Cloning And Expression Regulation Of Small Peptide Transporter PepT1 In Yak

Yak is a special livestock whose physical,physiological and ingestive characteristics are different from other species on the Qinghai-Tibetan ecological system.In recent years,many researches confirmed that yak had unique nitrogen metabolism mechanism to adapt to the malnutrition environment,which closely related to peptide nutrition.As the intermediate products of protein metabolism,the small peptides are transported by the small peptide transport carrier(Pep T1)in the gastrointestinal epithelium.Currently,many researches mainly focus on molecular basis and regulation mechanism for PepT1 in low altitude animals,such as chicken,pig,sheep and cattle;but it remains blank on yak.The objective of this study was investigating the PepT1 sequence,tissue distribution and active regulation in yak,and further explores the molecular basis and regulation mechanism of yak peptide absorption characteristic.The relative research achievements can not only enrich the protein nutrition theory and research methods and improve the feeding strategy in the alpine ruminants,but also minimize the environmental pollution caused by the imbalance of dietary nutrient supply.Therefore,the study mainly includes: 1)Cloning and biological analysis of the small peptide transport carrier PepT1 in yak;2)the distribution characteristics of grazing yak PepT1 and comparative study on the response of PepT1 mRNA relative expression to the different dietary nitrogen levels between yak and indigenous cattle;3)Comparative study on the effects of the small peptide types on PepT1 mRNA relative expression between yak and indigenous cattle in CHO-k1 cells.The results followed that:(1)The results of RT-PCR and RACE showed that the full-length full-length of yPepT1(Genbank: KT725253)cDNA is 2,805 bp and encode a putative protein of 707 AA.The yPepT1 cDNA include 5 putative extracellular N-glycosylation sites,2 putative intracellular protein kinase A sites,and 3 intracellular putative protein kinase C sites.It It is predicted adopt a putative 12-transmembrane-domain structure.The putative protein has a a pI of 7.1 and protein molecular weight of 78.4 kDa.It is a non-secretory protein with no signal signal peptide.The number of Val(9.1%)in yPepT1 is higher,and lower is the Trp(1.3%).The The yPepT1 is the hydrophilic AA,which the nonpolar AA(hydrophobic AA)accounted for 47.81%,and the polar AA(hydrophilic AA)accounted for 52.19%.Second-level structure prediction showed that the AA of yPepT1 contained ?-helix,?-sheet and random coil.Phylogenetic analysis suggested that the yak is correlated with cattle.Meanwhile,the homology of predicted AA sequence of yPepT1 with cattle,sheep,pig,human,rat and rabbit were 99%,95%,86%,83%,81% and 79%,respectively.Moreover,there is different from yPepT1 and bPepT1 in 258,512,536,544 AA residues.(2)The PepT1 mRNA of grazing yak distributed in all gastrointestinal,liver and breast tissues,its relative expression in jejunum was greater than other tissues(P < 0.001),followed by ileum and duodenum,lower in reticulum and breast,followed by rumen,omasum,colon,abomasum,caecum,liver and kidney,thereinto,the PepT1 mRNA in abomasum,caecum,liver and kidney was very low.(3)The comparison experiments of feeding and slaughter trial between yak and indigenous cattle showed that: the genotype,dietary nitrogen and the interaction between genotype and dietary nitrogen were significantly effects the PepT1 mRNA relative expression for yak and indigenous cattle.There was significant difference in the relative PepT1 mRNA expression of rumen,omasum,duodenum,jejunum,ileum,and liver tissues between the 2 genotypes(P < 0.05);moreover,the dietary nitrogen significantly effects the relative PepT1 mRNA expression in rumen,reticulum,duodenum,and ileum tissues.As dietary N content increased,the relative PepT1 mRNA expression increased linearly in rumen,omasum and ileum tissues(P < 0.05),but decreased in duodenum and liver tissues(P < 0.05).Moreover,when the 2 genotype animals were fed 10.3 g N/kg DM diet,the relative PepT1 mRNA expression was greater in omasum,duodenum,ileum and liver tissues(P < 0.05),but lower in jejunum tissue for indigenous cattle than yak(P < 0.01);when fed 19.5 g N/kg DM diet,the relative PepT1 mRNA expression was greater in rumen and reticulum,omasum,jejunum,and liver tissues(P < 0.05),but lower in ileum tissue for indigenous cattle than yak(P < 0.05);when fed 28.5 g N/kg DM diet,the relative PepT1 mRNA expression was greater in rumen,omasum,duodenum and ileum,liver tissues(P < 0.05),but lower in reticulum tissue for indigenous cattle than yak(P < 0.05);and the relative PepT1 mRNA expression was greater in duodenum and ileum tissues(P < 0.01),but lower in jejunum tissue for indigenous cattle than yak(P < 0.05)fed 37.6 g N/kg DM diet.These different on the relative PepT1 mRNA expression confirmed yak has a differ from indigenous cattle in small peptide absorption transport mechanisms,which indicate the adaptation for yak to survival in the alpine extreme environment.(4)Clone and construct yPepT1 gene and cattle PepT1(bPepT1)in eukaryotic expression vector and express it in Chinese hamster oocytes(CHO-k1)cells.compare the transport activity of recombinant between yPepT1 and bPepT1 mRNA relative expression.The result showed that the PepT1 mRNA expression was significant different in genotypes,small peptide treatments,and interactions between genotype and small peptide treatments.The PepT1 mRNA relative expressions were different from control treatment and small peptide treatment.In yak,the PepT1 mRNA relative expression in Met-Lys,Lys-Met,Let-Lys,Thr-Ser-Lys,Lys-Try-Lys and Met-Gly-Met-Met treatments were greater than control treatment(P < 0.05),but no significant differences between Met-Met,Leu-Ser-Phe or Lys-Thr-Ser treatment and control treatment(P > 0.05);in cattle,the PepT1 mRNA relative expressions in Met-Met,Met-Lys,Lys-Met,Lys-Lys,Leu-Ser-Phe,Lys-Thr-Ser and Thr-Ser-Lys treatments were greater than control treatment(P < 0.05),but no significant differences between Lys-Try-Lys or Met-Gly-Met-Met treatment and control treatment(P > 0.05).Moreover,in di-peptide treatment groups,the PepT1 mRNA relative expression in Met-Lys and Lys-Lys groups for indigenous cattle were greater than yak(P < 0.05);In tri-peptide treatment groups,the PepT1 mRNA relative expression in Leu-Ser-Phe and Lys-Thr-Ser groups for indigenous cattle were also greater than yak(P < 0.05),but in Lys-Lys group for yak was greater than indigenous cattle(P < 0.05).In conclusion,based on the difference between yak and indigenous cattle on PepT1 molecular characteristics,the response characteristics of dietary nitrogen and substrate regulation,the yak has evolved various small peptide absorption and transport mechanlism from indigenous cattle,which benefits to adapt the ecological environment on the Qinghai-Tibetan Plateau.