肉鸡心肌细胞中核转录因子Sp-1和AP-2及其结合序列研究

Research of Transcription Factors of Specificity Protein-1(Sp-1) and Activating Protein-2 (AP-2) in Heart Muscle Cell of Broiler

试验采用21日龄肉鸡的心肌组织作为研究对象用于抽提核蛋白,用蛋白质印记法和电泳迁移率变动分析法研究心肌细胞中与MnSOD基因转录相关的Sp-1和AP-2核转录因子及其结合序列。结果表明,在肉鸡心肌细胞的核蛋白中存有与MnSOD基因转录调控相关的核转录因子Sp-1和AP-2,且能与MnSOD基因启动子区特异性结合位点结合,其中在-79～-58有一个与人和大鼠相同的Sp-1结合位点核心序列(5'-GGGGCGGG-3'),在鸡MnSOD基因启动子区中AP-2结合位点核心序列与现有文献中报道的人和大鼠以及一些病毒AP-2结合位点核心序列不同,分别为位于-34～-14(5'-GGCGCAGGC-3')和-338～-319(5'-CCCAAGGTC-3')。上述结果提示,肉鸡心肌细胞中MnSOD基因的转录调控可能与核转录因子Sp-1和AP-2与MnSOD基因启动子区的特异性结合密切相关。

Nucleoprotein was obtained from heart muscle of 21--clay-age broilers to investigate the transcription factors related to manganese-containing superoxide ：dismutase （MnSOD） gene in broilers. The simple-stranded oligonucleotides specifically recognized by specificity protein-1 （Sp-1） and activating protein-2 （AP-2） were synthesized and end-labeled with Biotin-dUTP. The synthesized double-stranded oligonucleotides were used as a probe in electrophoretic mobility shift assays （EMSA） and western blot to identify the transcription factors Sp-1 and AP-2 in heart muscle. The results showed that there were transcription factors Sp-1 and AP-2 in heart,and they could bind with the specific motifs in the MnSOD promoter. There was a Sp-1 binding site （5＇-GGGGCGGG-3＇）in region -79 to -58,which was consensus with that found in human and rat. There were two AP-2 binding sites in both region -34 to -14 （5＇-GGCGCAGGC-3＇） and -338 to - 319 （5＇-CCCAAGGTC-3＇） ,which were not observed in human or rat. These results implied that transcriptional regulation of MnSOD gene might be related to transcription factors of Sp-1 and AP-2.