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809bp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-KappaB、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.%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 fireflyluciferase 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.