Cloning and expression characteristics of the HaHsp70 gene from Harmonia axyridis under temperature stress

Abstract

This study aimed to elucidate the physicochemical properties of the heat shock protein 70 (Hsp70) gene HaHsp70 in Harmonia axyridis and its expression patterns under temperature stress. This will provide a theoretical basis for investigating the function of this gene and the molecular mechanisms underlying thermal adaptation. The full-length cDNA of HaHsp70 was amplified using a polymerase chain reaction (PCR). Bioinformatic analyses, including sequence characterization, conserved domain prediction were performed, and a phylogenetic tree was constructed using the neighbor-joining method implemented in MEGA-X. HaHsp70 expression was analysed across different developmental stages and tissues, as well as under 4 °C cold and 37 °C heat stresses via real-time quantitative PCR (RT-qPCR). HaHsp70 protein (GenBank accession no. PX777786), identified in this study, is 2225 bp in full length, with a 1938 bp open reading frame encoding 645 amino acids. The encoded 70.74 kDa acidic protein (theoretical pI 5.61) contains the conserved cytoplasmic signature motif ‘EEVD’ at the C-terminus, confirming that it is a cytosolic Hsp70 protein. Phylogenetic analysis revealed that HaHsp70 showed the closest phylogenetic relationship with Coccinella septempunctata Hsp70 and clustered within the Coleoptera clade, indicating a high degree of evolutionary conservation among beetle Hsp70 proteins. In contrast,, HaHsp70 showed the most distant phylogenetic relationship with Hsp70 from Mamestra brassicae, a lepidopteran insect. RT-qPCR analysis showed that HaHsp70 is highly expressed in second-instar larvae (relative expression 14.67),female adult antennae (relative expression 7.82), and male adults (relative expression 368.84). Its expression peaked at 60 min under a 4 °C cold shock in both sexes, while a 37 °C heat shock induced a sharp upregulation that peaked at 60 min (1069.22) in males and at 90 min (751.67) in females. These results indicate that its expression patterns are dependent on sex and time. These findings indicate that HaHsp70 encodes a cytosolic Hsp70 that has the potential to act as a molecular chaperone. It may be associated with the cold stress response, and its expression pattern is consistent with a functional role in larval overwintering and extreme temperature tolerance in H. axyridis.