PepT1 Tissue Distribution And Impact Of Fasting As Well As Exogenous Protein On PepT1 MRNA Expression Levels In Procypris Rabaudi (Tchang)

The intestinal absorption of di- and tri-peptides generally occurs via the oligopeptide transporter(PepT1),which is an integral plasma membrane protein. In the course of the past few years, the study of intestinal peptide transport has rapidly evolved into a field of nutritional and biomedical applications. In particular, the effect of fasting and dietary protein on oligopeptide transporter 1(PepT1) expression and activity has been an area of active research in recent years.This paper selects Procypris rabaudi (Tchang) as experimental subject,and clone PepT1 cDNA fragment by regular PCR.In order to get information about PepT1 expression in Procypris rabaudi (Tchang), tissues distribution and impact of fasting as well as exogenous protein on PepT1 mRNA expression levels were done by real-time fluorescent quantification PCR.This paper has three sections:⒈PepT1 cDNA fragment clone of Procypris rabaudi (Tchang) and sequence analysis.The cDNA of Procypris rabaudi (Tchang) PepT1 was amplified by RT-PCR using primers designed based on conserved region of multiple alignment of the published sequences of PepT1 genes in GenBank. The amplified fragment was sequenced after purifying and T-A clone. The accession for the target fragment of 751bp is GU138157( not released yet).The results of BLAST showed the target fragment had 97% nucleotide identity with the corresponding segment of common carp,while 87% nucleotide identity with zebrafish.The deduced amino acids sequence encoded 250aa, covering 5 transmembrane regions.⒉Tissue distribution of PepT1 mRNA in Procypris rabaudi (Tchang).PepT1 mRNA tissue distribution was done in 11 tissues of Procypris rabaudi (Tchang) by real-time fluorescent quantification PCR(SYBR GreenⅠfluorescent dye),using 18S rRNA,EF1αand RFL13αas internal references.The 11 tissues were foregut,midgut,hindgut, spleen,eye,brain,muscle,heart,kidney,gill and liver.The results revealed the expression of PepT1 mRNA levels of foregut and midgut is significantly higher than other tissues which were no significant differences one another, and the expression of foregut is significant higher than midgut(P﹤0.05).⒊Impact of fasting and exogenous protein on PepT1 mRNA expression levels in Procypris rabaudi (Tchang) foregut.This study evaluated the impact of fasting and different concertration of soybean oligopeptide(SOP) solutions on PepT1 mRNA expression levels in Procypris rabaudi (Tchang) foregut.①The expression of PepT1 mRNA levels were measured after fasting 1d, 3d, 5d, 7d, 9d, 11d, 14d( fasting 1d is control).The results exhibited the expression of PepT1 mRNA levels was highest at fasting 5d.②100 juvenile Procypris rabaudi (Tchang) were divided into four groups at random with different concertration of SOP solutions (0,5%, 20%, 35%). The dose of force-feeding was 2ml/100g body weight. The control (that is 0 concertration of SOP solution) force-fed the same dose distilled water. The expression of PepT1 mRNA levels was quantified by RT-PCR after force-feeding 0.5,1.5,3.5,7.5,12h.The results showed there were no significanct differences between each groups at 1.5h;and the expression of PepT1 mRNA increased in 5%SOP group at 3.5h,which was significanctly higher than other groups at 3.5h (P﹤0.05); the expression of PepT1 mRNA increased in 5% and 20%SOP groups at 7.5h,which were significanctly higher than control (P﹤0.05); the expression of PepT1 mRNA increased in 20% and 35%SOP groups at 12h, which were significanctly higher than control (P﹤0.05). The results indicated that the expression of PepT1 mRNA in Procypris rabaudi (Tchang) foregut can induced by force-feeding SOP.