摘要
为克隆人类抵抗素样分子β(resistin-like molecule beta,RELMβ)基因的上游启动子序列,并观察其不同截短片段的启动子活性,以人类基因组DNA为模板,通过PCR扩增方法获得-871～+50bp、-729～+50bp、-471～+50bp、-438～+50bp、-371～+50bp大小的RELMβ启动子片段,将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子CDX-2结合位点的突变或缺失体.在阳离子脂质体的介导下,报告基因载体分别瞬时转染人胚肾293细胞、结肠癌HCT116和SW480细胞、宫颈癌HeLa细胞.结果发现,各RELMβ启动子片段在293、HCT116、SW480细胞中均有活性,但在HeLa细胞中活性缺失;-471～-438bp区存在RELMβ启动子的核心调控元件.针对该区域CDX-2转录因子结合位点进行突变,能导致RELMβ启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合CDX-2.结果提示,成功克隆了具有活性的RELMβ启动子序列,CDX-2为其重要的转录因子,为研究RELMβ基因的转录调控机制奠定了实验基础.
To clone the upstream,promoter sequence of human resistin-like molecule （RELMfl） beta and investigate the activities of its truncated segments, five segments of RELMβ promoter, -871 - ＋ 50 bp, -729 - ＋50 bp, -471 - ＋50 bp, -438- ＋ 50 bp and -371 - ＋ 50 bp, were obtained by PCR amplification from human genomic DNA and were subcloned into pGL3-Basic to construct luciferase reporter vectors. The mutation or deletion of transcription factor CDX-2 binding site within RELMβ promoter was prepared. The recombinant reporter vectors were transiently transfected into human embryonic kidney 293 cells, colon cancer HCT116 and SW480 cells, and cervical cancer HeLa cells by Lipofectamine 2000, respectively. Results showed that all these RELMβ promoters presented activities in 293, HCT116 and SW480 cells, but not in HeLa cells. The region of -471～ -438 bp contained the core element of RELMfl promoter. Mutation of CDX-2 binding site within this region resulted in significant decrease of promoter activities. EMSA demonstrated that this region could bind CDX-2. These results suggested that the activate promoter sequences of RELMβ were obtained successfully, and CDX-2 was an important transcription factor determining the activity of this promoter. These results established a basis for further studying transcriptional regulation mechanisms of RELMβ gene.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第7期548-553,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助项目(No.30200284
No.30600278)~~
