Cloning And Expression Of GS And ASCT2 From Ctenopharyngodon Idellus

Glutamine synthetase(GS)is considered a master enzyme that catalyses ATP-dependent biosynthesis of glutamine from glutamate.AlaSerCys Transporter 2(ASCT2),a glutamine/amino acid transporter,plays an important role in the absorption of neutral amino acids.In the present study,GS and ASCT2 from intestine in the grass carp(Ctenopharyngodon idellus)were completely cloned by means of TA and RACE methods.The GS and ASCT2 expression patterns in different tissues from grass carp were assayed.The characterization of GS expression in embryogenesis was assayed by real time RT-PCR.In order to study the effects of exogenous protein and nutrients on the molecular mechanism of grass carp,different protein sources and glutamine dipeptide were used in the diet.The GS and ASCT2 expressions with additive(glutamine dipeptide)in grass carp were studied.1.Cloning of full length cDNAs of GS and ASCT2 and biological information analysis.Using rapid amplification of cDNA ends(RACE),the full length cDNA sequences of GS and ASCT2 in grass carp Ctenopharyngodon idella were cloned and were analyzed by using BioEdit and DNAstar softwares.The full-length cDNA sequence of GS encoded a 371-amino-acid polypetide.Phylogenetic analysis of the C.idellus GS sequence revealed common carp(Cyprinus carpio)as its closest neighbor.ASCT2 encoded a 541-amino-acid protein.Phylogenetic analysis revealed that the ASCT2 sequence of grass carp clustered with the ASCT2 from Danio rerio.2.Determination of the tissue distribution and during early development of GS and ASCT2 transcripts.GS and ASCT2 mRNA expressions were performed using quantitative real-time PCR in different tissues from Ctenopharyngodon idellus.GS mRNA was differentially expressed in different tissues,with a high-to-low gradient expression in the intestine,brain,muscle,heart,gill,liver,pituitary gland,and spleen,respectively.ASCT2 mRNA with a gradient expression from high to low in tissues of the liver,gill,muscle,midgut,hypophysis,hindgut,kidney,heart,foregut,brain,spleen and gonad,respectively.Additionally,GS exhibited a dynamic pattern of expression during embryonic development,reaching maximal and minimal levels in the organ and hatching stages,respectively,and constant low levels from 7 to 28 days post-hatching.ASCT2 reached the maximum in the organ stage and was relatively stable after hatching stage.3.GS and ASCT2 expressions on dietary protein regulation of Ctenopharyngodon idellus.To determine the effect of diets with protein on GS and ASCT2 expression,two different protein sources and three different protein levels were designed.The effects of dietary protein sources on the GS and ASCT2 expression were analyzed by real-time PCR.The same amount of protein diets with different dietary protein sources were formulated.The results suggested that 22%crude protein(CP)and fish meal stimulated GS gene expression.Meanwhile,the quantitative real-time PCR results showed that 22%crude protein(CP)diets could significantly stimulate ASCT2 gene expression.In addition,ASCT2 was expressed in higher quantities in the soybean meal group than in the fish meal group.4.GS and ASCT2 expressions on dietary glutamine dipeptide regulation of Ctenopharyngodon idellus.Intestinal GS mRNA expression was significantly increased by the specific concentrations of glutamine dipeptide in vivo.The 2.5 g/kg glutamine dipeptide remarkably improved the expressions of GS and ASCT2 genes.