Cloning of the Pept1 gene and analysis of its expression in Ctenopharyngodon idellus tissues

To study the molecular mechanism of the small peptide transporter PepT1-mediated protein digestion and absorption,a full-length cDNA sequence of the PepT1 gene was cloned from Ctenopharyngodon idellus using RT-PCR and RACE techniques.The full-length cDNA sequence of PepT1 had 2762 nucleotides,including 141 nucleotides at 5′UTR and 479 nucleotides at 3′UTR.Its open reading frame had 2142 nucleotides encoding a 713-amino-acid peptide.The PepT1 gene sequences from C.idellus were most similar to those of Carassius au-ratus and Danio rerio at 77.6% and 74.0%,respectively;the deduced amino acid similarities were 78.0% and 76.7%,respectively;but the PepT1 gene sequence varied in similarity to other animals from 53.9% to 59.1%,and the deduced amino acid similarities were 60.5%—61.6%.The encoded protein molecular weight was predicted at 79.29 kD with pI at 5.87.The PepT1 protein had 11 helix trans-membrane regions;its amino acid sequence was highly homologous to those of other vertebrates.Phylogenetic analysis showed that the PepT1 gene sequence clustered with C.auratus and D.rerio as its closest neighbor.The abundances of PepT1 mRNA assayed by real-time PCR were differentially expressed by tissue type;the highest expression was in the foregut tissue and the second was in the muscle.However,PepT1 mRNA expression was relatively stable after incubation for 7 days.The PepT1 gene expressed rhythmically in the small intestine of C.idellus.Its expression was higher during the night and lower during the day.This work provides a theoretical basis for the molecular mechanism of protein digestion and absorption by the small peptide transporter PepT1,which mediates intestinal transport of small pep-tides in vivo.