Investigation of the Role of miR-1236-3p in Heat Tolerance of American Shad (Alosa sapidissima) by Targeted Regulation of hsp90b1

High temperatures are one of the most important abiotic stressors affecting the survival and growth of American shad (<i>Alosa sapidissima</i>). Building on previous omics sequencing studies of <i>A. sapidissima</i> liver and gills under high temperature stress, this study focused on investigating the regulatory role of miR-1236-3p and its target gene <i>hsp</i>90<i>b</i>1. The results indicate that the full-length cDNA of the <i>hsp</i>90<i>b</i>1 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 <i>hsp</i>90<i>b</i>1 sequence is highly conserved across species. In situ hybridization showed that <i>hsp</i>90<i>b</i>1 is mainly localized in the cytoplasm. Software prediction identified a potential binding site between miR-1236-3p and <i>hsp</i>90<i>b</i>1. Through the construction of wild-type and mutant 3'UTR <i>hsp</i>90<i>b</i>1 dual luciferase reporter plasmids, the targeted relationship between the two was confirmed. In addition, the spatiotemporal expression levels of the <i>hsp</i>90<i>b</i>1 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 <i>A. sapidissima</i> heat stress and offer data for subsequent molecular breeding studies.