| As a functional amino acid,arginine needs to enter the cell through the corresponding transporter to play its important role in fish growth,immunity and intestinal mucosa.The fish intestine is an important barrier to maintain the stability of the environment in the body,which is of great significance to the body's defense system.Therefore,it is of great significance to study the absorption and transport of arginine and its effects on the intestinal physics and immune barrier of fish.In this study,the whole length cloning of amino acid transporter CAT1 gene was used to study the absorption and transport mechanism of arginine at intestinal and the effects of different dietary arginine levels on the physical and immune barriers of the hybrid grouper intestinal.The brief summaries are as follows:1.In order to explore the transport mechanism of arginine,the full-length sequence of the c DNA of the amino acid transporter CAT1(SLC7A1)was cloned by the rapid amplification technology of c DNA ends,and the expression of CAT1 gene in different tissues of grouper was detected by realtime fluorescence quantitative PCR.The results showed that the total length of CAT1 gene was 2319 bp,including 1938 bp of the open reading frame(ORF)which encoding 645 amino acids,a 5-'UTR of 208 bp and a 3'-UTR of 173 bp.The predicted molecular weight was 69.7k Da and the theoretical isoelectric point was 5.31.The results of multiple sequence alignment showed that the CAT1 gene of hybrid grouper was highly conserved with that of other species.Smart software analyzed the protein sequence of CAT1 c DNA and found that it contains 14 transmembrane regions.Analysis of real-time quantitative PCR showed that the CAT1 gene was expressed in 12 tissues,including hindgut,brain,spleen,foregut,midgut,heart,head kidney,skin,gill,muscle,stomach and body kidney,except for in liver.2.Three isonitrogenous and isolipidic diets with arginine contents 1.96%,3.06%,and 3.74%(dry matter)were formulated to investigate the effects of dietary arginine on the growth,feed utilization,body composition and intestinal antioxidant capacity of hybrid grouper.There were five replicates per diet and 25 juvenile fish(initial weight: 20.79±0.04 g)per replicate.The fish were fed the experimental diets for 6 weeks.The results showed that dietary arginine had no effect on the survival rate(SR)(P>0.05).The weight gain rate(WGR),specific growth rate(SGR),protein efficiency ratio(PER)and condition factor(CF)of the group fed with 3.06% arginine were significantly higher than those of the 1.96% and 3.74% groups(P<0.05),and the feed coefficient ratio(FCR)was significantly lower than that of the groups fed with 1.96% and 3.74%(P<0.05).In body composition,dietary arginine had no effect on crude protein(CP),crude lipid(CL)and crude ash of hybrid grouper(P>0.05).The 3.06% arginine diet significantly enhanced the intestinal glutathione(GSH)contents and superoxide dismutase(SOD)activities and then decreased the malonaldehyde(MDA)contents(P<0.05).The activities of SOD,glutathione peroxidase(GSH-Px)and total antioxidant capacity(T-AOC)in the intestine of fish fed with 3.06% arginine showed the maximal value.Intestinal total nitric oxide synthase(T-NOS)activities and nitric oxide(NO)contents increased with the increasing dietary arginine levels(P<0.05).3.The effects of dietary arginine levels on intestinal immune barrier and disease resistance of hybrid grouper showed that the relative m RNA expression levels of intestinal toll-like receptor 22(TLR22)and heat shock protein 70(Hsp70)of the group fed with 3.06% arginine were significantly lower than those of the groups fed with 1.96% and 3.74%(P<0.05).The groups fed with 3.06% and 3.74% arginine showed had lower interleukin1?(IL-1?)and tumor necrosis factor-?(TNF-?)(proinflammatory cytokines)gene expression than the group fed with 1.96%.The relative expression of transforming growth factor-?(TGF-?)and interleukin 10(IL-10)m RNA of the group fed with 3.06% arginine were significantly higher than those of the groups fed with 1.96% and 3.74%(P<0.05).In order to test the resistance of arginine to hybrid grouper,a challenge test was performed with Vibrio harveyi.The challenge test indicated that the expression levels of intestinal TLR22 and My D88 m RNA in all treatment reached the highest value at 16 h post-injection(hpi)(P<0.05),and the expression levels of TLR22 and My D88 m RNA in the intestine of fish fed with 3.06% arginine group at 16 hpi were the lowest(P<0.05).The highest gene expression of TGF-? in the intestine of the groups fed with 1.96%,3.06% and 3.74% were observed at 12 hpi,8hpi and 4hpi,respectively.The highest gene expression of IL-10 in all treatment groups were occurred at 12 hpi.Gene expression of TGF-? and IL-10 in grouper intestine of the group fed with 3.06% arginine were significantly higher than those of the 1.96% and 3.74% groups after challenge.4.The effects of dietary arginine level on the intestinal morphology,physical barrier,and intestinal cell apoptosis of hybrid grouper showed that dietary arginine had significantly effects on intestinal fold height(FH)and muscle layer thickness(MT).The 3.06% arginine diet significantly enhanced the intestinal FH(P<0.05).The intestinal MT of the groups fed with 3.06% and 3.74% arginine were significantly higher than the group fed with 1.96%(P<0.05).Compared with the group fed with 3.06% arginine,the number of intestinal apoptotic cells in the groups fed with 1.96% and 3.74% arginine increased significantly(indicated by green particles).At the transcription level,gene expression of the caspase3 and p53 in intestine of fish fed 3.06% dietary arginine were lower than those in fish fed 1.96% and 3.74% arginine(P<0.05).Fish fed with 1.96% and 3.06% dietary arginine showed significantly lower gene expression of caspase9 in intestine compared with fish fed 3.74% dietary arginine(P<0.05).Gene expression of the claudin3,claudin12,claudin15 a and ZO-1 in intestine of the group fed with 3.06% dietary arginine were lower than those the groups fed with 1.96% and 3.74% arginine(P<0.05).In conclusion,the supplementation of appropriate arginine in the diet(3.06%)could increase the intestinal antioxidant capacity and immunity,reduced the apoptosis of intestinal cells and regulated the intestinal tight junction protein through PI3K/Akt/TOR signal pathway to promote the healthy growth of the fish. |
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