Abstract: In order to study the mechanism of stress-resistance, a heat shock protein gene hsp70 (named as Tmhsp70), was cloned from the larvae of Tenebrio molitor by PCR and RACE method, and the mRNA levels in developmental stages were detected by using semi-quantitative RT-PCR as well. The results showed the full sequence of Tmhsp70 cloned is 2 282 bp in length containing a 115 bp 5′ untranslated region (5′ UTR) rich in adenine, a 1 935 bp open reading frame and a 232 bp 3′ untranslated region (3′ UTR) rich in adenine and thymine. It also has seven repeats of the heat shock element nGAAn in its 5′ UTR and a 22 bp Poly (A) tail in the 3′ UTR. The deduced heat shock protein named as TmHSP70 contains three signature motifs of HSP70, i.e., IDLGTTYS, IFDLGGGTFDVSIL and IVLVGGSTRIPKIQQ as well as the terminal EEVD motif which is characteristic to cytoplasmic HSP70s. TmHSP70 has neither a signal peptide nor a transmembrane domain. It contains two main functional domains: a 42 kDa highly conserved N-terminal ATPase domain and a 18 kDa conserved C-terminal peptide-binding domain. The tertiary structure of ATPase domain is composed of two large globular subdomains and contains a nucleotide-binding core. Tertiary structure of the peptide-binding domain forms a sandwich of 2 four-stranded β-sheets and two α-helices, and includes a peptide-binding cavity. Furthermore, the expression of Tmhsp70 mRNA in T. molitor was characterized by heat-inducible and developmentalregulation feature. The overall increase in the levels of Tmhsp70 mRNA in different life stages when the larvae were exposed to 42℃ for 1 h, ranged from 1.4- to 26.9-fold on the basis of semi-quantitative RT-PCR analysis. At 25℃ Tmhsp70 mRNA expressed in 1-day old pupae was higher than that accumulated in other developmental stages, and after exposure to 42℃ for 1 h, Tmhsp70 mRNA expressed in 90-day old larvae became the most abundant, and was not only higher than that accumulated in 30- and 60-day old larvae but also higher than that accumulated in 15- and 30-day old adults. The results form a basis for further research on structure, function and expression regulation of HSPs from T. molitor as well as the relationship between HSPs and stress-resistance in the beetle.

Key words: Tenebrio molitor, heat shock protein (HSP), gene cloning, bioinformatics, mRNA expression, heat shock