ChCHK1 participates in the hyper-salinity stress response by arresting the cell cycle and regulating apoptosis in Crassostrea hongkongensis

Crassostrea hongkongensis (C. hongkongensis) is an important farmed oyster species living in estuaries that suffers frequent threats from salinity fluctuation. The specific strategy employed by C. hongkongensis to resist hyper-salinity stress remains unknown. In this study, a novel CHK1 gene was first characterized from C. hongkongensis . The ChCHK1 gene contained an open reading frame of 1458 bp, encoding a deduced peptide of 485 amino acids, with a conserved leucine-rich STKc-Chk1 domain. Multiple sequence alignment and phylogenetic tree analysis showed that ChCHK1 shared the highest identity (99.2%) with CHK1 protein of Crassostrea gigas . Tissue expression analysis showed that ChCHK1 had the highest expression in gill, gonad, and adductor, and was significantly up-regulated after hyper-salinity stress for 48 h. Second, ChCHK1 silencing significantly enhanced Caspase 3 and Caspase 8 activity and increased the apoptotic cell proportion, demonstrating that ChCHK1 responds to hyper-salinity stress by regulating apoptosis. Meanwhile, ChCHK1 silencing also reduced p53 phosphorylation, indicating that ChCHK1 regulates apoptosis by phosphorylating p53 to resist hyper-salinity stress. Moreover, transcript levels of ATM , CHK2 , p53 , and Caspase 3/8/9 all rose after ChCHK1 silencing, suggesting that the ATM–CHK2–p53 pathway is activated to compensate for ChCHK1 loss and induce apoptosis during hyper-salinity stress. Third, ChCHK1 silencing significantly promoted transcription of cell cycle-related genes, recovered cell cycle progression, and decreased CDC25A phosphorylation. This demonstrates that ChCHK1 arrests the cell cycle by phosphorylating CDC25A in response to hyper-salinity stress. The compelled recovery of the cell cycle after ChCHK1 silencing may increase the risk of aberrant DNA replication and lead to more apoptosis. Finally, interestingly, ChCHK1 silencing boosted oyster survival rate under hyper-salinity stress by 6.2-fold, nearly reaching the blank control level. This indicates that blocking ChCHK1 enhances salinity tolerance, providing a key strategy for anti-salt genetic breeding. Thus, these results demonstrate that ChCHK1 participates in the hyper-salinity stress response by arresting the cell cycle and regulating apoptosis in C. hongkongensis .