COMPLEMENTARY DNA CLONING AND TISSUE EXPRESSION PROFILE OF CHAPERONIN-CONTAINING T-COMPLEX POLYPEPTIDE 1-BETA IN PINCTADA MAXIMA EXPOSED TO COLD STRESS

Chaperonin-containing T-complex polypeptide 1 (CCT) is a molecular chaperone in the cytoplasm of eukaryotic cells involved in the correct folding and assembly of proteins, which plays an important role in cold-stress response. This study cloned two transcripts of Pinctada maxima CCT-beta composed of 1,829 bp (CCT-betal) and 2,289 bp (CCT-beta2), respectively, using rapid amplification of cDNA ends. Both transcripts possess 105 bp of 5' noncoding region and the same coding region, bul contain different 3' noncoding regions of 591 bp (CCT-betal) and 131 bp (CCT-beta2), respectively. The open reading framework (ORF) consisted of 1,593 bp that encodes 530 amino acids. The CCT-beta protein has three conserved functional domains (equatorial, apical, and intermediate domains), three CCT-family signature sequences, nine conserved ATP-binding sites, one DEAD box motif, and three protein-binding sites. The CCT-beta is highly conserved, with the cDNA homologies between P. maxima and Crassostrea gigas, Danio rerio, and Homo sapiens being 90.4%. 76.8%, and 75.8%, respectively. Real-time quantitative PCR showed that CCT-beta was differentially expressed in different organs of P. maxima, with mRNA contents declining from the gill, adductor muscle, foot, labial palps, mantle, and heart to hepatopancreas. Under cold stress, CCT-beta mRNA expression in various organs increased, but the patterns of increase varied among organs. The transcriptional level of CCT-beta peaked at 6h in the heart and hepatopancreas, and decreased significantly at 12h. In the adductor muscle, gill, and mantle, CCT-beta mRNA increased significantly at 12h and declined markedly at 24h in cold stress-exposed animals. These results indicate that CCT-beta is a cold-stress response gene, but the response pattern varies among organs. Organs with low CCT-beta basal expression may be more sensitive to cold stress than the organs with relatively high basal expression content. KEY WORDS: Pinctada maxima, CCT-beta, cDNA, cold stress
INTRODUCTION Chaperonin-containing T-complex polypeptide 1 (CCT) is a kind of type II chaperonin that is localized in the cytoplasm of eukaryotes (Frydman et al. 1992, Gao et al. 1992, Lewis [...]