关键词: 水牛; SOX2基因; 克隆; 调控序列

Abstract: To explore the transcriptional regulatory mechanism of buffalo SOX2 gene, its 5' regulatory region (2555 bp) were cloned, then five deletion mutants -2263, -1816, -1275, -660 and -407 bp were designed and constructed as EGFP reporter vectors respectively. The transcriptional activity of each deletion mutant was analysed by producing transgenic embryos and transfecting into buffalo fetal fibroblast (BFF). The results showed that the green fluorescent protein could be observed in all groups in pig embryos (4.5 d) except p-407-EGFP, and with the gradual reduction of the fragment, the activity of the deletion mutants had a highly significant decreasing trend(P<0.01). After transfecting into BFF 48 h, a small number of cells was able to see fluorescence in all groups except p-407-EGFP, and the transcriptional activity differences from each other were highly significant(P<0.01), the -2263 bp fragment had the highest activity, followed by -660 bp, then -1275 bp, and finally -1816 bp. No fluorescence was observed in p-407-EGFP group both embryo and BFF level. The results suggested that the -660~-407 bp was an integral part of the buffalo SOX2 gene basic promoter, -2263~-1816 bp contained the non-pluripotential cell-specific enhancer element, there were pluripotential cell-specific enhancer elements existed in -1816~-1275 bp and -1275~-660 bp.

Key words: buffalo; SOX2 gene; clone; regulatory sequence