Molecular Characterization of The Large Yellow Croaker (Larimichthys crocea) Wap65-2 Gene and Its Correlation Analysis upon Vibrio alginolyticus Infection
Abstract：Wap65-2 (warm temperature acclimation related 65 kD protein-2), a specific plasma glycoprotein in teleosts, plays a key role in fish immune response against pathogens. In the study, the large yellow croaker (Larimichthys crocea) Wap65-2 gene (LcWap65-2) was obtained with de-novo transcriptome sequencing of large yellow croaker liver. The cDNA sequence of LcWap65-2 (GenBank accession: KJ412463) was 1 585 nucleotides, which contained a large open reading frame that encoded a protein of 432 amino acids with a deduced molecular mass of 48.84 kD and a theoretical isoelectric point (pI) of 5.36. The N-terminus of LcWap65-2 contained a predicted signal peptide of 20 amino acids. Multiple sequence alignment of complete amino acid sequences showed that LcWap65-2 had the typical characters of fish Wap65-2. Sequence comparsion showed that LcWap65-2 shared the highest amino acid sequence identity (87.4%) with that of miiuy croaker (Miichthys miiuy). Phylogenetic tree analysis also confirmed that LcWap65-2 falled into the fish Wap65-2 cluster and was most closely related to that of miiuy croaker. Genome structure analysis showed that LcWap65-2 gene (GenBank accession: KJ412464) contained 10 exons and 9 introns. Real-time quantitative PCR (qRT-PCR) analysis showed that LcWap65-2 mRNA had the highest expression level in liver, and little amount in brain of the healthy large yellow croaker. Upon Vibrio alginolyticus infection, LcWap65-2 transcripts significantly upregulated in the liver, and reached its peak at 8 h. Then the mature peptide of LcWap65-2 (LcWap65-2m) overexpressed and the antiserum against LcWap65-2m was prepared. Western blot assay demonstrated that the serum LcWap65-2 showed no obvious difference at 4 h but significant increased at 8, 12, and 24 h in large yellow croaker infected with V. alginolyticus. In summary, our present study revealed a tight relationship between the LcWap65-2 expression and V. alginolyticus infection, which suggested that LcWap65-2 might play an important role in fish immune response against bacteria infection. The present study provides a theoretical basis for studying the functions of fish Wap65-2, and its molecular mechanism in regulating fish immune response to pathogens.
