Expression and function analysis of two heat shock protein 70 genes ( Hsp70s) involved in different high-temperature stresses in Zeugodacus cucurbitae (Diptera: Tephritidae)

Abstract

The melon fly, Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae), is an important quarantine pest prevalent worldwide. Heat shock protein 70s (Hsp70s) are crucial proteins involved in thermal stress tolerance. In the current study, we identified and cloned four Hsp70 genes from melon fly, namely ZcHsp70-1, ZcHsp70-2, ZcHsp70-3 and ZcHsp70-4. Phylogenetic analysis showed that they had a close genetic relationship with Hsp70s from Diptera insects. Their spatio-temporal expression showed that they were highly expressed in the midgut of 5-d-old adults. After a moderate high-temperature (38°C) induction, only ZcHsp70-1 and ZcHsp70-2 were found to be up-regulated by 6.10-fold and 7.76-fold, respectively. After extreme high-temperature (42°C) induction, only ZcHsp70-1 was found to be up-regulated to a very high level by 117.87-fold. ZcHsp70-1 and ZcHsp70-2 were heterologously expressed in Escherichia coli, and the ATPase activity of the purified recombinant proteins was significantly increased upon 38°C and 42°C treatment. These recombinant proteins also effectively improved the tolerance of E. coli in response to high-temperature stress. In vivo, separate suppression of ZcHsp70-1 or ZcHsp70-2 expression did not affect the mortality of melon fly to high-temperature stress of 38°C, whereas suppression of both genes increased the mortality. In addition, only suppression of ZcHsp70-1 expression increased the mortality to extremely high-temperature stress of 42°C. Our data show that ZcHsp70-1 and ZcHsp70-2 play essential roles in high-temperature tolerance but differ in their relationship to the extreme high-temperature tolerance of the melon fly.