Molecular cloning,tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding

Mucins are important components of mucus, which form a natural, physical, biochemical and semipermeable mucosal layer on the epidermis offish gills, skin, and the gastrointestinal tract. As the first step towards characterizing the function of Muc2, we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp, and analyzed its tissue-specific expression pattern by quantitative real-time PCR （qPCR）. The obtained sequence comprised 41 bp 5＇-untranslated region （5＇-UTR）, 2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains （VWD-C8-TIL-VWD-C8） and had high homology with mucins from other vertebrates. Among four candidate reference genes （β-Actin, RP113a, RPII, 18S） for the qPCR, RPII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine, in the order （highest to lowest） middle-intestine 〉 fore-intestine 〉 hind-intestine. Muc2 was expressed relatively poorly in other organs （brain, liver, kidney, spleen, skin and gill）. Furthermore, after 20-days of starvation, M. amblycephala Muc2 expressions after refeeding for 0 h, 3 h, 16 h, 3 d, and 10 d were significantly decreased in the three intestinal segments （P〈0.05） at 16 h, and were then upregulated to near the initial level at 10 d.

Mucins are important components of mucus,which form a natural,physical,biochemical and semipermeable mucosal layer on the epidermis of fish gills,skin,and the gastrointestinal tract. As the first step towards characterizing the function of Muc2,we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp,and analyzed its tissue-specific expression pattern by quantitative real-time PCR(qPCR). The obtained sequence comprised 41 bp 5′-untranslated region(5′-UTR),2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains(VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes(β- Actin,RPI13α,RPII,18S) for the qPCR,R PII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine,in the order(highest to lowest) middle-intestine > fore-intestine > hind-intestine. Muc2 was expressed relatively poorly in other organs(brain,liver,kidney,spleen,skin and gill). Furthermore,after 20-days of starvation,M. amblycephala Muc2 expressions after refeeding for 0h,3 h,16 h,3 d,and 10 d were significantly decreased in the three intestinal segments(P <0.05) at 16 h,and were then upregulated to near the initial level at 10 d.