-Li, JIANG Wen-Ping, CHENG Shun. THE RELATIONSHIP OF SOX9 EXPRESSION AND ITS CPG ISLAND METHYLATION IN CULTER ALBURNUS[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(3): 473-478. DOI: 10.7541/2019.058
Citation: JIA Yong-Yi, ZHENG Jian-Bo, GU Zhi-Min, CHEN Li-Qiao, LUO Chen, CHI Mei-Li, LIU Shi-Li, JIANG Wen-Ping, CHENG Shun. THE RELATIONSHIP OF SOX9 EXPRESSION AND ITS CPG ISLAND METHYLATION IN CULTER ALBURNUS[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(3): 473-478. DOI: 10.7541/2019.058

翘嘴鲌Sox9基因的克隆及CpG岛甲基化与基因表达的关系

THE RELATIONSHIP OF SOX9 EXPRESSION AND ITS CPG ISLAND METHYLATION IN CULTER ALBURNUS

  • 摘要: 为了揭示翘嘴鲌(Culter alburnus)性别决定与分化的作用机制, 进而更好地发展性别控制育种技术, 研究重点分析了Sox9基因在翘嘴鲌性腺分化过程中的作用。通过RT-PCR和RACE方法获得了翘嘴鲌2个旁系同源基因Sox9aSox9b的cDNA序列: Sox9a全长1642 bp, 编码458个氨基酸; Sox9b全长1673 bp, 编码456个氨基酸。序列分析表明两者相似度达到73.95%, 编码HMG盒区域极其保守。蛋白质次级结构预测显示Sox9a和Sox9b除了保守的HMG盒结构域外, 还存在2个核定位信号; 两者的三维结构都存在多个螺旋结构。系统进化树分析发现翘嘴鲌Sox9a与罗非鱼关系最近, 但Sox9b形成单独的一支。利用实时荧光定量PCR技术分析了翘嘴鲌Sox9aSox9b基因在各成体组织中的表达水平, 结果显示Sox9a在脑和精巢中表达量最高, 其次是肌肉、鳍条、眼睛和卵巢, 在肾脏、脾脏、肝脏中相对较低; Sox9b只在脑、鳍条、眼睛和精巢中检测到一定水平的表达。通过重亚硫酸氢盐DNA测序方法分析了翘嘴鲌性腺组织Sox9a启动子CpG岛甲基化修饰模式, 结果显示在精巢中CG位点几乎不发生甲基化, 然而卵巢中的甲基化程度非常高。这些结果表明启动子CpG甲基化可以调控Sox9a的性别异形表达, 表观遗传修饰在翘嘴鲌性腺发育过程中可能具有重要的生物学功能。

     

    Abstract: In order to elucidate the mechanisms of sex determination and differentiation in Culter alburnus and to control breeding, this study analyzed the role of C. alburnus Sox9 during gonad development. The cDNA sequence of two homologous genes Sox9a and Sox9b were obtained. The full-length cDNA of Sox9a was 1642 bp, encoding 458 amino acids; the full-length cDNA of Sox9b was 1673 bp, encoding 456 amino acids. Sequence analysis revealed that the similarity between them was 73.95% and the HMG motif regions were highly conservative. Protein second structural prediction showed that Sox9a and Sox9b contained a HMG domain and two nuclear localization signal (NLS) sequences; their three-dimensional structure contained multiple spiral structures. Phylogenetic tree analysis discovered that C. alburnus Sox9a was the closest to Oreochromis niloticus, while Sox9b formed a separate branch. Sox9a was highly expressed in brain and testes, followed by muscle, fins, eyes and ovaries, and rarely detected in kidney, spleen and liver. The expression of Sox9b were only detected in brain, fins, eyes and testes. Promoter CpG methylation analysis of Sox9a showed that CpGs were not methylated in the testes, whereas CpGs were hypermethylated in the ovaries. These results suggested that promoter CpG methylation can regulate sexual dimorphic expression of sex-related genes, and epigenetic modification may play an important role in the gonad development of C. alburnus.

     

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