Molecular characterization of Calreticulin: A biomarker for temperature stress responses of the giant tiger shrimp Penaeus monodon

Abstract

In crustaceans, calcium signaling plays important roles in growth, reproduction and molting. Calreticulin (CRT) is a main protein involved in calcium homeostasis of eukaryotes. The full-length cDNA of CRT in the giant tiger shrimp ( Penaeus monodon), identified by RACE-PCR, was 1682 bp in length, containing an ORF of 1221 bp corresponding to a deduced protein of 406 amino acids. Genomic sequence of PmCRT spanned 3006 bp, composing of 4 exons (85, 119, 187 and 830 bp) and 3 introns (411, 231 and 120 bp). Semi-quantitative RT-PCR revealed that PmCRT in hemocytes of juvenile P. monodon was up-regulated at 0 and 1 h post treatment (hpt) at 35 °C for 3 h ( P < 0.05). However, expression levels of PmCRT in gills and hepatopancreas after the temperature stress (0–48 hpt) were not significantly different ( n = 3 for each group; P > 0.05). Quantitative real-time PCR confirmed the expression profile of PmCRT in hemocytes and illustrated that this transcript was up-regulated at 0 and 3 hpt for approximately 25 fold ( n = 5; P < 0.05), reduced to about 5 fold between 3 and 12 hpt ( P < 0.05) and returned to the baseline level at 24 and 48 hpt ( P > 0.05). Recombinant PmCRT was successfully expressed in vitro and exhibited an ability to form a complex with recombinant Endoplasmic Reticulum protein 57 of P. monodon (rPmERp57). Results from this study strongly suggested that PmCRT can be regarded as a biomarker for temperature stress responses in P. monodon.