Molecular Cloning and Expression Analysis of C-Type Lectin from Scapharca broughtonii

Abstract:

The current study cloned the full-length cDNA of C-type lectin (Sb-Lec1) using RACE (Rapid amplification of cDNA ends) method from Scapharca broughtonii with 700 bp that includes a 5′ UTR of 29 bp and 3′ UTR of 167 bp. The 504 bp open reading frame (ORF) encodes a polypeptide of 167 amino acids, including a signal peptide of 23 amino acids, one carbohydrate-recognition domain (CRD) motif of 129 amino acids and 6 cysteines involved in the formation of disulfide bond. The predicted protein molecular weight is 19.11 kDa, with a theory isoelectric point of 4.74. Multiple sequences alignment and phylogeny analysis showed that the identity of Sb-Lec1 gene shared with Crassostrea gigas, Mytilus galloprovincialis, and Argopecten irradians was 38%~40%, 34%~35%, and 38%~39%, respectively. The amino acids of CRD motif had many similarities with other species such as 4 conserved Cys. Phylogenetic analysis revealed two main branches including all C-type lectin of molluscs and the C-type lectin of vertebrate, and that the deduced polypeptide of Sb-Lec1 had the characteristics of the C-type lectin family. Quantitative real-time PCR (qRT-PCR) was used to assess the mRNA expression in all tested tissues, including hemocytes, foot, adductor muscle, mantle, gill, and hepatopancreas. The highest and lowest Sb-Lec1 mRNA were in hepatopancreas and adductor muscle, respectively. Vibrio anguillarum challange induced Sb-Lec1 mRNA expression in all tested tissues (P<0.05). These results showed that Sb-Lec1 gene may play an important role in immune defense.