牦牛GLUT1、GLUT3和GLUT8基因的克隆及其在乳腺上皮细胞中的表达

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摘要乳腺中葡萄糖摄取主要依赖葡萄糖转运蛋白(glucose transporter, GLUT)，不同GLUT对葡萄糖摄取所起作用不同。为研究GLUT1、GLUT3和GLUT8参与牦牛(Bos grunniens)泌乳机能调节的分子机制，本研究采用胰酶消化法离体培养牦牛乳腺上皮细胞，通过免疫细胞化学方法检测角蛋白18、β-酪蛋白和波形蛋白；从纯化的细胞中分别克隆牦牛GLUT1、GLUT3和GLUT8基因，并分析其相关生物学特性；采用qRT-PCR和Western blot分别从基因和蛋白水平分析三者在乳腺上皮细胞表达差异性，间接免疫荧光检测其在乳腺上皮细胞中的分布。结果显示，角蛋白18和β-酪蛋白在分离得到的细胞中表达，波形蛋白不表达，所分离的细胞为乳腺上皮细胞。成功克隆出包含完整编码区的牦牛GLUT1、GLUT3和GLUT8基因序列(GenBank登录号分别为: KU902419, KX094556和KX268646)，其编码区核苷酸长度分别为1 479、1 485和1 437 bp，分别编码492、494和478个氨基酸；三者在进化过程中均有较高的保守性，编码的蛋白具有相似理化性质，均具有12个跨膜螺旋区的疏水性膜蛋白。在乳腺上皮细胞中，GLUT1的表达量最高，GLUT8的表达量次之，GLUT3的表达量最低，且三者之间的表达差异极显著(P＜0.01)。牦牛乳腺上皮细胞胞质和胞核均有GLUT1、GLUT3和GLUT8蛋白表达，且主要分布在细胞核。研究结果为了解牦牛GLUT生理学功能提供了重要信息，为进一步研究其调控牦牛泌乳机能的生物学作用提供了新的理论依据。

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	李月琴
	潘阳阳
	田奎丽
	赵天
	张译夫
	樊江峰
	崔燕
	余四九

关键词 ：牦牛, 葡萄糖转运蛋白1(GLUT1), GLUT3, GLUT8, 乳腺上皮细胞

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.

Key words： Yak Glucose transporter 1 (GLUT1) GLUT3 GLUT8 Mammary epithelial cells

收稿日期: 2016-09-02 出版日期: 2016-12-24

ZTFLH:

S857.2

基金资助:甘肃省生物技术专项;马麝病毒性出血症的病原分离鉴定及其生物学特性研究

通讯作者: 余四九 E-mail: sjyu@163.com

引用本文:

李月琴潘阳阳田奎丽赵天张译夫樊江峰崔燕余四九. 牦牛GLUT1、GLUT3和GLUT8基因的克隆及其在乳腺上皮细胞中的表达[J]. , 2017, 25(1): 21-31.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I1/21

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