Cloning Of Porcine Cationic Amino Acid Transporters CDNA And Its MRNA Expression And Regulation

Cationic amino acid transporters play an important role in transporting cationic amino acid through the cell membrane, of which Systems b0,+, y+L and y+ shoulder the mission of transporting most cationic amino acids. The field of research in porcine cationic amino acid transporter system function has drawn progressing attention these years. In order to investigate the molecular mechanism of porcine cationic amino acid absorption, five experiments were carried out as follows.Experiment 1: The cDNA silicon cloning of the porcine 4F2hc and rBAT genes and the sequence validation by RT-PCR. Some porcine ESTs were obtained by searching the NCBI EST database (None Human & Mouse). These cDNAs were cloned using the bioinformatics software package and validated by RT-PCR.(1) The results showed that the full-length of porcine rBAT cDNA was 2487 bp, including a 2049 bp CDS, a 5' untranslated region (UTR) and a 3' UTR. The porcine rBAT cDNA sequence shared a 77.4% identity with human sequence, especially in coding sequence (CDS) regions (85.7%), which manifests a 87% identity of amino acid sequence with that of human.(2) A 1730 bp porcine partial mRNA sequence of 4F2hc gene has been cloned. The partial porcine mRNA sequence had 80.2% homology with known human sequences.(3) The RT-PCR results showed that the sequences obtained by electro-clone were reliable. The partial porcine mRNA sequence had 98.4%, 99.4%, 99.5% and 98.2% homology with electro-clone sequences.Experiment 2: Molecular cloning of porcine b0,+AT and y+LAT1 amino acid transporters. Primers of porcine partial cDNA sequence were designed based on the EST sequences deposited in the GenBank Data Library. Full-length of porcine b0,+AT and y+LAT1 amino acid transporter cDNA sequences has been cloned by RACE and RT-PCR using porcine intestines mRNA template.(1) Two full-length cDNAs encoding porcine b0,+AT-1,2 were isolated. Porcine b0,+AT-1 cDNA is a 1680bp sequence, including an 1464bp ORF encoding a 487 amino acid residues protein, 90 bp of 5'UTR and 126 bp of 3'UTR with a consensus AATAAA polyadenylation signal sequence at 7–12 nt upstream of a poly(A) stretch. Porcine b0,+AT-2 gene is a 1488bp sequence, including an 1488bp ORF encoding a 423 amino acid residues protein. The CDS part of porcine b0,+AT-2 gene is 192bp less than that of b0,+AT-1, which encodes 64 amino acid residues. Sequence analysis demonstrated that the CDS part of porcine b0,+AT-2 sequence shared 75.1%, 71.9% and 86.6% degree of sequence identity with those of human, mouse b0,+AT and porcine b0,+AT-1 respectively.(2) Porcine y+LAT1 cDNA includes an 1536 bp ORF encoding a 511 amino acid residues protein, a 134 bp 5' UTR, a 441 bp 3'UTR with a consensus AATAAA polyadenylation signal squence at 15–20 nt upstream of a poly(A) stretch. BLASTn analysis demonstrated that the porcine y+LAT1 cDNA sequence shared a high degree of sequence identity with cattle, human, mouse and rat y+LAT1, respectively, especially in CDS regions (91%, 90%, 87% and 87%), which manifest a high identity in amino acid sequences (93.2%, 92.4%, 88.5% and 88.9%).Experiment 3: The ontogenetic expression of rBAT and 4F2hc mRNAs in porcine intestine. The rBAT and 4F2hc mRNAs expression abundances were investigated by real-time quantitative polymerase chain reaction using tissue samples of duodenum, jejunum and ileum collected from Lantang and Landrace pigs at 1-, 7-, 26-, 30-, 60-, 90- and 150-day-old (150d). The results showed that as follows:(1) The expression of rBAT and 4F2hc mRNAs abundance could be regulated by developmental stage, breed and segment of intestine in pigs. There was, however, no difference in ontogenetic expression of rBAT and 4F2hc mRNAs of Lantang and Landrace pigs during post-weaning (weaning on 28d) period.(2) The ontogenetic expression of 4F2hc mRNA in duodenum and jejunum of Lantang and Landrace pigs were in a similar way. The 4F2hc mRNA level of Lantang pigs reached the peak on day 7 while that of Landrace on day 90 in the duodenum and jejunum. (3) There were different ontogenetic expression patterns of rBAT and 4F2hc mRNA in porcine duodenum, jejunum and ileum with no Linearity between them.Experiment 4: Study on the tissue distribution of b0,+AT,y+LAT1,rBAT and 4F2hc mRNAs. The tissue distribution patterns of b0,+AT, y+LAT1, rBAT and 4F2hc mRNAs were determined by real-time Q–PCR using the heart, liver, lung, kidney, brain, muscle and intestine total RNA. The results showed as follows:(1) Porcine 4F2hc mRNA was highly expressed in intestine and then in lung and brain. Besides, the mRNA of 4F2hc was also expressed in liver, kidney, heart and muscle but with low levels.(2) Porcine rBAT mRNA was highly expressed in intestine and then in brain, muscle and kidney. Besides, the mRNA of rBAT was lowly expressed in the liver and lung. However, there was no detectable levels of rBAT b0,+ AT mRNA expression in the heart.(3) The porcine small intestine had the highest b0,+AT mRNA abundance while the muscle had the lowest. Porcine b0,+AT mRNA was also expressed a small quantity in kidney and heart. However, there was no detectable levels of b0,+AT mRNA expression in the brain, lung and liver.(4) The porcine small intestine had the highest y+LAT1 mRNA abundance while the lowest in kidney, lung and heart. However, there was no detectable levels of y+LAT1 mRNA expression in the liver, muscle and brain.Experiment 5: Study on the in vitro porcine IEC cationic amino acid transporter mRNA expression under the influence of different lysine concentration. The primary culture of porcine IEC system was established by mechanical and enzymatic (collagenaseⅡ) methods using small intestines removed from colostrum-deprived newborn piglets. The IEC cultivated in vitro in Dulbecco's minimum essential medium–Hanks'F-12 (DMEM–F12). The IEC were adherent in 1~2 days and formed cell clone in 5~6 days, and then the monolayers were confluent in 9-11days. The IEC was identified by alkaline phosphatase and immunological methods. Total RNA was isolated from IEC samples after treatment with 0.5 mM, 2mM and 8mM lysine for 96 hours. The cationic amino acid transporters mRNA expression abundances were determined by real-time Q–PCR. The results showed as follows: (1) High concentrations of lysine have significant up-regulation effect on the CAT-1 mRNA expression and there was an obvious dose-dependent effect (P<0.01).(2) Lysine treatment had no distinct effect on the CAT-2 mRNA expression abundance. CAT-2 mRNA expressions of 8mM and 2mM lysine treatment were lower than that of 0.5mM lysine group, but with no significant difference (P>0.05).(3) Lysine treatment had no significant effect on the y+LAT1 mRNA expression abundance (P>0.05).(4) High concentrations of lysine could enhance 4F2hc mRNA expression. 4F2hc mRNA expression abundances of 0.5 mM and 2mM lysine groups were significantly lower than that of 8 mM lysine group (P<0.05). There was no significant difference between 0.5mM and 2mM group (P>0.05).