Cloning of the heat shock protein 60 gene from the stem borer, Chilo suppressalis, and analysis of expression characteristics under heat stress

Abstract

Heat shock protein 60 is an important chaperonin. In this paper, hsp60 of the stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), was cloned by RT-PCR and rapid amplification of cDNA end (RACE) reactions. The full length cDNA of hsp6 degrees Consisted of 2142 bp, with an ORF of 1719 bp, encoding 572 amino acid residues, with a 5'UTR of 158 bp and a 3'UTR of 265 bp. Cluster analysis confirmed that the deduced amino acid sequence shared high identity with the reported sequences from other insects (77%-86%). To investigate whether hsp60 in C. suppressalis responds to thermal stress, the expression levels of hsp60 mRNA in larval haemocytes across temperature gradients from 31 to 39 degrees C were analysed by real-time quantitative PCR. There was no significant difference for hsp60 expression from 28 to 31 degrees C. he temperatures for maximal induction of hsp60 expression in haemocytes was close to 36 degrees C. Hsp60 expression was observed by using flow cytometry. These results revealed that thermal stress significantly induced hsp60 expression and Hsp60 synthesis in larval haemocytes, and the expression profiles of Hsp60 at the mRNA and protein levels were in high agreement with each other from 33 to 39 degrees C.

Figure 1.

The complete cDNA sequence and predicted amino acid sequence of Hsp60 of Chilo suppressalis. A classical mitochondrial Hsp60 signature is shown in box, and a typical GGM repeat motif at the C terminus is underlined. The stop codon is marked with an asterisk. High quality figures are available online.

Figure 2.

Phylogenetic tree based on amino acid sequences of mitochondrial Hsp60 (constructed using the Clustal W programme). The GenBank accession numbers for amino acid sequence data were shown in the brackets. High quality figures are available online.

Figure 3.

Expression analysis of hsp60 mRNA of haemocytes from Chilo suppressalis under different heat-shock temperature by Real-time PCR. Values are means ± S.D. of three independent experiments in triplicate. **p < 0.05 vs. control, **p < 0.01 vs. control. High quality figures are available online.

Figure 4

Expression levels of Hsp60 of haemocytes from Chilo suppressalis under different heat-shock temperature by using Flow cytometry. Values are means ± S.D. of three independent experiments in triplicate. *p < 0.05 vs. control. **p < 0.01 vs. control. High quality figures are available online.