Genome-Wide CRISPR/Cas9 Screening Reveals BmM-ALP Orchestrates the Antioxidant Response and Metabolic Adaptations for Heat Resistance in Bombyx mori.

Climate change-induced heat stress represents an increasing threat to biodiversity, engendering the need to understand evolutionary adaptations to thermal extremes. Using Bombyx mori as an economically important insect and research model, we employed genome-wide CRISPR/Cas9 screening integrated with high-throughput sequencing to systematically identify adaptations for heat resistance. The analysis identified the Bombyx mori membrane-bound alkaline phosphatase-like gene (BmM-ALP) as a vital thermoregulator. BmM-ALP triggered a pleiotropic protective cascade by significantly decreasing reactive oxygen species (ROS), suppressing apoptosis, and reprogramming mitochondrial metabolism via the phosphorylation of Vitamin B1 (VB1), suggesting a potential role in sustaining oxidative phosphorylation and enhancing energy metabolism under stress. Transgenic validation experiments confirmed the consistency of this strategy, and the BmM-ALP-OE strain displayed significantly enhanced thermal tolerance and prolonged survival under high-temperature stress. Beyond establishing BmM-ALP as a key gene of heat resistance in silkworms, this study uncovered a regulatory axis linking BmM-ALP to Vitamin B1 metabolism, illustrating a novel connection between energy homeostasis and thermal adaptation. The findings provide new insights that can be applied to breeding resistant strains in agriculture and biodiversity conservation in the context of global warming.