| The expression patterns of amino acids and glucose transporters have a very close relationship with muscle growth and meat quality. The nutritional substrate transported by amino acids and glucose transporters is very important for skeletal muscle growth and development. And factors of breed, age, muscle characteristics and nutrition could cause expression changes for amino acids and glucose transporter, which demonstrated its inherent physiological significance. Accordingly, the following five experiments were carried out to reveal the development pattern of some pig skeletal muscle glucose and amino acid transporters mRNA expression as well as the influence of nutritional factors and genetic factors, and explore differences in muscle development of Lantang and Landrace pigs and effect mechanisms of Lactobacillus on meat quality from the molecular level.Experiment 1:The cDNA cloning of the porcine GLUT-1The 792bp porcine partial mRNA sequence of the GLUT-1 gene has been cloned. The partial porcine mRNA sequence had 92.7% and 90.7% homology with known human and rat GLUT-1.Experiment 2:Molecular cloning of porcine amino acid transporter CAT-2 and glucose transporter GLUT-4Primers were designed based on the EST sequences and the porcine partial cDNA sequence which have been deposited in the GenBank Data Library. The full-length cDNA sequences of porcine amino acid transporter CAT-2 and glucose transporter GLUT-4 have been cloned by RACE and RT-PCR based on muscle mRNA template from landrace at the age of 6 d. The results are showed as follows:(1) The full-length cDNA encoding porcine CAT-2 from porcine was isolated. Sequence analysis of the 3147bp porcine CAT-2 cDNA revealed an ORF of 1974bp that would encode a protein of 657 amino acid residues,85 bp of 5'untranslated region (UTR) and 1087 bp of 3'UTR. Sequence analysis demonstrated that the porcine sequence shared a high degree of sequence identity (91.3%,88.1%,92.1% and 89.2%), with those of human CAT-2A,B and mouse CAT-2A,B. Hydrophobicity prediction suggested 14 putative membrane-spanning domains within porcine CAT-2, similar to other mammalian CATs. (2) Sequence analysis of the porcine GLUT-4 cDNA revealed that it had an ORF of 1530 bp that would encode a protein of 509 residues,34 bp of 5'UTR,927 bp of 3'UTR. The porcine sequence shared a high degree of sequence identity, both in the nucleotide sequences, especially in coding sequence (CDS) regions (90.3%,90.3% and 91.0%), and in the deduced amino acid sequences (94.5%,95.1% and 93.1%), with the human, rabbit and cattle GLUT-4, respectively. Hydrophobicity prediction suggested 12 putative membrane-spanning domains within porcine CAT-2, and similar to other species.Experiment 3:Study on the tissue distribution of amino acid transporter CAT-2 and glucose transporter GLUT-1/4 mRNAsThe tissue distribution patterns of CAT-2 and GLUT-1/4 mRNAs were determined by real-time Q-PCR using the heart, liver, lung, kidney, brain, muscle and intestine total RNA. The results showed that as follows:(1) Porcine CAT-2 mRNA was expressed in the brain, lung, liver, kidney, skeletal muscle, intestine and heart. Porcine CAT-2 mRNA was expressed highly in heart, and then in liver, skeletal muscle, lung, brain and intestine while the lowest in kidney.(2) Porcine GLUT-1 mRNA was expressed in the brain, lung, liver, kidney, muscle, intestine and heart. Porcine GLUT-1 mRNA was expressed highly in heart, and then in liver, kidney, brain, muscle and intestine while the lowest in lung.(3) Porcine GLUT-4 mRNA was expressed in the brain, lung, liver, kidney, muscle, intestine and heart. Porcine GLUT-4 mRNA was expressed highly in heart, and then in liver, muscle and brain while the lowly in lung, kidney and intestine.Experiment 4:Development comparisons of mRNA expressions of some glucose and amino transporter and insulin receptor in skeletal muscles from Lantang (LT) and Landrace pigs (LD)LT and LD at the ages of 1,7,26,30,60,90 and 150 days were selected to determine the expression patterns of GLUT-1/4 and cationic amino acid transporter at different skeletal muscle tissues by the method of real-time fluorescence quantitative PCR. Results:(1) LT and LD exhibited similar development of mRNA expressions of GLUT-1 in the longissimus dorsi muscles (LM), semimembranosus muscle (SM) and semitendinosus muscle (SD) from 1 to 150 days of age with highest level at the age of 1 day. The relative mRNA expression of GLUT-1 in the SM of LT was higher than that of LD with same age of 60 d (P<0.10). And the relative mRNA expression of GLUT-1 in the SD of LT was significantly higher than that of LD with same age of 150 d (P<0.05). (2) LT and LD exhibited waveform variations of mRNA expressions of GLUT-4 from 1 to 150 days of age with breed differences in the speed and extend of variations. At the age of 1 day the relative mRNA expression of GLUT-4 in the LM of LT was lower than that of LD with same ages (P<0.10) while they were significantly higher than that of LD with same age of 60 d (P<0.05). At the age of 7 and 60 days the relative mRNA expression of GLUT-4 in the SM of LT was significantly higher than that of LD with same ages (P<0.05). At the age of 1 day the relative mRNA expression of GLUT-4 in the SD of LT was significantly lower than that of LD with same ages (P<0.05) while they were higher than that of LD at other ages. And at the age of 150 day the relative mRNA expression of GLUT-4 in the SD of LT was 48.14% more than that of LD with same ages(P<0.10).(3) LT and LD exhibited higher level of the mRNA expressions of IR in the LM, SM and SD at the age of 1 and 30 days, while there were not significant difference among 7,60,90 and 150 days of age. There was no significant difference between LT and LD with same ages.(4) The relative mRNA expression of CAT-2 in the LM, SM and SD of LT and LD gradually increased after 1 day of age, and reached the highest level at the age of 90 days, which was significantly higher than that at other ages. And then it gradually declined.Experiment 5:Effect of plant-derived lactobacillus on mRNA expressions of glucose and amino acid transporters in skeletal muscles of finished Landrace x Yorkshire pigsA total of 54 steer boar pigs of Landrace×Yorkshire were randomly divided into two groups, including control group (group CON) and treatment group of Lactobacillus (group TREAT). Each group had 3 replicates, and each replicate had 9 pigs. At the end of the trial two steer boar pigs with average live weight per replicate were selected and bleed to death. Tissue samples were collected from the thoracolumbar of the LM, the middle of the SM and psoas muscle (PM) by anatomical location within 2 hours post-slaughter to determine the expression patterns of GLUT-1/4 and cationic amino acid transporter at different skeletal muscle tissues by the method of real-time fluorescence quantitative PCR. Results:(1) Dietary supplement plant-derived lactobacillus had no significant effect on the concentration of total glycogen. But it had a trend of improving the concentration of proglycogen (PG) in the LM (P<0.10).(2) The relative mRNA expression of GLUT-1/4 and IR in the PM was higher than that in the LM and SD. Dietary supplement plant-derived lactobacillus had no significant effect on the relative mRNA expression of GLUT-1/4 and IR.(3) Dietary supplement plant-derived lactobacillus had no significant effect on the relative mRNA expression of CAT-2, but it had trend to lower the expression of CAT-2 in the PM.
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