鸡肠道中转录因子GATA3对SGLT1和GLUT5调控机制影响的研究

发布时间：2018-05-12 17:59

本文选题：鸡小肠上皮细胞 + GATA3　；参考：《山西农业大学》2015年硕士论文

【摘要】：糖类是动物生长必不可少的主要营养物质之一,饮食中糖类的消化吸收对动物的正常生长发育起着重要作用。糖类的消化主要在小肠中进行,位于肠上皮细胞刷状缘膜上的糖类营养转运蛋白SGLT1和GLUT5负责单糖的转运。所以调控两种基因的表达可以影响糖类的吸收,进而影响动物的生长发育。转录因子GATA3是GATA锌指转录因子家族的一员,它对许多组织细胞中基因的表达都有调控作用。本研究为探索鸡肠道中转录因子GATA3对sGLT1和GLUT5的调控机制。首先对鸡小肠上皮细胞的体外分离培养的适宜条件和营养因子(接种密度和血清种类)进行筛选,通过筛选出的最佳条件培养细胞,并对培养出的细胞进行鉴定,然后利用生物信息学软件寻找SGLT1和GLUT5基因5’上游GATA3基因的结合位点,在此基础上通过脂质体转染法将实验室已成功构建的真核表达载体pcDNA3.1-GATA3转染鸡小肠上皮细胞,最后利用实时荧光定量PCR技术检测转染后鸡小肠上皮细胞中基因GATA3、SGLT1和GLUT5的绝对表达量。研究结果：体外分离培养鸡小肠上皮细胞的最佳接种密度为6.5×105/mL左右；与添加胎牛血清的培养液相比,添加鸡血清的培养液更适宜鸡小肠上皮细胞的体外分离培养；通过筛选得到的最佳适宜条件和营养因子成功体外分离培养出鸡小肠上皮细胞,并通过鉴定得到肯定；生物信息学软件发现糖类营养转运蛋白SGLT1和GLUT5基因5’上游含有多个GATA3基因的结合位点；转染组GATA3的表达量极显著高于空白对照组和阴性对照组(P0.01),说明利用脂质体转染法将真核表达载体pcDNA3.1-GATA3已成功转染进入鸡小肠上皮细胞；鸡小肠上皮细胞中GATA3过表达后,转染组GLUT5的表达量极显著高于空白对照和阴性对照组(P0.01),而转染组SGLT1的表达量与对照组之间无明显的差异(P0.05)。以上结果揭示了在原代培养的鸡小肠上皮细胞中转录因子GATA3的过表达对糖类转运蛋白基因GLUT5的表达起到正调控作用,而对糖类营养转运蛋白基因SGLT1则没有影响,为研究鸡肠道功能以期选育高饲料转化率的优良鸡种提供参考依据。
[Abstract]:Carbohydrate is one of the essential nutrients for animal growth. The digestion and absorption of sugar in diet play an important role in the normal growth and development of animals. The digestion of carbohydrates mainly takes place in the small intestine, and SGLT1 and GLUT5, which are located on the brush-edge membrane of intestinal epithelial cells, are responsible for the transport of monosaccharide. Therefore, regulating the expression of two genes can affect the absorption of sugar, and then affect the growth and development of animals. Transcription factor GATA3 is a member of GATA zinc finger transcription factor family. It regulates gene expression in many tissue cells. The aim of this study was to investigate the regulatory mechanism of GATA3 on sGLT1 and GLUT5 in chicken intestinal tract. The optimal conditions and nutritional factors (inoculation density and serum species) for isolation and culture of chicken small intestinal epithelial cells in vitro were first selected. Then the binding sites of SGLT1 and GLUT5 gene 5'upstream GATA3 gene were found by bioinformatics software, and then the successfully constructed eukaryotic expression vector pcDNA3.1-GATA3 was transfected into chicken small intestinal epithelial cells by liposome transfection. In the end, the absolute expression of GATA3GLT1 and GLUT5 in transfected chicken small intestinal epithelial cells was detected by real-time fluorescence quantitative PCR. The results showed that the optimal inoculation density of chicken small intestinal epithelial cells was about 6.5 脳 105/mL, and the medium supplemented with fetal bovine serum was more suitable for isolation and culture of chicken small intestinal epithelial cells in vitro. Chicken small intestinal epithelial cells were successfully isolated and cultured in vitro under the optimum conditions and nutrition factors, and confirmed by identification. Bioinformatics software found that SGLT1 and GLUT5 gene 5'upstream contained multiple binding sites of GATA3 gene. The expression of GATA3 in the transfected group was significantly higher than that in the blank control group and the negative control group, indicating that the eukaryotic expression vector pcDNA3.1-GATA3 had been successfully transfected into the chicken intestinal epithelial cells by liposome transfection, and the expression of GATA3 in the chicken intestinal epithelial cells was over-expressed. The expression of GLUT5 in the transfected group was significantly higher than that in the blank control group and the negative control group, but there was no significant difference in the expression of SGLT1 between the transfected group and the control group. These results suggest that the overexpression of transcription factor GATA3 plays a positive role in the expression of carbohydrate transporter gene GLUT5 in primary cultured chicken small intestinal epithelial cells, but has no effect on the carbohydrate nutrition transporter gene SGLT1. To study the intestinal function of chicken in order to select good chicken breeds with high feed conversion rate.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

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