鹅PIT-1基因启动子区转录调控的研究  

Analysis of Transcription Regulation in the Promoter Region of PIT-1 Gene in Goose

作　　者：赵荣雪[1] 赵文明[1] 徐琪[1] 段修军 董飚 孙国波 毕瑜林[1] 李秀[1] 张扬[1] 黄正洋[1] 陈国宏[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009 [2]国家级水禽基因库,泰州225300

出　　处：《畜牧兽医学报》2012年第2期220-225,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：现代农业产业技术体系建设专项资金(nycytx-45-04);优质高产水禽新品种选育国家科技部支撑计划重大专项资金(2006BDA01A09);国家科技支撑计划重点项目(2006BDA14B06);江苏省属高校自然科学基础研究面上项目(07KJB230138)

摘　　要：为研究垂体特异性转录因子(Pituitary transcription factor 1,PIT-1)启动子的转录调控机制,寻找该基因转录调控的顺式作用元件、反式作用因子和基本转录单位。利用前期染色体步移技术扩增得到鹅PIT-1基因的启动子区序列,经PCR扩增,定向亚克隆至荧光素酶表达载体pGL3-Basic中,把目的片段连接到荧光素酶报告基因载体(pGL-Basic),制备了一系列启动子缺失体(-1 485/-1bp、-1 293/-1bp、-1 014/-1bp、-775/-1bp、-561/-1bp、-353/-1bp和-206/-1bp),并通过限性内切酶酶切、PCR及测序进行鉴定,构建了含有正确目的基因的报告基因重组体,然后瞬时转染GH3细胞,利用荧光素酶报告基因检测试剂盒检测。本试验所克隆的PIT-1基因启动子区具有明显的启动子活性,其中-561/-1bp的启动活性最强,该区域存在有PIT-1、POU3F2、myogenin和GR等多个转录因子结合位点。利用系列缺失法成功构建了荧光素酶报告基因真核表达载体,并且验证了克隆的启动子具有启动活性,找到了正负调控区域及核心启动子区,为进一步研究其转录调控机制奠定了基础。The aim of this study was to study the possible regulation mechanism of PIT-1 promot- er by searching for the cis-regulatory elements, trans-acting factors and basic transcription units of the transcription regulation. The goose promoter of PIT-1 gene was cloned by genome walking and subcloned into the luciferase expression vector pGL3-Basic directly. A series of promoter missing mutants were constructed （- 1 485/- 1 bp, - 1 293/- 1 bp, -1 014/- 1 bp, - 775/ - 1 bp, - 561/- 1 bp, - 353/- 1 bp, - 206/- 1 bp）, and identified by restrictive endonuclease enzyme cutting, PCR and sequencing. The recombinant plasmids were detected by Steady-Glo^⑧ Luci{eraseAssay system after transient transfecting GH3 cell. This study indicated that the PIT-1 gene promoter cloned had obviously promoter activity. All of them, -561/-1 bp had highest ac- tivity, and many possible transcription factor binding sites such as （PIT-1,POU3F2, myogenin and GR） were predicted. The pGL-PIT-1 promoter was constructed successfully by series miss- ing method. The promoter cloned in the study had activity, the positive and negative regulation areas and core promoter region were found. Furthermore, the result provided the foundation foranalyzing the promoter activity and transcriptional regulation mechanism.

关 键 词：鹅 PIT-1基因 启动子 荧光素酶基因表达载体 转录调控 

分 类 号：S835[农业科学—畜牧学] S831.2[农业科学—畜牧兽医]