High-throughput screening of thermal tolerant candidate genes in the ivory shell (Babylonia areolata) based on the yeast strain INVSc1

Global climate warming and frequent heatwaves present significant threats to the growth and survival of marine organisms. The ivory shell, Babylonia areolata, plays a crucial role in marine aquaculture as a widely distributed mollusk in tropical and subtropical seas. However, limited research has been conducted on its molecular mechanisms in response to heat stress. This study aims to explore thermal-tolerant related genes and regulatory pathways by constructing a cDNA library under heat stress and using a yeast-based high-throughput screening method. Following exposure of three populations to acute heat stress, a heat stress cDNA library was constructed with a capacity of 1.104 × 10⁸, containing 2.208 × 10⁸ clones. Subsequently, the library was transformed into yeast INVSc1 and underwent high-temperature screening at 39 °C. All positive clones were then subjected to next-generation sequencing (NGS) for rapid identification of 1148 candidate genes associated with thermal tolerance. Enrichment analysis revealed that these genes were significantly enriched in seven KEGG pathways, including Protein processing in endoplasmic reticulum, Ribosome and Ubiquitin mediated proteolysis. Additionally, through first-generation sequencing of 96 randomly selected positive clones at 39 °C, we identified 51 unique sequences associated with heat stress which included previously reported genes like EEF2, HSPB1, UBC and HSPA4. Subsequent yeast heat tolerance experiments further validated the essential role played by these 51 genes in response to thermal stress conditions. Finally, RNA-seq data provided evidence for upregulated expression levels of these genes during exposure to elevated temperatures. This study successfully constructed the first cDNA library for B. areolata under heat stress conditions, identified key pathways and candidate genes associated with thermal tolerance, and demonstrated the applicability of yeast high-throughput screening methods in investigating stress resistance traits in invertebrates. These findings contribute to a deeper understanding of the strategies employed by B. areolata to respond to heat stress, and provide technical support for studying the molecular mechanisms underlying abiotic stress responses in aquatic organisms.