Cloning of down-regulated genes under cold stress and identification of important genes related to cold tolerance in zebrafish (Danio rerio).

Low-temperature stress poses a significant risk to the survival of both cultivated and wild fish populations. Existing studies have found that the pre-acclimation of fishes to moderate cold stress can stimulate the activation of acclimation pathways, thereby enhancing their tolerance to cold stress. The fitness of fish relies heavily on appropriately controlled transcriptional reactions to environmental changes. Despite previous characterization of gene expression profiles in various fish species during cold acclimation, the specific genes responsible for essential functions in this process remain largely unknown, particularly the down-regulated genes induced by cold acclimation. To investigate the genes involved in cold acclimation, this study employed real-time quantitative PCR (RT-qPCR), molecular cloning, microinjection techniques, and cold stress experiments to determine the genes that play an essential part in cold acclimation. Consequently, 18 genes were discovered to be down-regulated in larval zebrafish experiencing cold stress. All 18 genes successfully detected overexpression in zebrafish at 96 and 126 hpf (fold change ≥3), which declined with the growth of zebrafish. Following microinjection, it was observed that her8a, cyp51, lss, txnipb, and bhlha9 had an adverse impact on the survival rate of zebrafish larvae under cold stress. These genes have been identified to play significant roles in various biological processes. For instance, bhlha9 has been found to be involved in both limb development and temperature sensing and her8a has been implicated in neural development. Additionally, cyp51 and lss have been identified as participants in the cholesterol synthesis pathway. Txnipb has been reported to induce cell apoptosis, thereby potentially influencing the survival rate of zebrafish larvae under cold stress. These findings offered crucial data for the analysis of molecular processes related to cold tolerance and the development of cold-resistant fish breeding.