On of GLUT1, GLUT12, sweet1 and amino acid transporter gene expression by RT-qPCRThe mRNA expression of GLUT1 was increased by 80 in the GT1-GMGE cells when compared with the GMGE cells 10781694 (Fig. 2), whereas no difference in GLUT12 expression was observed. Because glucose is required as a substrate for protein synthesis, we also detected the expression of three amino acid transporters: SLC1A5, SLC3A2 and SLC7A5. The mRNA of SLC7A5 and SLC3A2 in the GT1-GMGE cells increased by 20 and 25 , respectively, but was not significant when compared with the GMGE cells (Fig. 2). In the GT12-GMGE cells, the mRNA expression of GLUT12 was nearly increased by 40-fold compared with the GMGE cells, whereas GLUT1 expression was significantly decreased (P,0.01). Furthermore, the mRNA expression levels of the three amino acid transports were all significantly increased (P,0.01). Sweet1 is a recently reported sugar transporter involved in intercellular exchange and nutrition. The GMGE cells were found to express sweet1 RNA, yet this expression was not influenced by the overexpression of GLUT1 or GLUT12.Primers names RPS6-F RPS6-R RPL4-F RPL4-R RPL22-F RPL22-R RPL35-F RPL35-R EEF1A1-F EEF1A1-R EEF2-F EEF2-R PIK3C3-F PIK3C3-R RHEB-F RHEB-R PRLR-F PRLR-R STAT5B-F STAT5B-R ?actin-F ?actin-RType Forward Reverse Forward Reverse Forward Reverse Forward 16985061 Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward ReverseSequences 5′-CTGGGTGAAGAATGGAAGGG-3′ 5′-CGAACTCTGCCATGGGTCA-3′ 5′-AAGATGGCGCCGAAGAAAG-3′ 5′-TTTCCCGAATCAAAAATTCCA-3′ 5′-TCGAAGCTAGGACGTGGTGG-3′ 5′-TTGGCTCCTGTGTTGTCAGC-3′ 5′-TTGGAAACATGTGTCGTGGG-3′ 5′-GCAGATGGCGTATCGCTTCT-3′ 5′-CATCCCAGGCTGACTGTGC-3′ 5′-TGTAAGCCAAAAGGGCATGC-3′ 5′-GGGCTGGTGTCTACTGGCC-3′ 5′-TCAGGATTGTCCTCTGGATCG-3′ 5′-GCCAAGCATTGTTGAAGGGT-3′ 5′-GCACCAGCCGATCTACAAAAG-3′ 5′-GCTAAGATGCCGCAGTCCA-3′ 5′-CGTCAACGAGGATTTCCCC-3′ 5′-ATAGCATGGTGACCTGCATCC-3′ 5′-TCTTCGGACTTGCCCTTCTC-3′ 5′-GCAGCTCCAGAACACGTACG-3′ 5′-CATTGTTGGCTTCTCGGACC-3′ 5′-CCAACCGTGAGAAGATGA-3′ 5′-CAGAGTCCATGACAATGC-3’Glucose consumption and lactose yield in GMGE, GT1GMGE and GT12-GMGE cellsGlucose consumption was detected to verify the biological function of the goat GLUT1 and GLUT12 genes. Compared to the GMGE cells, the glucose consumption of the GT1-GMGE cells (2.0230960.02674) was significantly increased in comparison to the GMGE cells (1.7713960.00346; P,0.05) at 24 h and reached 3.1984360.24797 at 48 h (P,0.01; Fig. 3). The mammary gland Title Loaded From File absorbs glucose to synthesize lactose, and the lactose concentration in the GT1-GMGE culture (65.4435865.69766) was significantly higher than that in the GMGE culture (49.9257864.19244) (P,0.05; Fig. 3). There was no difference in the glucose consumption Title Loaded From File between the GT12GMGE and GMGE cells at 24 h; however, the glucose consumption increased significantly (P,0.01) at 48 h in the GT12-GMGE cells. The lactose concentrations between the GT1GMGE/GT12-GMGE and GMGE culture media were not statistically significant at 48 h.doi:10.1371/journal.pone.0065013.tSequence analysis of goat GLUT1 and GLUTThe prediction of the transmembrane helices in goat GLUT1 and GLUT12 by the TMHMM Server v. 2.0 revealed that the distribution pattern of its hydrophobic and presumed membranespanning segments generally favored the proposed secondary structure of GLUTs, a 12-helix model. Goat GLUT1 is predicted to present a large exoplasmic loop between TMs 6 and 7 (loop 6) and a glycosylated extracellular loop between TMs.On of GLUT1, GLUT12, sweet1 and amino acid transporter gene expression by RT-qPCRThe mRNA expression of GLUT1 was increased by 80 in the GT1-GMGE cells when compared with the GMGE cells 10781694 (Fig. 2), whereas no difference in GLUT12 expression was observed. Because glucose is required as a substrate for protein synthesis, we also detected the expression of three amino acid transporters: SLC1A5, SLC3A2 and SLC7A5. The mRNA of SLC7A5 and SLC3A2 in the GT1-GMGE cells increased by 20 and 25 , respectively, but was not significant when compared with the GMGE cells (Fig. 2). In the GT12-GMGE cells, the mRNA expression of GLUT12 was nearly increased by 40-fold compared with the GMGE cells, whereas GLUT1 expression was significantly decreased (P,0.01). Furthermore, the mRNA expression levels of the three amino acid transports were all significantly increased (P,0.01). Sweet1 is a recently reported sugar transporter involved in intercellular exchange and nutrition. The GMGE cells were found to express sweet1 RNA, yet this expression was not influenced by the overexpression of GLUT1 or GLUT12.Primers names RPS6-F RPS6-R RPL4-F RPL4-R RPL22-F RPL22-R RPL35-F RPL35-R EEF1A1-F EEF1A1-R EEF2-F EEF2-R PIK3C3-F PIK3C3-R RHEB-F RHEB-R PRLR-F PRLR-R STAT5B-F STAT5B-R ?actin-F ?actin-RType Forward Reverse Forward Reverse Forward Reverse Forward 16985061 Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward ReverseSequences 5′-CTGGGTGAAGAATGGAAGGG-3′ 5′-CGAACTCTGCCATGGGTCA-3′ 5′-AAGATGGCGCCGAAGAAAG-3′ 5′-TTTCCCGAATCAAAAATTCCA-3′ 5′-TCGAAGCTAGGACGTGGTGG-3′ 5′-TTGGCTCCTGTGTTGTCAGC-3′ 5′-TTGGAAACATGTGTCGTGGG-3′ 5′-GCAGATGGCGTATCGCTTCT-3′ 5′-CATCCCAGGCTGACTGTGC-3′ 5′-TGTAAGCCAAAAGGGCATGC-3′ 5′-GGGCTGGTGTCTACTGGCC-3′ 5′-TCAGGATTGTCCTCTGGATCG-3′ 5′-GCCAAGCATTGTTGAAGGGT-3′ 5′-GCACCAGCCGATCTACAAAAG-3′ 5′-GCTAAGATGCCGCAGTCCA-3′ 5′-CGTCAACGAGGATTTCCCC-3′ 5′-ATAGCATGGTGACCTGCATCC-3′ 5′-TCTTCGGACTTGCCCTTCTC-3′ 5′-GCAGCTCCAGAACACGTACG-3′ 5′-CATTGTTGGCTTCTCGGACC-3′ 5′-CCAACCGTGAGAAGATGA-3′ 5′-CAGAGTCCATGACAATGC-3’Glucose consumption and lactose yield in GMGE, GT1GMGE and GT12-GMGE cellsGlucose consumption was detected to verify the biological function of the goat GLUT1 and GLUT12 genes. Compared to the GMGE cells, the glucose consumption of the GT1-GMGE cells (2.0230960.02674) was significantly increased in comparison to the GMGE cells (1.7713960.00346; P,0.05) at 24 h and reached 3.1984360.24797 at 48 h (P,0.01; Fig. 3). The mammary gland absorbs glucose to synthesize lactose, and the lactose concentration in the GT1-GMGE culture (65.4435865.69766) was significantly higher than that in the GMGE culture (49.9257864.19244) (P,0.05; Fig. 3). There was no difference in the glucose consumption between the GT12GMGE and GMGE cells at 24 h; however, the glucose consumption increased significantly (P,0.01) at 48 h in the GT12-GMGE cells. The lactose concentrations between the GT1GMGE/GT12-GMGE and GMGE culture media were not statistically significant at 48 h.doi:10.1371/journal.pone.0065013.tSequence analysis of goat GLUT1 and GLUTThe prediction of the transmembrane helices in goat GLUT1 and GLUT12 by the TMHMM Server v. 2.0 revealed that the distribution pattern of its hydrophobic and presumed membranespanning segments generally favored the proposed secondary structure of GLUTs, a 12-helix model. Goat GLUT1 is predicted to present a large exoplasmic loop between TMs 6 and 7 (loop 6) and a glycosylated extracellular loop between TMs.
