National Demonstration Center for Experimental Fisheries Science Education, Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai 201306, China.
National Demonstration Center for Experimental Fisheries Science Education, Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai 201306, China.
Gene. 2023 Apr 30;862:147262. doi: 10.1016/j.gene.2023.147262. Epub 2023 Feb 8.
Rad21/Rec8 family proteins are vital for sister chromatid segregation in mitosis and homologous recombination in meiosis, but no molecular data are available in crustacean species. In this study, a germ cell-specific Rad21 named EsRad21 was identified in the crab Eriocheir sinensis. EsRad21 mRNA has an open reading frame of 2310 base pairs (bp) encoding a 769 amino acids (aa) protein. RT-PCR showed that EsRad21 mRNA was particularly expressed in testis and ovary. The RT-qPCR results further revealed that the EsRad21 mRNA exhibited similar expression pattern in gonads at various developmental stages. EsRad21 mRNA expression level was the highest in testis at early spermatogenesis stage and ovaries at previtellogenesis stage, thereafter decreased significantly at middle spermatogenesis and vitellogenesis, and finally reach the lowest level at late spermatogenesis and vitellogenesis. In situ hybridization (ISH) analysis showed that EsRad21 mRNA was exclusively expressed in germline cells, but not in gonadal somatic cells. Notably, hybridized signal was detected on chromosomes of metaphase spermatocytes. EsRad21 is thus an underlying helpful indicator of the early phases of germ cell development. RNAi knockdown of EsRad21 downregulated the expression of other meiosis-related genes like Smc5-Smc6 and SPO11 and resulted in high mortality of individuals after 24 h post injection of EsRad21 dsRNA. Taken together, our results showed a potential role for EsRad21 in early meiosis of oocytes and spermatocytes in E. sinensis. This is the first report on the molecular characterization of the Rad21 transcript in a crustacean species.
Rad21/Rec8 家族蛋白对于有丝分裂中的姐妹染色单体分离和减数分裂中的同源重组至关重要，但在甲壳动物物种中没有分子数据。本研究在中华绒螯蟹（Eriocheir sinensis）中鉴定了一种生殖细胞特异性的 Rad21，命名为 EsRad21。EsRad21 mRNA 具有一个开放阅读框，长 2310 个碱基对（bp），编码一个 769 个氨基酸（aa）的蛋白质。RT-PCR 显示 EsRad21 mRNA 特别在睾丸和卵巢中表达。RT-qPCR 结果进一步表明，EsRad21 mRNA 在不同发育阶段的性腺中具有相似的表达模式。EsRad21 mRNA 的表达水平在早期精母细胞期的睾丸和前卵黄发生期的卵巢中最高，随后在中精母细胞期和卵黄发生期显著下降，最后在晚期精母细胞期和卵黄发生期达到最低水平。原位杂交（ISH）分析表明，EsRad21 mRNA 仅在生殖细胞中表达，而不在性腺体细胞中表达。值得注意的是，在中期精母细胞的染色体上检测到杂交信号。因此，EsRad21 是生殖细胞早期发育的潜在有用指标。EsRad21 的 RNAi 敲低下调了其他减数分裂相关基因如 Smc5-Smc6 和 SPO11 的表达，并导致在注射 EsRad21 dsRNA 24 小时后个体死亡率很高。总之，我们的研究结果表明 EsRad21 在中华绒螯蟹卵母细胞和精母细胞的早期减数分裂中起重要作用。这是在甲壳动物物种中首次报道 Rad21 转录本的分子特征。
