School of life sciences, Liaoning Normal University, Dalian 116081, China.
Department of Biology, Dalian Medical University, Dalian 116081, China.
Genes (Basel). 2019 Sep 30;10(10):768. doi: 10.3390/genes10100768.
The cell cycle checkpoint protein RAD9 is a vital cell cycle regulator in eukaryotic cells. RAD9 is involved in diverse cellular functions by oligomer or monomer. However, the specific mechanism of its activity remains unknown in crustaceans, especially in embryonic diapause resumption of the brine shrimp .
In the present article, a 1238 bp full-length cDNA of -RAD9 gene, encoding 376 amino acids, was obtained from . The expression pattern of -RAD9 was analyzed by qPCR and Western blot. The mRNA expression level climbs to the top at the 10 h stage of embryo development, while the protein expression pattern is generally consistent with qPCR results. Moreover, the -RADd9 related signaling proteins, -RAD1, -HUS1, -RAD17, and -CHK1, were also detected. Immunofluorescence assay showed that the location of -RAD9 did not show tissue or organ specificity, and the intracellular expression was concentrated in the cytoplasm more than in the nucleus. We also explored the amount of -RAD9 under the stresses of cold and high salinity, and the results indicate that -RAD9 is a stress-related factor, though the mechanisms may be different in response to different stresses. Knocking down of the -RAD9 gene led to embryonic development delay in .
All these results reveal that -RAD9 is necessary for post-diapaused embryonic development in .
细胞周期检查点蛋白 RAD9 是真核细胞中一种重要的细胞周期调控因子。RAD9 通过寡聚体或单体参与多种细胞功能。然而，其在甲壳类动物中的具体活性机制尚不清楚，特别是在卤虫胚胎休眠的恢复过程中。
本文从卤虫中获得了全长为 1238bp 的 -RAD9 基因 cDNA，编码 376 个氨基酸。通过 qPCR 和 Western blot 分析了 -RAD9 的表达模式。mRNA 表达水平在胚胎发育的 10 小时阶段达到峰值，而蛋白质表达模式与 qPCR 结果基本一致。此外，还检测到了 -RADd9 相关信号蛋白 -RAD1、-HUS1、-RAD17 和 -CHK1。免疫荧光分析表明，-RAD9 的位置没有组织或器官特异性，细胞内表达主要集中在细胞质中，而不是细胞核中。我们还研究了在冷胁迫和高盐胁迫下 -RAD9 的含量，结果表明 -RAD9 是一种应激相关因子，尽管在应对不同应激时机制可能不同。 -RAD9 基因的敲低导致 在 中的胚胎发育延迟。
这些结果表明，-RAD9 是 在 中休眠后胚胎发育所必需的。
