Molecular Characterization And Expression Research Of Oligopeptide Transporter PepT1in Siniperea Chuatsi

In animal nutrition, protein is the most critical nutrient. Improvement of the utilization of protein not only can reduce farming costs, but also can reduce nitrogen pollution to the environment. Utilization of protein usually relies on the action of digestive enzymes: digestive enzymes devide proteins into smaller peptides and free amino acids, and then peptide transporter (PepTl) transports small peptides to cells. Studies have shown that intestinal epithelial cells whose function is to absorb the protein plays an important role, which is reveals the absorption mechanism of protein.PepTl is a transporter with low-affinity and high capacity peptide, which plays an important role in the peptide absorption. PepTl expression has important physiological significance in animal tissues, quantitative expression of PepTl gene expression both reflects the amount of protein in the cell, but also reflects the PepTl transport activity. In this study, we cloned the complete sequence for PepTl gens in Siniperca chuatsi, we analyzed PepTl gene expression in different tissues, different parts of the intestine, and the different stages of growth and development of the embryo, and we studied the short-term starvation refeeding PepTl gene expression impact; we also studied the effects of the cycle of hunger on digestive enzyme activity in intestinal tissue structure and on the digestive tract of Siniperca chuatsi, The main findings are as follows:(1) In order to reveal the molecular mechanism of PepTl-mediated protein digestion and absorption, the PepTl gene of the Siniperca chuatsi was cloned by using RT-PCR and RACE techniques. The full-length cDNA sequence of the PepTl was2480bp, including43nucleotides at5’UTR and232nucleotides at3UTR and2205nucleotides as an open reading frame encoding a735-amino-acid peptide. The PepTl amino acid sequence was determined and it shared similarity to those of other mammalian homolog protein, which included12helix trans-membrane regions and existed a large outer-ring between9th and10th of the transmembrane region. Homologous analysis of the PepTl showed that the amino acid of the PepTl was highly homologous to those of other vertebrates and it showed high percentages of similarities (at89%) to Epinephelus aeneus and Dicentrarchus labrax. In addition, the PepTl amino acid sequence varied in similarity to other vertebrates--from46%to56%. The encoded protein molecular weight was predicted at64.8kD with pi at8.97. (2) Using qRT-PCR method to study the PepTl gene expression in Siniperca chuatsi different tissues and parts and at different growth stages. The results showed that the highest expression of the PepTl gene occurred in the intestinal, in addition to expression in the intestine, expression were also detected in the heart, liver, brain, kidney, and red and white muscle; we also found that, PepTl in foregut had the highest expression, which gradually degraded along the longitudinal axis of the intestine. The study of the expression of the PepTl gene in Siniperca chuatsi of different weights showed that, PepTl expresssion peaked when body weight reaches lOg (P<0.05), and then decreased with weight increased and stabilized after the weight reached50g. In light of this, we speculate that when the fish about10g, mandarinfish’s ability to absorb the peptide peaks at the weight of10g, which makes it the best stage for rapid growth with adequate nutrition.(3) During the experimental cycle of hunger, we gave different experimental groups different starvation time (2d,4d,7d) and different recovery time (12d,10d,7d), after two cycles the study of fish growth indexes showed that the group4d (group C) reached full compensation growth, while group B and group D effect is not ideal, with only a part of the growth effect of compensation reached. We conclude that, in the actual production of Siniperca chuatsi, hungry treatment of no more than4d has the best effect of improving the efficiency of Siniperca chuatsi growth and reducing nutrition costs. Hunger treatment for longer than7d will cause significant damage to Siniperca chuatsi’s intestinal tissue structure.(4) Using the quantitative RT-PCR Method to study the effects on PepTl gene of refeedingafter starvation, Experimental results show that, The first, with starvation time (2d,4d,7d) increased, PepTl expression became significantly higher. The Second, that short-term starvation2d (group B) could cause significant reduction in PepTl expression. For example, after the end of the second cycle of hunger, group C’s PepTl gene expression was significantly higher than that of group B and D, and significantly lower than that of the control group A. The third, Sufficient feeding after starvation of7d showed that the expression in groups of lh-24h decreased at varying degrees:the expression in groups of24h-48h increased gradually; the expression peaked at48h, then gradually decreased for48h-96h, and stabilized after96h.