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祝骏贤, 陈辰, 刘晓莉, 王亚坤, 雷骆, 洪孝友, 于凌云, 徐红艳, 李伟, 朱新平. 中华鳖组蛋白H2A变体克隆及其在卵母细胞中的表达分析[J]. 水生生物学报, 2021, 45(6): 1207-1213. DOI: 10.7541/2021.2020.167
引用本文: 祝骏贤, 陈辰, 刘晓莉, 王亚坤, 雷骆, 洪孝友, 于凌云, 徐红艳, 李伟, 朱新平. 中华鳖组蛋白H2A变体克隆及其在卵母细胞中的表达分析[J]. 水生生物学报, 2021, 45(6): 1207-1213. DOI: 10.7541/2021.2020.167
ZHU Jun-Xian, CHEN Chen, LIU Xiao-Li, WANG Ya-Kun, LEI Luo, HONG Xiao-You, YU Ling-Yun, XU Hong-Yan, LI Wei, ZHU Xin-Ping. CLONING AND EXPRESSION ANALYSIS OF HISTONE H2A VARIANT IN OOCYTES OF CHINESE SOFT-SHELLED TURTLE (PELODISCUS SINENSIS)[J]. ACTA HYDROBIOLOGICA SINICA, 2021, 45(6): 1207-1213. DOI: 10.7541/2021.2020.167
Citation: ZHU Jun-Xian, CHEN Chen, LIU Xiao-Li, WANG Ya-Kun, LEI Luo, HONG Xiao-You, YU Ling-Yun, XU Hong-Yan, LI Wei, ZHU Xin-Ping. CLONING AND EXPRESSION ANALYSIS OF HISTONE H2A VARIANT IN OOCYTES OF CHINESE SOFT-SHELLED TURTLE (PELODISCUS SINENSIS)[J]. ACTA HYDROBIOLOGICA SINICA, 2021, 45(6): 1207-1213. DOI: 10.7541/2021.2020.167

中华鳖组蛋白H2A变体克隆及其在卵母细胞中的表达分析

CLONING AND EXPRESSION ANALYSIS OF HISTONE H2A VARIANT IN OOCYTES OF CHINESE SOFT-SHELLED TURTLE (PELODISCUS SINENSIS)

  • 摘要: 为研究组蛋白H2A对龟鳖动物生殖细胞发育分化作用和机制, 克隆了中华鳖(Pelodiscus sinensis)组蛋白H2A变体的同源物(命名为PsH2A), 分析其转录本的表达模式及在卵巢发育成熟过程中的细胞定位。PsH2A cDNA序列全长575 bp, 5′端非编码区68 bp, 3′端非编码区108 bp, 开放阅读框399 bp, 编码133个氨基酸。氨基酸序列比对结果显示其与龟类的H2A变体同源性更高, 与哺乳类同源性较低。RT-qPCR和RT-PCR结果显示, PsH2A转录本在1冬龄、2冬龄和3冬龄的中华鳖卵巢中高表达(P<0.01), 而在精巢和其他成体组织中几乎检测不到。化学原位杂交结果显示, PsH2A mRNA在卵母细胞中特异性表达, 其中初级卵母细胞中表达信号最强, 且均匀的分布在细胞质中。随着卵母细胞发育成熟进入到生长期和成熟期后, 目的信号逐渐减弱, 并且主要在核周区域表达。此外, PsH2A mRNA的相对表达量也表现出中华鳖卵巢发育的季节性变化。综上, 研究结果表明PsH2A在中华鳖卵母细胞发育过程中可能发挥着重要作用。

     

    Abstract: Histone H2A and its variants play an important role in many cellular processes. However, their role in the germ cells differentiation of turtle has not been well characterized yet. Here, we cloned the Chinese soft-shell turtle (Pelodiscus sinensis) PsH2A and analyzed its expression pattern and cellular location during oogenesis. The 575 bp PsH2A cDNA was isolated from P. sinensis, which contains an open reading frame (ORF) of 399 bp encoding a protein of 133 amino acid residues, a partial 5′ untranslated region (5′-UTR) of 68 bp and a partial 3′ untranslated region (3′-UTR) of 108 bp. Real-time quantitative PCR (RT-qPCR) result showed that PsH2A mRNA expression level was higher in ovary than other tissues (P<0.01), such as heart, liver, spleen, kidney, brain, muscle, and testis. Reverse transcription PCR (RT-PCR) result showed that PsH2A mRNA only detected in ovary. Moreover, in the male and female gonads of 1-year, 2-year and 3-year-old P. sinensis, the PsH2A mRNA was only significantly expressed in ovary (P<0.01). The results of chemical in situ hybridization (CISH) showed that PsH2A mRNA was specifically expressed in oocytes. When primary oocytes developed to stage Ⅲ, strong signal could be detected and evenly distributed in the cytoplasm. From growing follicular stage (stage Ⅶ oocytes) to mature follicular stage (stage Ⅸ oocytes), the PsH2A mRNA signal gradually weakened and spread in the cytoplasm around the nucleus. The signal was distributed in the perinuclear cytoplasm, but was undetected in the peripheral cytoplasm. Furthermore, the relative expression level of PsH2A mRNA also responded to seasonal change in ovarian development in P. sinensis. In 1-year-old and 3-year-old ovaries, the relative level of PsH2A mRNA increased as ovary developing. However, in 2-year-old ovary, the relative level of PsH2A mRNA increased first and then decreased, reaching a peak level in April. In addition, in different season, the relative expression of PsH2A mRNA showed a downward trend with age increasing. In summary, these findings suggest that PsH2A may play an important role in oogenesis of P. sinensis.

     

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