Molecular Identification and mRNA Expression Profiles of Galectin-9 Gene in Red Sea Bream (Pagrus major) Infected with Pathogens

Abstract

Galectin (Gal) is a member of a family of β-galactoside-binding lectin. The members of this family play important roles in the recognition of carbohydrate ligands and in various other biological processes. In this study, we identified the gene encoding Gal-9 in Pagrus major (PmGal-9) and analyzed its expression in various tissues after pathogen challenge. Alignment analysis revealed that the two galactose-binding lectin domains of the deduced protein were highly conserved among all the teleosts. Phylogenetic analysis revealed that PmGal-9 is most closely related to the Gal-9 gene of gilthead sea bream. PmGal-9 was ubiquitously expressed in all tissues analyzed but was predominantly expressed in the spleen, head kidney, and intestine. After challenges with major microbial pathogens (Edwardsiella piscicida, Streptococcus iniae, or red sea bream iridovirus) of red sea bream, PmGal-9 mRNA expression was significantly regulated in most immune-related tissues. These results suggested that PmGal-9 not only plays an important role in the immune system of red sea bream but is also a possible inflammatory marker for pathogenic diseases.

Keywords: Edwardsiella piscicida; Streptococcus iniae; aquaculture; gene profiling; iridovirus.

Figure 1

Multiple sequence alignment analysis of the deduced amino acid sequence of PmGal-9 with fish Gal-9 sequences. This analysis is based on the following sequence data: rock bream (ANN46244), gilthead sea bream (XP_030261557), large yellow croaker (XP_010754381), Nile tilapia (XP_003458375), rainbow trout (ACO08221), Atlantic salmon (ACI67584), pig (NP_999097), cattle (NP_001034266), and human (CAB93851). The predicted galactose-binding lectin domains are indicated by the box. Black boxes: identity = 100%; gray boxes: 80% ≤ identity < 100%; light gray boxes: 50% ≤ identity < 80%.

Figure 2

A phylogenetic tree of PmGal-9 and other known Gal-9 homologs based on the neighbor-joining (NJ) method. The scale bar indicates a branch length of 0.2. Numbers are bootstrap percentiles from 1000 replicates. This analysis is based on the following sequence data: Human Gal-1 (NP_002296), Human Gal-2 (NP_006489), Human Gal-3 (BAA22164), Human Gal-4 (NP_006140), House mouse Gal-6 (AAI60275), House mouse Gal-7 (NP_032522), Human Gal-8 (AAF19370), Gilthead sea bream Gal-9 (XP_030261557), Large yellow croaker Gal-9 (XP_010754381), Rock bream Gal-9 (ANN46244), Nile tilapia Gal-9 (XP_003458375), Atlantic salmon Gal-9 (ACI67584), Rainbow trout Gal-9 (ACO08221), Cattle Gal-9 (NP_001034266), Human Gal-9 (CAB93851), Pig Gal-9 (NP_999097), Human Gal-10 (NP_001819), Cattle Gal-11 (CBX54571), Human Gal-12 (NP_001136007), Human Gal-13 (ACR09640), Human Gal-14 (ACR09644), Sheep Gal-15 (NP_001009238), and Human Gal-16 (NP_001177370).

Figure 3

Expression level of PmGal-9 mRNA in various tissues of healthy red sea bream. The PmEF-1α gene was used to normalize the RT-qPCR results. The expression level is expressed as the fold change compared to the expression level of PmGal-9 mRNA in the brain. An agarose gel image of the PCR products is shown at the bottom. All data are presented as the mean ± SD from three independent cDNA samples with three replicates per sample.

Figure 4

Expression level of PmGal-9 mRNA in the gill, kidney, liver, and spleen of red sea bream after infection with (A): Streptococcus iniae, (B): Edwardsiella piscicida, or (C): red sea bream iridovirus (RSIV). Gene expression levels and their significance are represented as the mean ± SD. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01) versus the control group.