|
|
Cloning of GLUT1, GLUT3 and GLUT8 Genes and Their Expression in Mammary Epithelial Cells of Yak (Bos grunniens) |
|
|
Abstract Glucose uptake in the mammary gland mainly depends on the glucose transporter (GLUT), and the different GLUT has different effects on glucose uptake. The aim of this study was to investigate the molecular mechanism of glucose transporter that participate in lactation regulation of yak (Bos grunniens). Mammary epithelial cells were cultured in vitro using pancreatic enzyme digestion method, and the immunocytochemistry method was used to appraise mammary epithelial cells by detecting the expression of cytokeratin 18, β-casein and vimentin; GLUT1, GLUT3 and GLUT8 gene were cloned by PCR from purified cells and its biological characteristics were also analyzed. The mRNA and protein expression of GLUT1, GLUT3 and GLUT8 was detected by qRT-PCR and Western blot, and their location in yak mammary epithelial cells were detected by indirect immunofluorescence. The results showed that cytokeratin 18 and β-casein could be detected in the isolated cells, and vimentin was negative, which indicated the cells were mammary epithelial cells. The GLUT1, GLUT3 and GLUT8 (GenBank accession number: KU902419, KX094556 and KX268646) gene were successfully cloned from mammary epithelial cells of yak, which contained a complete coding sequence, the length of nucleotide were 1 479, 1 485 and 1 437 bp, respectively, and encoded 492, 494 and 478 amino acids, respectively. Nucleotide sequence analysis revealed that GLUT1, GLUT3 and GLUT8 genes were conserved. The physical and chemical properties of protein encoded by 3 genes were similar, all of them were hydrophobic membrane proteins including 12 transmembrane helical region. The expression of GLUT1, GLUT3 and GLUT8 in mammary epithelial cells of yak were extremely significant difference (P<0.01), GLUT1 was the highest, followed by GLUT8, while GLUT3 was the lowest. GLUT1, GLUT3 and GLUT8 protein were mainly located in the nucleus of in mammary epithelial cells of yak. The results provide important information for understanding the physiological function of yak glucose transporters and a new theoretical basis for further studying the biological function of yak lactation.
|
Received: 02 September 2016
Published: 24 December 2016
|
|
|
|
|
何翃闳, 崔燕, 潘阳阳, 等. 2015. 牦牛HSP27基因的克隆及其在雌性生殖器官中的表达[J]. 中国农业科学, 48(20):4178-4187. (He H H, Cui Y, Pan Y Y, et al. 2015. Cloning of Bos grunniens HSP27 gene and its expression in the female yak reproductive organs [J]. Scientia Agricultura Sinica, (20):4178-4187.) 郝明超, 刘犇, 樊江峰, 等. 2013. 牦牛乳腺上皮细胞系的建立与生物学特性[J]. 兽类学报, 33(2):157-163. (Hao M C, Liu B, Fan J F, et al. 2013. Establishment and characterization of yak mammary epithelial cell line [J]. Acta Theriologica Sinica, 33(2):157-163.)李文清, 王加启, 南雪梅, 等. 2012. 奶牛乳糖合成及泌乳相关基因和细胞信号通路的研究进展[J]. 中国畜牧兽医, 39(11):104-111. (Li W Q, Wang J Q, NAN X M, et al. 2012. Research progress on genes and signal pathways associated with lactose synthesis and lactation in dairy cows [J]. China Animal Husbandry & Veterinary Medicine, 39(11):104-111.)李楠, 刘荣立, 柯浩, 等. 2013. 水牛GLUT3基因的克隆与序列分析[J]. 黑龙江畜牧兽医, 19(19):4-6. (Li N, Liu R L, Ke H, et al. 2013. Cloning and sequence analysis of GLUT 3 gene in swamp buffalo [J]. Heilongjiang Animal Science and Veterinary Medicine, 19(19):4-6.)王汝, 余四九, 崔燕. 2009. 幼龄牦牛甲状腺的显微结构和超微结构观察[J]. 中国兽医科学, 39(4):357-361. (Wang R, Yu S J, Cui Y. 2009. Observation of microstructure and ultrastructure of the thyroid gland in juvenile yak[J]. Chinese Veterinary Science, 39(4):357-361.)王华, 张正线, 柴建华, 等. 1995. 促葡萄糖转运蛋白基因家族的分子进化研究[J]. J遗传学报, 22(6):413-423. (Wang H, Zhang Z X, Chai J H, et al. 1995. Studis on molecular evolution of Glucose transporters genes family [J]. Acta Genetica Sinica, 22(6):413-423.)郑红飞, 潘阳阳, 刘鹏刚, 等. 2015. 牦牛Tp53基因特征分析及其在不同发情时期卵巢中的表达[J]. 华北农学报, 30(6):70-76. (Zheng H F, Pan Y Y, Liu P G, et al. 2015. Characteristics of yak tumor protein p53gene (Tp53) and its expression in different periods of estrus ovaries [J]. Acta Agriculturae Boreali-Sinica, 30(6):70-76.)赵珂. 2011. 奶牛乳腺上皮细胞葡萄糖摄取的调控及其对乳成分合成的影响研究[D]. 浙江大学. (Zhao k. Studies on the regulation of glucose transport and utilization in boving mammary epithelial cells [D]. Zhejiang University.) 周美菊, 张夫千, 杨延周, 等. 2008. 奶牛泌乳性状相关基因的研究进展[J]. 安徽农业科学, 36(18):7657-7658. (Zhou M J, Zhang F Q, Yang Y Z,et al. 2008. Research progress on the interrelated gene of milk production traits in cow [J]. Journal of Anhui Agricultural Sciences, 36(18):7657- 7658.)张译夫, 潘阳阳, 崔燕, 余四九. 2015. 牦牛输卵管蛋白(Ovn)的cDNA克隆及其在发情周期中的差异性表达[J]. 农业生物技术学报, 23(9): 1217-1225. (Zhang Y F, Pan Y Y, Cui Y, et al. 2015. cDNA Cloning of Yak (Bos grunniens) Oviductin (Ovn) and Its different expression during the estrous cycle [J]. Journal of Agricultural Biotechnology, 23(9): 1217-1225.)Bionaz M, Loor J J. 2011. Gene networks driving bovine mammary protein synthesis during the lactation cycle [J]. Bioinformatics & Biology Insights, 5(5):83-98. Deng D, Xu C, Sun P, et al. 2014. Crystal structure of the human glucose transporter GLUT1 [J]. Nature, 510(7503):121-5. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap [J]. Evolution, 39:4(39):783-791. Joost H G, Thorens B. 2001. The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics,and potential function of its novel members (review) [J]. Molecular Membrane Biology, 18(4):247-256. Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2? ΔΔCT method [J]. methods, 25(4): 402-408.Miller P J, Finucane K A, Hughes M, et al. 2005. Cloning and expression of bovine glucose transporter GLUT12 [J]. Mammalian Genome Official Journal of the International Mammalian Genome Society, 16(11):873-883. Macheda M L, Williams E D, Best J D, et al. 2003. Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland [J]. Cell & Tissue Research, 311(1):91-97.Madon R J, Martin S, Davies A, et al. 1990. Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland [J]. Biochemical Journal, 272(272):99-105.Nemeth B A, Tsang S W, Geske R S, et al. 2000. Golgi targeting of the GLUT1 glucose transporter in lactating mouse mammary gland [J]. Pediatric Research, 47(1):444-50. SCOTT R A, Bauman D E, Clark J H. 1976. Cellular Gluconeogenesis by Lactating Bovine Mammary Tissue [J]. Journal of Dairy Science, 59(1):50-6. Shennan D B, Peaker M. 2000. Transport of milk constituents by the mammary gland [J]. Physiological Reviews, 80(3):925-51. Tamura K, Dudley J, Nei M, et al. 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology & Evolution, 24(8):1596-1599.Wilson-O'Brien A, Dehaan C S. 2008. Mitogen-stimulated and rapamycin-sensitive glucose transporter 12 targeting and functional glucose transport in renal epithelial cells [J]. Endocrinology, 149(3):917-924.Wood I S, Trayhum P. 2007. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins [J]. British Journal of Nutrition, 89(1):3-9. Zhao F Q, Keating A F. 2007b. Expression and regulation of glucose transporters in the bovine mammary gland [J]. Journal of Dairy Science, 90:E76-E86.Zhao F Q, Miller P J, Wall E H, et al. 2004. Bovine glucose transporter GLUT8: cloning, expression, and developmental regulation in mammary gland [J]. Biochimica et Biophysica Acta, 1680(2):103–113.Zhao F Q, Glimm D R, Kennelly J J. 1988. Distribution of mammalian facilitative glucose transporter messenger rna in bovine tissues [J]. International Journal of Biochemistry, 25(12):1897-1903. |
[1] |
ZHAO Zhi-Dong, TIAN Hong-Shan, JIANG Yan-Yan, SHI Bin-Gang, LIU Xiu, LI Xu-Peng, WANG Deng-Zhe, CHEN Jin-Lin, HU Jiang. Polymorphisms of ACSL1 Gene Promoter and Their Association Analysis with Milk Quality Traits in Yak (Bos grunniens)[J]. 农业生物技术学报, 2019, 27(9): 1596-1603. |
|
|
|
|