文章摘要
张涛,张子平,贾锡伟,王淑红,王国栋,王艺磊.杂色鲍HSP90基因启动子的功能分析[J].水产学报,2017,41(4):490~497
杂色鲍HSP90基因启动子的功能分析
Functional analysis of the promoter in Haliotis diversicolor HSP90 gene
投稿时间:2016-03-15  修订日期:2016-10-16
DOI:10.11964/jfc.20160310311
中文关键词: 杂色鲍  HSP90  启动子  瞬时转染  功能分析
英文关键词: Haliotis diversicolor  HSP90  promoter  transient transfection  functional analysis
基金项目:国家自然科学基金(41176152);集美大学创新团队基金(2010A001)
作者单位E-mail
张涛 集美大学水产学院, 农业部东海海水健康养殖重点实验室, 福建 厦门 361021  
张子平 福建农林大学动物科学学院, 福建 福州 350002  
贾锡伟 集美大学水产学院, 农业部东海海水健康养殖重点实验室, 福建 厦门 361021  
王淑红 集美大学水产学院, 农业部东海海水健康养殖重点实验室, 福建 厦门 361021  
王国栋 集美大学水产学院, 农业部东海海水健康养殖重点实验室, 福建 厦门 361021  
王艺磊 集美大学水产学院, 农业部东海海水健康养殖重点实验室, 福建 厦门 361021 ylwang@jmu.edu.cn 
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中文摘要:
      为探索启动子在热休克蛋白90表达调控中的作用,本研究在课题组已有杂色鲍HSP90基因cDNA的基础上,通过Genome walking、Tail-PCR和常规PCR等技术克隆获得该基因的5'调控区序列。在翻译起始位点(ATG)和第一外显子(长度94 bp)之间有一个809 bp的内含子,第一外显子之前的5'调控区共2800 bp,从预测的转录起始位点(A)起,共2811 bp。在转录起始位点(A)上游-30 bp处存在TATA box。潜在的转录因子结合位点包括ATF、TBP、Sp1、Oct-1、C/EBPalpha、NF-1、NF-κappaB、GATA-1、Sox-2等。CpG岛预测软件分析其含1个CpG岛,长度为131 bp。实验构建了8个启动子缺失片段的萤火虫荧光素酶表达载体,通过瞬时转染293T细胞并进行双荧光素酶报告基因活性检测,确定杂色鲍HSP90基因核心启动子区位于-98~83 bp。在-624~-539 bp,Oct-1、C/EBPalpha、NF-1这3个转录因子都起到一定的抑制作用。
英文摘要:
      Heat shock protein 90 (HSP90), a molecular chaperone in cell, which can regulate multiple signaling pathways, plays a key role in the process of cell differentiation, development and transportation. To explore the role of the promoter in regulating the expression of heat shock protein 90, based on the HSP90 gene cDNA of Haliotis diversicolor from our lab, its 5' flanking region was cloned by genome walking and Tail-PCR techniques. Results showed that there is an 809 bp intron between translation initiation site (ATG) and first exon (94 bp). The length of 5' flanking region is 2800 bp before the first exon and 2811 bp before the predicted transcriptional start site (A). A TATA-box was located in the upstream -30 bp of the transcriptional start site (A). Potential transcription factor binding sites include ATF, TBP, Sp1, Oct 1, C/EBPalpha, NF-1, NF-kappaB, GATA-1, and Sox-2, etc. A CpG island was found by the CpG island prediction software, whose length is 131 bp. Eight firefly-luciferase reporter gene vectors with different deletions of HdHSP90 gene were constructed and transiently transfected into 293T cells, and the activity of dual-luciferase reporter gene was detected. The results showed that the core promoter is located between -98 to 83 bp, and the three transcription factors between -624 to -539 bp, Oct-1, C/EBPalpha, and NF-1, can inhibit the gene transcription.
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