| This study aimed to evaluate the arginine synthesis from glutamate in juvenile hybrid grouper(Epinephelusfuscoguttatus ♀× Epinepheluslanceolatus ♂)and the regulating mechanism of arginine in growth and immunity of this species.1.The synthesis of glutamate to arginine in juvenile hybrid grouper.Four cell culture experiments were conducted,and cells used were intestine and body kidney tissues.The basic medium in these four experiments was L-15 medium consisted of 15%FBS,100 IU ml-1 penicillin,100 μg ml-1 streptomycin and had no glutamate,ornithine,citrulline and arginine.In experiment 1,1 mmol/L ornithine were added to the basic medium(medium A),and intestine cells were cultured for 30 mins.In experiment 2,1 mmol/L citrulline were added in the basic medium(medium B),and body kidney cells were cultured for 30 mins.In experiment 3,0,2.00,3.47,5.00mmol/L arginine and 5 mmol/L glutamate were added in the basic medium(medium C,D,E,F),intestine cells were cultured for 30 mins.In experiment 4,the of,0,2.00,3.47,5.00mmol/L arginine and 5 mmol/L glutamate were added in the basic medium(medium G,H.I,J),and intestine cells together with body kidney cells were cultured for 30 mins.After the end of cell culture,the contents of free glutamate,ornithine,citrulline and arginine in the cells and mediums of these four experiments were measured.In experiment 1,few citrulline was measured after intestine cells were cultured in the 1 mmol/L ornithine contained medium for 30 mins.In experiment 2,no arginine was measured after body kidney cells were cultured in the 1mmol/L citrulline contained medium for 30 mins.In experiment 3,after intestine cells were cultured in the 5 mmol/L glutamate contained mediums together with 0,2.00,3.47,5.00 mmol/L arginine for 30 mins,free glutamate concentration in the group of 0 mmol/L arginine was significantly lower than that in the group of 2.00,3.47 or 5.00 mmol/L arginine.The free ornithine concentration in the group of 5.00 mmol/L arginine was significantly higher than that in the group of 2.00 or 3.47 mmol/L arginine.Free arginine was not detected in the group of 0 mmol/L arginine.In experiment 4,after intestine cells and body kidney cells were together cultured in the 5 mmol/L glutamate contained mediums together with 0,2.00,3.47,5.00 mmol/L arginine for 30 mins,free glutamate concentration in the group of 5.00 mmol/L arginine was significantly higher than that in the group of 2.00,3.47 or 5.00 mmol/L arginine.The concentrations of free ornithine in groups of 0.00 and 2.00 mmol/L arginine were significantly lower than those in the groups of 3.47 or 5.00 mmol/L arginine.Free arginine was not detected in the group of 0.00 mmol/L arginine.2.The regulating mechanism of arginine in growth and immunity of hybrid grouper.A 9-week growth trial was conducted to obtain the muscle,liver and head kidney samples,and then transcriptomics were analyzed to evaluate the regulating mechanism of arginine in growth and immunity of hybrid grouper.Two isoenergetic(350 kcal per 100 g dry matter),isoproteic(53.15%of dry matter)and isolipidic(7%of dry matter)experimental diets were formulated.Dietary arginine levels were 2.05 and 3.62%of dry matter.The overall amino acid(AA)composition except for arginine in the diets was adjusted to the reference A A profile established in our previous study.Aspartic acid and glycine(1:1)were used to replace part of arginine to maintain the content of crude protein in the dietary.Groups of fifteen fish(average initial body weight:11.2 g)were randomly distributed into six glass tanks.At the end of the growth trial,muscle,liver as well as head kidney samples were obtained for the analysis of transcriptomics sequencing.A total of 108464 Unigenes were assembled.By Blast software,42242 Unigenes were annotated,and the numbers of genes obtained from the annotation of the four databases were:41912(Nr),19064(keg),27806(Swissport),25810(KOG),and 66222 Unigenes were uncommented.After assembly,56682 genes and 51979 genes were detected in muscle of fish fed 2.05%and 3.62%dietary arginine,respectively.Compared to fish fed 2.05%dietary arginine,373 different genes were expressed in muscle of fish fed 3.62%dietary arginine,among which 265 genes were up-regulated and 108 genes were down-regulated.There were 26 significant enrichment pathways in KEGG and GO of DEGs,including amino acid metabolism,fatty acid metabolism,terpenoid backbone biosynthesis,carbon metabolism,and these pathways were closely related to growth.Expression of 110 genes were significantly different between these two kinds of muscles,including stearoyl-CoA desaturase 1a(SCD1a),fatty acid-binding protein(FABP),very long-chain acyl-CoA synthetase-like(acyl-CoA),muscle phosphoglyceratemutase 2(PGAM2),glycine amidinotransferase(GATM),ornithine aminotransferase(OAT),aspartate aminotransferase(Got1),creatine kinase M-type-like(CKM),pyrroline-5-carboxylate reductase 2-like(PYCR2),glutaminase liver isoform(GLS2),cysteinyl leukotriene receptor 1-like(CYSLTR1),etc.After assembly,43875 genes and 48712 genes were detected in liver of fish fed 2.05%and 3.62%dietary arginine,respectively.Compared to fish fed 2.05%dietary arginine,98 different genes were expressed in muscle of fish fed 3.62%dietary arginine,among which 48 genes were up-regulated and 50 genes were down-regulated.There were 26 significant enrichment pathways in KEGG and GO of DEGs,including Oxidative phosphorylation,Cardiac muscle contraction,oxidation-reduction process,electron transport chain,generation of precursor metabolites and energy,regulation of peptidase activity,which were involved in the transport and metabolism of cellular substances and energy,and these pathways were closely related to metabolism.Expression of 25 genes were significantly different between these two kinds of livers,including NADH dehydrogenase subunit 4(nad4),cytochrome c oxidase subunit Ⅲ(C03),phosphoenolpyruvatecarboxykinase(PCK1),glutathione peroxidase 3 precursor(Gpx3),MHC class Ⅱ antigen beta chain(Epak-DAA),myosin-7B-like(MYH7B),etc.After assembly,70393 genes and 71620 genes were detected in head kidney of fish fed 2.05%and 3.62%dietary arginine,respectively.Compared to fish fed 2.05%dietary arginine,110 different genes were expressed in muscle of fish fed 3.62%dietary arginine,among which 54 genes were up-regulated and 56 genes were down-regulated.There were 6 significant enrichment pathways in KEGG and GO of DEGs,including lyase activity,hydro-lyase activity,carbon-carbon lyase activity,carbon-oxygen lyase activity,immunoglobulin binding,protein complex binding,and these pathways were closely related to immunity.Expression of 25 genes were significantly different between these two kinds of head kidneys,including calcium/calmodulin-dependent protein kinase Ⅱ(CAMK2D),MHC class Ⅱantigen(Epco-DAA),Methylmalonate-semialdehyde dehydrogenase(mmsA),etc.Comaprision in transcriptomics analysis of muscle,liver and head kidney tissues between fish fed 2.05%and 3.62%dietary arginine showed that the arginine supplementations to the diets could regulate the growth and immunity of juvenile hybrid grouper through participating in amino acid metabolism,protein synthesis,fat metabolism,energy metabolism,extracellular matrix composition and immune response. |
PDF Full Text Request