Distribution Of Intestine Mucous Cells And Molecular Cloning, Tissue Expression Of Two Mucin Genes In Megalobrama Amblycephala

Mucus is one kind of sticky liquid with high water content that was based on mucins as framework. The skin, gills and intestine of fish are covered by a layer of mucus which is largely secreted by mucous cells. As mucus plays an important role in protecting fish against pathogen invasion, signaling events, regulating osmotic pressure and so on, the research about mucus has been paid wide attention.Using tissue section technique and staining method of AB-PAS (Alcian blue and periodic acid Schiffs reaction), the types and distribution of mucous cells in the intestine of healthy M. amblycephala were studied. And the numbers of intestine mucous cells in different parts were analyzed statistically. The result showed that there were four types of mucous cells in the digestive tracts of M. amblycephala:Type â… , â…¡, â…¢ and â…£.Type â…  was pure red, AB negative and PAS positive containing neutral mucoprotein. Type â…¡ was pure blue, AB positive and PAS negative containing acidic mucoprotein. Type â…¢ was purple reddish, both AB and PAS positive, PAS positive more than AB containing acidic mucoprotein. Type IV was blue purple, both AB and PAS positive, AB positive more than PAS containing mainly acidic mucoprotein and a few neutral mucoprotein. The order of mucous cells density was H-intestine> M-intestine> F-intestine> esophagus. The result could provide a reference for the study of M. amblycephala intestine.According to the sequences of other known vertebrate mucins and the transcriptome datebase of our laboratory in M. amblycephala, we cloned the Muc2 and Muc5b partial mRNA sequences of M. amblycephala. An appropriate housekeeping (HK) gene was selected for the study of gene expression by qPCR, and tissue distribution of Mucin genes were examined in M. amblycephala. The results showed that M. amblycephala Muc2 and Muc5b had high homology with other vertebrates. The predicted amino acid sequence of M. amblycephala Muc2 and Muc5b showed a common VWD-C8-TIL-VWD-C8 secreted mucin module domains. In this study, the RPâ…¡ was the best reference gene in the nine tissues of M. amblycephala. M. amblycephala Muc2 was mainly expressed in intestine, and its expression in M-intestine> F-intestine> H-intestine; relative higher expression of Muc5b was in gills and skin, lower expression in brain, spleen, kidney, almost no expression in liver and intestine.According to the results of tissue expression of mucin genes, the variation of M. amblycephala Muc2 after re-feeding was detected in intestine, and the variation of M. amblycephala Muc5b after catching-stress was detected in gills and skins. The results showed that Muc2 expression was sharply decreased in all segments of intestine (P< 0.05), and after 16h, it recovered near initial level. Muc5b expression was significantly reduced in gills (P< 0.05) at 1h after catching-stress, and recovered to initial level at 24h; On the contrary, Muc5b expression was increased in the skin at 4h(t-test, P<0.05), then recovered to the initial level.