Molecular cloning and expression of the calreticulin gene of the Pacific white shrimp, Litopenaeus vannamei, in response to acute hypo-osmotic stress

Abstract

Calreticulin is a ubiquitous and highly conserved calcium-binding protein that is located in the endoplasmic reticulum (ER). In the present study, the cDNA of the Litopenaeus vannamei calreticulin (LvCRT) gene was cloned using the rapid amplification of cDNA ends (RACE) technique. Its full-length cDNA was 1866bp in length, which included a 1221-bp open reading frame and encoded a 406-amino acid. In addition, a 222-bp 5′ untranslated region (UTR) and a 423-bp 3′ UTR with a polyadenylation [poly(A)] signal (AATAAA) located 27bp upstream of the poly(A) tail were identified. The predicted molecular weight and isoelectric point of the protein were 45.3kDa and 4.90, respectively. Protein analysis revealed that LvCRT contains a conserved calreticulin family signature sequence (KHEQNIDCGGGYLKVF), a signal peptide (MKTWVFLALFGVVLVES), and a conserved endoplasmic reticulum HDEL retention sequence. Furthermore, NCBI BLASTX and phylogenetic analyses showed that LvCRT has a high degree of sequence homology with CRT proteins from Fenneropenaeus chinensis and Penaeus monodon. In addition, qRT-PCR analysis indicated that the highest level of LvCRT expression was observed in the intestines, whereas the lowest was in the muscle. When the shrimps were transferred from salinity 30 to 2psu diluted seawater, the expression level of LvCRT in the hepatopancreas and hemocytes sharply increased at 6h and 12h (which were about 3.23-fold and 2.01-fold of that in the 0h control group), and then, significantly decreased at 96h and 24h (p<0.05). The expression of LvCRT in the intestines and gills was not affected by the low-salinity stress (p>0.05). In conclusion, the responses of L. vannamei to changes in salinity via the calreticulin protein facilitate a better understanding of its function in relation to stress.