Overexpression of small heat shock protein 21 protects the Chinese oak silkworm Antheraea pernyi against thermal stress

Abstract

Small heat shock proteins (sHSPs) usually act as molecular chaperones to prevent proteins from being denatured in extreme conditions. We first report the sHSP21 gene, named as Ap-sHSP21, in the Chinese oak silkworm Antheraea pernyi (Lepidoptera: Saturniidae). The full-length cDNA of Ap-sHSP21 is 976 bp, including a 5'-untranslated region (UTR) of 99 bp, a 3'-UTR of 316 bp and an open reading frame (ORF) of 561 bp encoding a polypeptide of 186 amino acids. The deduced A. pernyi sHSP21 protein sequence reveals the percent identity is 82-93% in comparison to other sHSPs from insects. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis shows that Ap-sHSP21 expression is higher in testis than that in other examined tissues and significantly up-regulated after heat shock. In addition, prokaryotic expression and purification of the Ap-sHSP21 protein were performed. SDS-PAGE and Western blot analysis demonstrated that a 25 kDa recombinant protein was successfully expressed in Escherichia coli cells and the purified recombinant protein was also confirmed to protect restriction enzymes from thermal inactivation. The expression of Ap-sHSP21 was significantly down-regulated after RNA interference, which was confirmed by qRT-PCR and Western blot analysis. All together, these results suggest that Ap-sHSP21 play a key role in thermal tolerance.

Keywords: Antheraea pernyi; Expression; Heat shock protein; Temperature.