Molecular characterization and expression analysis of calreticulin cDNA from Hyriopsis cumingii

Abstract

In order to study genes related to pearl formation in freshwater pearl mussel and their regulation mechanism,a 1 437 bp cDNA sequence of calreticulin gene from Hyriopsis cumingii(HcCRT)was obtained by rapid amplification of cDNA ends(RACE).It consisted of a 231 bp 5′-untranslation region(UTR),a 615 bp 3′UTR and a 591 bp open reading frame(ORF).The inferred amino acids sequence was composed of 196 amino acids,including a signal peptide of 21 amino acids and a mature peptide of 175 amino acids.The molecular weight of the peptide was predicted to be 22.4 ku,with a theoretical isoelectric point of 5.01.Amino acid sequence analysis showed that there was no obvious amino acid sequence of membrane domain.Results of the ProtScale online analysis showed that the protein was hydrophilic protein.Homology analysis indicated that HcCRT amino acid had a conservative sequence of calreticulin family and was highly conserved with Danio rerio(77%),Crassostrea gigas(70%),Pinctada fucata(70%).The sequence analysis showed that HcCRT shared two potential calreticulin family signature motifs with CRT from other species,KHEQNIDCGGGYLKVF and IMFGPDICG.The prediction results of H.cumingii HcCRT protein secondary structure and tertiary structure indicated that the protein contains the alpha helix and beta folding.Real-time quantitative PCR(RT-PCR)showed that HcCRT is expressed in a wide range of tissues including the mantle,blood,gill,foot,liver,kidney,intestine and muscle,with the highest level of transcripts in mantle,followed by blood,while there is rare expression in other tissues.These data suggested that HcCRT might be involved in pearl formation of H.cumingii.This study which was first time to obtain HcCRT gene from H.cumingii played an important role in exploring pearl formation of H.cumingii.