Abstract
High temperatures are one of the most important abiotic stressors affecting the survival and growth of American shad (Alosa sapidissima). Building on previous omics sequencing studies of A. sapidissima liver and gills under high temperature stress, this study focused on investigating the regulatory role of miR-1236-3p and its target gene hsp90b1. The results indicate that the full-length cDNA of the hsp90b1 gene is 2023 bp and comprises a 5' end of 58 bp, a 3' end of 84 bp, and a coding region of 1881 bp, encoding 626 amino acids. Sequence alignment and phylogenetic tree analysis reveal that the hsp90b1 sequence is highly conserved across species. In situ hybridization showed that hsp90b1 is mainly localized in the cytoplasm. Software prediction identified a potential binding site between miR-1236-3p and hsp90b1. Through the construction of wild-type and mutant 3'UTR hsp90b1 dual luciferase reporter plasmids, the targeted relationship between the two was confirmed. In addition, the spatiotemporal expression levels of the hsp90b1 was found to be highest in the multicellular stage and liver tissue at a cultivation temperature of 27 °C; miR-1236-3P was highly expressed in the hatching stage and heart tissue at 30 °C. These findings provide a theoretical foundation for further investigating the regulatory role of non-coding RNA in A. sapidissima heat stress and offer data for subsequent molecular breeding studies.