Molecular cloning and characterization of two heat shock proteins in Thitarodes pui (Lepidoptera: Hepialidae).

Heat shock proteins (HSPs) show transient expression in response to rapid temperature increase. The larvae of Thitarodes insects are the host of Ophiocordyceps sinensis, with high cold-tolerance. In order to study the adaptive mechanisms to temperature change, we cloned and sequenced the full-length cDNAs of two HSP genes (designated as tp-hsp90 and tp-hsp70) using the technique of rapid amplification of cDNA ends (RACE) from T. pui. The complete cDNA sequences of tp-hsp90 and tp-hsp70 are 2,842 bp and 2,169 bp long, encoding polypeptides of 712 and 651 amino acids with molecular weights of 81.57 and 71.27 kDa respectively. They show significant sequence similarity to homologous genes in insects. The inferred amino acid sequences of tp-hsp90 and tp-hsp70 are characterized by conserved features of HSP family: the proteins contain five signature sequences of HSP90 and three signatures of HSP70, respectively. Real-time quantitative reverse transcription-PCR (qRT-PCR) analyses show that tp-hsp90 expression is up-regulated in October and December, followed by a gradual rebound in January, March and May; while tp-hsp70 expression does not change significantly during the same period. These results suggest that tp-hsp90, rather than tp-hsp70, responds to temperature changes and should play a key role in cold tolerance in Thitarodes pui.