畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (2): 220-225.doi:

• 遗传繁育 • 上一篇    下一篇

鹅PIT1基因启动子区转录调控的研究

赵荣雪1,赵文明1,徐琪1,段修军2,董飚2,孙国波2,毕瑜林1,李秀1,张扬1,黄正洋1,陈国宏1*   

  1. 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
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-02-15 发布日期:2012-02-15
  • 通讯作者: 陈国宏

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

ZHAO Rongxue1, ZHAO Wenming1, XU Qi1, DUAN Xiujun2, DONG Biao2,
SUN Guobo2,BI Yulin1,LI Xiu1, ZHANG Yang1, HUANG Zhengyang1, CHEN Guohong1*
  

  1. 1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; 2. National Native Waterfowl Gene Pool, Taizhou 225300, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2012-02-15 Published:2012-02-15
  • Contact: CHEN Guohong

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/-1 bp、-1 293/-1 bp、-1 014/-1 bp、-775/-1 bp、-561/-1 bp、-353/-1 bp和-206/-1 bp),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/-1 bp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。

Abstract: The aim of this study was to study the possible regulation mechanism of PIT1 promoter 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 LuciferaseAssay 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 activity, 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 missing 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 for analyzing the promoter activity and transcriptional regulation mechanism.