MOLECULAR CLONING, CHARACTERIZATION AND EXPRESSION OF CHAPERONIN CONTAINING T-COMPLEX POLYPEPTIDE SUBUNIT (ZETA) TRANSCRIPT FROM ABALONE HALIOTIS DISCUS HANNAI INO IN RESPONSE TO DIETARY ZINC

Chaperonin containing T-complex polypeptide (CCT) is believed to play crucial role in mediating protein folding and correlating with better cell growth in the physiological processes. In this study, the chaperonin containing T-complex polypeptide subunit (zeta) was cloned from abalone Haliotis discus hannai Ino (HdhCCTζ) by homology cloning with degenerate primers and RACE techniques. The full length of HdhCCTζ cDNA was 1960 bp with a 1599 bp open reading frame (ORF) encoding 532 amino acids with a calculated molecular mass of 58.46 kD and a predicted isoelectric point of 6.53. BLAST analysis revealed that HdhCCTζ shared high identities with other known CCTs from mammal, bird and fish, etc. Sequence characterization revealed that HdhCCTζ contained three characteristic signature motifs (35RTNLGPKGTLKML47, 56LTKDGNVLLHEMQIQHP72 and 84QDDITGDGT92). And there was a putative adenosine triphosphate-binding domain (89GDGTTS94) in HdhCCTζ. In Addition, three potential N-glycosylation sites (23NISA26, 128NKSL131 and 234NVSL237) were also identified in HdhCCTζ. Relatively higher levels mRNA expression were detected in gonad, haemocytes and hepatopancreas by real-time PCR assays. And the relative expression levels of HdhCCTζ mRNA in hepatopancreas and haemocytes were detected by real-time PCR in abalone fed with four different diets containing graded levels of zinc (6.69, 33.85, 710.63 and 3462.5 mg/kg) for 20 weeks, respectively. The results showed that the expression levels of HdhCCTζ mRNA were higher at dietary zinc (33.85 mg/kg, 710.63 mg/kg and 3462.5 mg/kg) than these at zinc-deficient diets. But HdhCCTζ mRNA expression levels were down-regulated by excessive concentration of dietary zinc (3462.5 mg/kg) compared with optimal dietary zinc (33.85 mg/kg). It is implied that the expression levels of HdhCCTζ could be affected by dietary zinc in the hepatopancreas and haemocytes, and the relative higher expression of HdhCCTζ was potentially involved in the anti-stress responses in Pacific abalone H. discus hannai.