牦牛GLUT1、GLUT3和GLUT8基因的克隆及其在乳腺上皮细胞中的表达
发布时间：2018-08-10 07:15
【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q RT-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(Gen Bank登录号分别为:KU902419,KX094556和KX268646),其编码区核苷酸长度分别为1479、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(P0.01)。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。
[Abstract]:Glucose uptake in mammary glands is mainly dependent on glucose transporter glut, and different GLUT have different effects on glucose uptake. In order to study the molecular mechanism of GLUT3 and GLUT8 involved in the regulation of lactation function of yak (Bos grunniens), the mammary epithelial cells of yak were cultured by trypsin digestion in vitro. Keratin 18, 尾 -casein and vimentin were detected by immunocytochemistry. The GLUT3 and GLUT8 genes were cloned from purified cells, and their biological characteristics were analyzed. The difference of expression of GLUT3 gene and GLUT8 gene in mammary epithelial cells was analyzed by Q RT-PCR and Western blot from gene and protein levels, respectively. Indirect immunofluorescence was used to detect its distribution in mammary epithelial cells. The results showed that keratin 18 and 尾-casein were expressed in the isolated cells, but vimentin was not expressed. The cells isolated were mammary epithelial cells. The complete coding region of Yak GLUT1G UT3 and GLUT8 gene were cloned successfully. The (Gen Bank accession numbers of GLUT3 and GLUT8 gene were: 1: KU902419 KX094556 and KX268646). The nucleotide length of the coding region was 1479bp1 485 and 1 437bprespectively, encoding 492494 and 478amino acids, respectively. The three proteins are highly conserved in the evolution process and have similar physicochemical properties and 12 hydrophobic membrane proteins with 12 transmembrane helical regions. The highest expression of GLUT1 was found in mammary epithelial cells and the lowest was GLUT8, and the difference among them was significant (P0.01). GLUT1, GLUT3 and GLUT8 protein were expressed in cytoplasm and nucleus of mammary epithelial cells of yak, and were mainly distributed in the nucleus. The results provide important information for understanding the physiological function of yak GLUT, and provide a new theoretical basis for further study on the biological function of regulating the lactation function of yaks.
【作者单位】： 甘肃农业大学动物医学院/甘肃省牛羊胚胎工程技术研究中心;
【基金】：国家自然科学基金(No.31472244) 甘肃省生物技术专项(GNSW-2013-23)
【分类号】：S823.85
[Abstract]:Glucose uptake in mammary glands is mainly dependent on glucose transporter glut, and different GLUT have different effects on glucose uptake. In order to study the molecular mechanism of GLUT3 and GLUT8 involved in the regulation of lactation function of yak (Bos grunniens), the mammary epithelial cells of yak were cultured by trypsin digestion in vitro. Keratin 18, 尾 -casein and vimentin were detected by immunocytochemistry. The GLUT3 and GLUT8 genes were cloned from purified cells, and their biological characteristics were analyzed. The difference of expression of GLUT3 gene and GLUT8 gene in mammary epithelial cells was analyzed by Q RT-PCR and Western blot from gene and protein levels, respectively. Indirect immunofluorescence was used to detect its distribution in mammary epithelial cells. The results showed that keratin 18 and 尾-casein were expressed in the isolated cells, but vimentin was not expressed. The cells isolated were mammary epithelial cells. The complete coding region of Yak GLUT1G UT3 and GLUT8 gene were cloned successfully. The (Gen Bank accession numbers of GLUT3 and GLUT8 gene were: 1: KU902419 KX094556 and KX268646). The nucleotide length of the coding region was 1479bp1 485 and 1 437bprespectively, encoding 492494 and 478amino acids, respectively. The three proteins are highly conserved in the evolution process and have similar physicochemical properties and 12 hydrophobic membrane proteins with 12 transmembrane helical regions. The highest expression of GLUT1 was found in mammary epithelial cells and the lowest was GLUT8, and the difference among them was significant (P0.01). GLUT1, GLUT3 and GLUT8 protein were expressed in cytoplasm and nucleus of mammary epithelial cells of yak, and were mainly distributed in the nucleus. The results provide important information for understanding the physiological function of yak GLUT, and provide a new theoretical basis for further study on the biological function of regulating the lactation function of yaks.
【作者单位】： 甘肃农业大学动物医学院/甘肃省牛羊胚胎工程技术研究中心;
【基金】：国家自然科学基金(No.31472244) 甘肃省生物技术专项(GNSW-2013-23)
【分类号】：S823.85
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