Molecular cloning and expression of the stress gene HSP70 in the marine crab Charybdis japonica (A. Milne-Edwards, 1861) (Decapoda: Brachyura: Portunidae) in response to ammonia-N, nitrite-N, and sulfide exposure

A full-length complementary DNA sequence of heat-shock protein 70 (HSP70) was cloned from the hepatopancreas of Charybdis japonica (A. Milne-Edwards, 1861) through reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The expression profile of HSP70 in different tissues of C. japonica and its expression upon exposure to ammonia-N, nitrite-N, and sulfide was also investigated. The full-length cDNA of HSP70 was 2203 bp long and contained a 107 bp 5' untranslated region (UTR), a 143 bp 3' UTR, and a 1953 bp open reading frame, which distributed 108-2060 bp and encoded a polypeptide of 650 amino acids (aa) with a calculated molecular mass of 71.24 kDa and an isoelectric point of 5.34. Three signature sequences of the HSP70 family, IDLGTT-S-V (residues 918), IFDLGGGTFDVSIL (197-210 aa), and IVLVGGSTRIPKIQK (334-348 aa) were detected in the predicted amino-acid sequence. The HSP70 gene of C. japonica showed the highest similarity (>90%) to HSP70 from other crustacean species. The HSP70 gene was expressed in all tissues tested, and its abundance in these tissues can be arranged as hepatopancreas > stomach > gill > heart > ova > muscle. The expression of HSP70 was significantly induced after the stress of ammonia-N, nitrite-N, and sulfide, and the induced degree showed a positively correlation with increasing concentrations. The HSP70 expression reached a peak after 3 or 6 h of stress, which suggests that the HSP70 expression was affected quickly by the changes of environmental factors.