Tissue Expression And Regulation Of Fasting As Well As Exogenous Protein On Cationic Amino Acid Teansporter Y + LAT1 In Rock Carp, Procypris Rabaudi(tchang)

Protein is mainly absorbed by the form of small peptides and free amino acids in the small intestine. The uptake of amino acid is mediated by the amino acid transporters. The number and activity of transporters is effected by hormones, diet nutrition, and other neuroendocrine matters. The expression of transporters can reflect the ability of animal adapting to change of environmental conditions. The fish have ten kinds of essential amino acids which have three kinds of cationic amino acids. The uptake of cationic amino acid is related to the growth of fish. Cationic amino acid transporter is responsible for the transmembrane transporting of the cationic amino acids in the cell membrane.Among them,b^0,+ and y⁺L and y⁺system transporting the most of cationic amino acids.In the research, the cationic amino acids transporter y⁺LAT1 in rock carp was cloned and sequenced. To understand the absorption of cationic amino acids, tissues distribution and effect of fasting as well as exogenous protein on y⁺LAT1 mRNA expression levels were done by real-time fluorescent quantification. The main results as follows:1.The molecular cloning and sequence analysis of cationic amino acid transporter y⁺LAT1 in rock carp, Procypris rabaudi(Tchang)The cDNA of rock carp cationic amino acid transporter y⁺LAT1 was amplified by RT-PCR using primers designed based on multiple alignment of the published sequences of y⁺LAT1 genes in genbank.The amplified segment was sequenced after purifying.The landing sequence number for the fragment of 512bp is GU138158. Homologous analysis indicated that the sequenced segments of y⁺LAT1 had 92.2% nucleotide identity with the corresponding segment of zebra fish.The sequence encoded 169 amino acid residues, relative molecular mass of 18.334kDa, including 8 strongly cationic amino acid residues,8 acidic amino acid residues, 81 hydrophobic amino acid residues, 51 polar amino acid residues. 2.Segmental distribution of cationic amino acid transporter y + LAT1 expression in rock carpThe y⁺LAT1 abundance was determined by real-time quantitative RT-PCR using SYBR Greenâ… RT-PCR mix kit.Amplification of 18s,EF1a and RF13a cDNA were performed in order to confirm the steady-state level of expression of a housekeeping gene allowing an internal control for y⁺LAT1 gene expression in eleven different tissues of the rock carp.Results indicated that expression abundance of y⁺LAT1 in foregut is the hignest in the eleven tissues,which was followed by spleen,midgut,kidney,and trace of Y+LAT1 expression was observed in the liver and muscle.3.The expression regulation of cationic amino acid transporter y⁺LAT1 mRNA in rock carpThis chapter studied the expression regulation of cationic amino acid transporter y⁺LAT1 by fasting and force-feeding of soybean oligopeptide in rock carp.(1) The expression level of y⁺LAT1 gene was measured after hunger 24h, 32h, 38h, 2d, 3d, 4d, 5d, 6d, 7d, 8d, 9d, 10d. The results showed that the control treatment was significantly higher than other groups (P<0.05). (2)The 120 of rock carp juveniles were randomly divided into four treatments with different levels of soybean oligopeptide solution (control,5%,20%,35%).The levels of force-feeding is 1ml/10g body weight .The treatment of control force-fed the same dose of distilled water irrigation. The expression level of y⁺LAT1 gene was quantified by RT-PCR after force-feeding 0.5,1.5,3.5,7.5,12h.Fish fed of soybean oligopeptide (5% and 20%)exhibited significantly increased y⁺LAT1 gene expression in foregut compared to the control treatment(0%) after force-feeding 1.5h and 7.5h. Fish fed of soybean oligopeptide(20% and 35%) exhibited significantly increased y⁺LAT1 gene expression in foregut compared to the control treatment(0%) after force-feeding 3.5h (P<0.05).Fish fed of soybean oligopeptide(35%) exhibited significantly increased y⁺LAT1 gene expression in foregut compared to the control treatment(0%) and no significant difference among the other treatments after force-feeding12h.The results suggest that amino acid transporter y⁺LAT1 mRNA expression is influenced by soybean oligopeptide in foregut of rock carp.