Full-length CDNA Cloning Of PepT1and PepT2and Tissue Expression Of PepT2Gene From Grass Carp

PepTl and PepT2are two kinds of different small peptides transporter belong to POT (Proton-coupled oligopeptide transporter) family.The responsible for the uptake of dietary di-and tripeptides in cells. It transports peptides against a concentration gradient in a pH-dependent manner.PepTl is considered predominantly expressed in small intestine, it transports small peptides from dietary digestive;and PepT2mainly expressed in kidney and brian,it responsible for the reabsorption of di-and tripeptides and neuropeptide, Also, they transport a variety of peptidomimetics such as β-lactam antibiotics, aminopeptidase.This paper design primer according to the gene conserved sequence of Danio rerio. Sea bass、 Salmon、Homo、Mouse、Rabbit. Cloning the part of sequence of PepTl and PepT2, The homolog compare with program of BLASTN. BLASTX and TBASTX in National Center for Biotechnology for establish distinct gene.Then, design5’,3’Gene-Specific Primers,get5’and3’ race products by RACE-PCR technique.The results show that the full length of Grass carp PepT2gene cDNA sequence is2733bp,its ORF(open reading frame)is2169bp,encoding722amino acids, with82bases of5’-untranslated region (5’-UTR), and482bases of3’-UTR.The homolog of the predicted grass carp PepT2amino acid sequence results show that grass carp PepT2exhibited86%identity with the Danio rerio.also exhibited a higher percentage of identity with mammalian, between55%and58%.It has a lowest55%identity with mouse. the full length of Grass carp PepTl gene cDNA sequence is2456bp, its ORF is2160bp, encoding719amino acid, with135bases of5’-UTR, and161bases of3’-UTR; The result of amino acid homolog shows that grass crap PepTl has a highest80%identity with carassius, has a lowest53%identity with mouse.Compared the predicted grass carp PepT2and PepTl amino acid sequence show only48%identity.According to the grass carp PepT2gene design specific RT (reverse transcription) primer, use the β-actin as control gene,Study the distribution of PepT2mRNA in grass crap different tissues by Semi-quantitative RT-PCR technique.The result shows that Expression level of PepT2mRNA in different tuisses of grass carp from high to low is kidney、intestine、brain、the head of the kidney、liver、muscle,PepT2mRNA was not detected in heart and gills.