畜牧兽医学报2013，Vol.44Issue(3)：387-394,8.
沼泽型水牛NANOG基因5'调控序列克隆与功能分析
Cloning and Function Analysis of Swamp Buffalo NANOG Gene 5′ Regulatory Region
摘要
Abstract
To investigate the expression regulation mechanism of NANOG gene in swamp Buffalo, the 5' regulatory region of NANOG gene was cloned and analyzed. Four deletion mutants fragments-1 213,-45,-425 and-312 bp were designed and constructed as EGFP reporter vectors respectively. Using the reporter vectors, the transgenic buffalo and pig embryos were produced by micro-injection method. The result showed that EGFP could only be observed in inner cells mass(ICM). In the 4. 5 d pig embryos, the EGFP could be observed in all reporter vectors. After tranfecting the four reporter plasmids into buffalo fetal fibroblast about 48 h, fluorescence could be observed in a small number of cells in all groups. QRT-PCR analysis showed that the differences of the transcriptional activity from each other were highly significant in pig 4. 5 d embryo cells, the -745 bp fragment had the highest activity, followed by -425 bp, then -1 213 bp and finally-312 bp. In buffalo fetal fibroblasts, the activity of -425 bp fragment was significantly higher than that of the others, there was no significant difference between -1 213 and -745 bp, and the both were significantly higher than that of -312 bp fragment. These results indicated that, in buffalo blastocyst the - 1 213 bp fragment can mediate specific expression of buffalo NANOG gene in ICM. Based on the above results, we predict that there is pluripotent cell-specific inhibition element existed in - 1 213-- 745 bp and -745--425 bp contains pluripotent cell-specific enhancer element, and -425--312 bp contains non-pluripotent cell-specific enhancer element.关键词
沼泽型水牛/NANOG/调控序列/克隆Key words
swamp buffalo/ NANOG/ regulatory sequence/ clone分类
农业科技引用本文复制引用
崔奎青,张会娜,刘帅,刘晓华,杨素芳,刘庆友,石德顺..沼泽型水牛NANOG基因5'调控序列克隆与功能分析[J].畜牧兽医学报,2013,44(3):387-394,8.基金项目
国家高技术研究发展计划(863)项目(2011AA100607) （863）
国家转基因重大专项(2011ZX08007-003) （2011ZX08007-003）
国家自然科学基金(30960251) （30960251）
