Molecular Cloning And Expression Analysis Of Cationic Amino Acid Transporters Y~+LAT1and Y~+LAT2in Ctenopharyngodon Idellus

Transport of amino acid across intestinal epithelial cell membranes is mediated by multiple transporters with different specificities. These transporters belong to different transport systems in fish. Cationic amino acid (CAA) transporters are involved in CAA transport across cellular membranes. b0,+, y+and y+L system transport most of the CAA. The number and activity of transporters is affected by development phase, intestinal physiological status, diet composition and other neuroendocrine factors.Due to changes in environmental conditions, individual differences and unequal distribution of food, fish often suffers from starvation. Study on response of gene expression of grass carp cationic amino acid transporter to starvation is helpful to understand the ecological strategies of starvation which has the important theory significance. The research will also provide an important guide to the management of fishery resources, fish breeding, aquaculture and compensatory growth to obtain economic benefits.In this study, we described cDNA cloing, mRNA tissue distribution and gene expression of two cationic amino acid transporters y+LAT1and y+LAT2in Ctenopharyngodon idellus. The research laid a foundation for further study on the metabolism of fish amino acid absorption and transport.The results are as follows:1. y+LAT1and y+LAT2gene cDNA cloning and analysisThe full-length of y+LAT1cDNA was2688bp encoding501amino acids. The GenBank accession number was JX013494. The deduced amino acid had similar structure function area to other vertebrate y+LAT1s, including one glycosylation site, three consensus sites for protein kinase C phosphorylation, etc., which were highly conserved among vertebrates. Amino acid sequence analysis showed that sequence homology of grass carp was95.0%identity with the sequence of danio rerio. The phylogenetic tree of amino acid sequence built from y+LAT1homology of grass carp and other animals was consistent with that of the traditional morphology results. The length of y+LAT2cDNA was1849bp encoding456amino acids. The3’end sequence of length was478bp. The GenBank accession number was KC206055. The deduced amino acid had similar structure function area to other vertebrate y+LAT2s, including one glycosylation site, three consensus sites for protein kinase C phosphorylation and etc which were highly conserved among vertebrates. Amino acid sequence analysis showed that sequence homology of grass carp was96.5%identity with the sequence of danio rerio. The phylogenetic tree of amino acid sequence built from y+LAT2homology of grass carp and other animals was consistent with that of the traditional morphology results.2. y+LAT1and y+LAT2tissues expression analysisThe y+LAT1and y+LAT2mRNA were detected in muscle, brain, gill, heart, kidney, liver, hindgut, midgut, foregut and spleen by Real-time PCR. The results showed that y+LAT1mRNA was expressed the highest in midgut (p<0.05), then in foregut and spleen and weakly in kidney, hindgut, gill, heart, liver, muscle and brain in turn. y+LAT2mRNA was expressed highest in foregut (p<0.05), then in spleen, and weakly in foregut, kidney, heart, muscle, liver, hindgut, gill and brain in turn.3. Effect of starvation on the transcription of y+LAT1and y+LAT2The y’LAT1and y+LAT2mRNA affected by starvation in grass carp were detected in kidney, spleen, foregut and midgut by Real-time PCR. The results showed that the expression patterns of y+LAT1in spleen, kidney and foregut, midgut were different. In foregut and midgut, the mRNA level of y+LAT1decreased after fasting for1day and then increased gradually in midgut whereas seemed to increase without a clear pattern in foregut. However in spleen and kidney y+LAT1expressed inconsistently. The mRNA level of y+LAT1in kidney gradually decreased after fasting for14days while the level in spleen decreased and kept low (p>0.05) after fasting for14days. The expression patterns of y+LAT2in spleen, kidney and foregut, midgut were different. In spleen and kidney the mRNA level of y+LAT2decreased to the lowest after fasting for2days and3days, respectively. Then the level seemed to change without a clear pattern. In foregut and midgut the y+LAT2expressed inconsistently, the level trended to decrease from fasting for0day to5days. Then the level increased significantly (p<0.05), to the peak.