Abstract: 【Aim】 This study aims to explore the role of heat shock protein 70 (HSP70) genes in the process of resisting high temperature stress and to provide a theoretical basis for revealing the expansion mechanism of Hyphantria cunea and predicting its potential distribution area. 【Methods】 HSP70 genes of H. cunea were cloned by PCR and subjected to bioinformatical analysis. The expression characteristics of HSP70 genes in the day-2 4th instar newly molted larvae of H. cunea under 25, 30, 35 and 40℃ were detected by qPCR. The prokaryotic expression vector of HSP70 of H. cunea was constructed and induced to express in Escherichia coli BL21. The protein was purified by Ni²⁺-His column and verified by Western blot. Then, the ATPase activity of the recombinant protein obtained by prokaryotic expression was determined by in vitro experiments. 【Results】 The two HSP70 genes of H. cunea including HcHSP70 (GenBank accession no.: MT995848) and HcHSC70 (GenBank accession no.: MT261583) were cloned and sequenced. Their ORFs are 1 917 and 2 061 bp in length, encoding 637 and 687 amino acids with the predicted molecular weights of about 69.66 and 74.96 kD, and the isoelectric points of 5.90 and 5.96, respectively. The structure prediction conformed to the characteristics of the heat shock protein 70 family, which contains three highly conserved regions GIDLGTTYS, IFDLGGGTFDVSIL, and VGGSTRIPKVQ. The 3D structure of the two HSP70 proteins is composed of the N-terminal ATPase functional domain and C-terminal substrate binding domain. The phylogenetic tree showed that HcHSP70 and other members of the HSP70 family of Lepidoptera were clustered into one branch, while HcHSC70 and other members of the HSC70 family of Lepidoptera were clustered into another branch. The qPCR results showed that the expression of HcHSP70 in the day-2 4th instar newly molted larvae of H. cunea was significantly upregulated under heat stress and reached the peak under 35℃ for 2 h, while HcHSC70 had a weak expression response under heat stress. The prokaryotic expression vector of HcHSP70 was successfully constructed, and HcHSP70 was expressed in vitro. The purified recombinant protein HcHSP70 had ATPase activity, which was stable under high temperature stress. 【Conclusion】 In this study, HcHSP70 and HcHSC70 of H. cunea were cloned, and their expression characteristics under high temperature were confirmed. The prokaryotic expression and purification of HcHSP70 were successfully performed. The recombinant HcHSP70 has stable ATPase activity under high temperature, suggesting that it may play an important role in the response of H. cunea to high temperature stress.

Key words: Hyphantria cunea, heat shock protein 70, heat stress, prokaryotic expression, molecular chaperone