Molecular Regulation Mechanism Of Tributyrin On PepT1 Gene Expression In Ctenopharyngodon Idellus

In the present study,the in vivo and in vitro experiments were conducted to study the regulatory effect of tributyrin on the expression of PepT1 gene in the intestine of grass carp through transcription factors CDX2 and Sp1,and to reveal the regulatory mechanism of the expression of PepT1 gene in grass carp.1)The effect of tributyrin on the expression of PepT1,CDX2 and Sp1 genes in the gut of grass carp.Breeding experiments: In the present study,320 grass carps with an initial body weight of 43.50±0.5g were fed for 4 weeks.0.0 g/kg tributyrin was used as the control group,0.5g/kg,1.0 g/kg,and 1.5 g/kg tributyrin were used as the experimental groups.There were 4 treatment groups which contained 4 replicates and 20 carps for each replicate.The results show that:(1)Tributyrin can significantly increase the weight gain rate and specific growth rate of Ctenopharyngodon idellus.The weight gain rate and specific growth rate of the 1.0 g/kg level were significantly higher than those of the control group and other treatment groups(P<0.05).(2)The expression level of PepT1 gene firstly increased and then decreased with the increase of tributyrin levels.The expression levels of mRNA of PepT1,CDX2 and Sp1 at 0.5g/kg level were higher than those in other levels.It shows that tributyrin can affect the expression of PepT1,CDX2 and SP1 genes in the gut of Ctenopharyngodon idellus.Cell experiments: In the present study,Ctenopharyngodon idellus intestinal cells were cultured,and tributyrin at concentrations of 0 mM,5 mM,10 mM,15 mM,and 20 mM were added to the culture medium,mixed well,then added to the six-well plate.The experiment was divided into 5 treatment groups which contained 4 replicates.The intestinal cells were harvested at 24 h after tributyrin challenge for gene expression analysis.The results showed that:(1)The intestinal cells of Ctenopharyngodon idellus showed a ring shape,connected tightly,arranged neatly,paved with stones,individual cells were independent,the boundaries were obvious,and there were 1-2 nucleoli within.(2)The highest expression levels of PepT1,CDX2 and Sp1 mRNA in the treatment group with 5 mM tributyrin,which was significantly higher than that in other groups(P<0.05).These results indicated that tributyrin can affect the expression of PepT1,CDX2,and Sp1 genes of intestinal cells in Ctenopharyngodon idellus.Thus,the appropriate amount of tributyrin can improve the growth performance of Ctenopharyngodon idellus,and can increase the expression of PepT1,CDX2 and Sp1 mRNA of intestinal tissues and intestinal cells Ctenopharyngodon idellus.We may speculate that fish may have similar regulatory mechanisms as mammalswhich the grass carp CDX2 and Sp1 can regulate the expression of PepT1 in response to tributyrin treatment in vivo and in vitro.2)The regulation of PepT1 gene expression by transcription factor CDX2/Sp1.(1)The Genome Walking technique was used to clone the promoter of PepT1 gene from grass carp.Bioinformatics analysis revealed that the binding sites of CDX2 and Sp1 existed on the promoter of PepT1 in grass carp,which revealed that transcription factors CDX2 and Sp1 may play a possible role in regulating PepT1 expression.(2)Subcellular co-localization analysis showed that CDX2 and Sp1 could co-localize in the nucleus of HEK293 T cells.Mammalian two-hybrid experiments indicated that CDX2 may directly interact with Sp1 protein in live cells.(3)To futher investigate the effects of CDX2/Sp1 overexpression on PepT1 promoter,pGL3-PepT1 and CDX2/Sp1 eukaryotic expression vector were constructed and co-transfected into HEK293 T cells.The luciferase reporter gene experiments found that grass carp transcription factor CDX2 can significantly increase the promoter activity of PepT1(P <0.05),while Sp1 has no significantly effects on the promoter of PepT1(P>0.05),which was futher verified that the grass carp transcription factor CDX2/Sp1 could regulate the expression of PepT1 gene.