【作者】 谢伟；

【导师】 张镜宇；

【作者基本信息】 天津医科大学 ， 生物医学工程， 2011， 硕士

【摘要】 目的：研究甲状腺激素受体新亚型TRβ△通过甲状腺激素应答元件(thyroid response element, TRE)增强靶基因转录的作用,判断TRβ△是否具有转录因子活性。利用两种突变方式改变TRβ△的DBD (DNA-binding domain, DBD)中P-box的氨基酸序列,分析TRβ△在突变之后是否仍具有与DNA结合后开启转录激活作用的功能。从而阐明保持DBD中TRPA氨基酸序列的保守性对于P-box受体结合DNA和激活转录的功能的重要性。方法：利用基因重组技术,分别以TRβ△和pETDuet-TRβ1为模板,扩增pETDuet-TRβ△和TRβ1的cDNA插入到载体pcDNA3.1中,构建pcDNA3.1-TRβ1和pcDNA3.1-TRβ△真核表达载体。设计一对特异引物,利用定向突变技术,将作为TRE类型之一的PAL (palindromic) TRE,即回文序列TRE插入到载体pGL3-Promoter中,构建含有PAL的pGL3-Promoter报告基因载体。将pcDNA3.1-TRβ1和pcDNA3.1-TRβ△分别与含有PAL的pGL3-Promoter报告基因载体瞬时共转染至COS-7细胞中,并用加有T3的培养基和不加T3的培养基培养细胞后,检测荧光素酶的活性。同时设立pcDNA3.1-TRβ△和pcDNA3.1-TRβ1与不含PAL的pGL3-Promoter共转染及含有PAL的pGL3-Promoter单独转染的对照组。利用定向点突变技术,将pcDNA3.1-TRβ△的DBD中P-box氨基酸序列分别通过突变改换不同的氨基酸。突变后产生的蛋白和其与RXR蛋白杂二聚化后的复合物分别与标记后的DR4进行EMSA实验。突变后的载体分别与含有PAL的pGL3-Promoter报告基因载体瞬时共转染至COS-7细胞中,并用加有T3的培养基和不加T3的培养基培养细胞后,检测荧光素酶的活性。结果：PCR证实成功构建了pcDNA3.1-TRβ1和pcDNA3.1-TRβ△真核表达载体,测序及酶切结果完全正确。双酶切及测序证实成功构建了含有PAL的pGL3-Promoter报告基因载体。pcDNA3.1-TRβ1和pcDNA3.1-TRβ△分别与含有PAL的pGL3-Promoter报告基因载体瞬时共转染48小时后,可以检测到很强的荧光素酶比活性,而且pcDNA3.1-TRβ1与含有PAL的pGL3-Promoter共转染后用加有T3的培养基培养细胞,同时间点的荧光素酶比活性(RLUs/mg蛋白)比不加T3的培养基培养的细胞升高至16倍,pcDNA3.1-TRβ△与含有PAL的pGL3-Promoter共转染后用加有T3的培养基培养细胞,荧光素酶比活性升高至14倍。测序证实成功构建了pcDNA3.1-TRβ△mut1突变P-box,将DBD中P-box的第三位氨基酸P突变为G,突变后的P-box的氨基酸序列变为与TRβ1一致的CEGCKG。成功构建了pcDNA3.1-TRβ△Amut2突变P-box,将DBD中P-box的氨基酸序列最重要的两个氨基酸EG突变为GA,突变后的P-box的氨基酸序列变为CGACKP。TRβ△mut1蛋白和其与RXR蛋白杂二聚化后的复合物均可与标记后的DR4结合。而TR(3Amut2蛋白自身不能与DR4结合,但RXR可与DR4结合,所以TR(3Amut2蛋白与RXR蛋白杂二聚化后才可与标记后的DR4结合。pcDNA3.1-TRβ△mut1和pcDNA3.1-TRβ△mut2分别与含有PAL的pGL3-Promoter报告基因载体瞬时共转染48小时后,检测荧光素酶比活性,发现前者荧光素酶比活性是后者的7倍,两者同时间点的荧光素酶比活性比不加T3的培养基培养的细胞分别升高至7倍和2.5倍。结论：1.新发现的甲状腺激素受体亚型TRβ△与已发现的TRβ1一样能通过甲状腺激素应答元件增强靶基因转录。2.结果显示报告基因表达水平均可被T3提高。证实TRβ△是一个受配体(T3)诱导的功能性的转录因子。3.新发现的甲状腺激素受体亚型TRβ△的DBD中P-box氨基酸序列与TRβ1的原始DBD中P-box氨基酸序列均与转录激活作用有关,把TRβ△受体的P-box氨基酸序列CEGCKP变为和TRβ1一致的CEGCKG,在其后比TRβ1多30几个氨基酸的情况下仍可通过TRE对报告基因产生转录激活作用,报告基因表达水平均可被T3提高,TRβ△mut1蛋白和其与RXR蛋白杂二聚化后的复合物均可与标记后的DR4结合。但是改变了DBDP-box氨基酸序列中最重要的两个氨基酸,则无法产生转录激活作用,TRβ△mut2蛋白自身不能与DR4结合。说明保持DBD中P-box氨基酸序列的保守性对于TRβ△受体结合DNA和激活转录的功能十分重要。更多还原

【Abstract】 Objective:To study the transactivation of a novel thyroid hormone receptor isoform, TRβ△, on target genes through thyroid hormone response element(TRE),and whether TRβ△had the characteristics of transcription factor. Used the two way of mutation to change the P-box amino acid sequence in the DBD (DNA-binding domain, DBD) of TRβ△, and then research whether TRβ△still had the function of binding DNA and transactivation after mutation.At last it clarified that it was important for the binding DNA and transactivation of TRβ△receptor to maintain the conservation of its P-box amino acid sequence in DBD.Methods:The thyroid hormone receptorβ1 andβ△gene were amplified respectively from pETDuet-TRβ1 and pETDuet-TRβ△by PCR and sub-cloned into pcDNA3.1. The multi-copy recombinant eukaryotic expression plasmid pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△were constructed. A short of sequence of the pGL3-Promoter would mutate to a kind of TRE-PAL (palindromic) with designing a pair of special primers. The reporter gene vector plasmid pGL3-Promoter-PAL TRE was constructed. The recombinant reporter gene vector pGL3-promoter-PAL TRE was transiently co-transfected into COS-7 cells with pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△respectively. Cells were treated with DMEM with or without T3, then the activity of luciferase was detected.The control group is that:the reporter gene vector pGL3-promoter was transiently co-transfected into COS-7 cells with pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△respectively, pGL3-promoter-PAL TRE was transfected into COS-7 cells lonely.The DBD P-box amino acid sequence in TRβ△was mutated respectively to different sequence used the way of site-directed mutation. EMSA was performed after the labeled DR4 respectively combined with two kinds of mutation proteins and the dimerization complex of mutation proteins with RXR protein.The recombinant reporter gene vector pGL3-promoter-PAL TRE was transiently co-transfected into COS-7 cells with two kinds of mutant plasmids. Cells were treated with DMEM with or without T3, then the activity of luciferase was detected. Results:The new multi-copy recombinant eukaryotic expression plasmid pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△were successfully constructed, which was confirmed by PCR. The result of sequencing and double digesting of recombined plasmid were completely correct. The reporter gene vector plasmid pGL3-Promoter-PAL was successfully constructed, which was confirmed by the result of sequencing and double digesting of recombined plasmid. The recombinant reporter gene vector pGL3-promoter-PAL TRE was transiently co-transfected into COS-7 cells with pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△respectively. It can be detected very high activity of luciferase after transfection for 48h.The expression of reporter gene could be respectively increased up to 16 and 14 times by T3 after pGL3-promoter-PAL TRE with pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△co-transfected into cells.The mutant plasmids pcDNA3.1-TRβ△mutl and pcDNA3.1-TRβ△mut2 were successfully constructed, which were confirmed by the result of sequencing. The DBD P-box amino acid sequence in pcDNA3.1-TRβ△mutl was mutated to CEGCKG,the same as TRβ1. The P-box amino acid sequence in pcDNA3.1-TRβ△mut2 was mutated to CGACKP. TRPAmutl protein and the dimerization complex of TRβ△mutl protein with RXR protein can all combine with labeled DR4. The TRβ△mut2 protein itself can not combine with labeled DR4, but RXR protein can combine with DR4, only the dimerization complex of TRβ△mut2 protein with RXR protein can combine with DR4.pGL3-promoter-PAL TRE was transiently co-transfected into COS-7 cells with pcDNAS.l-TRβ△mutl and pcDNA3.1-TRβ△mut2 respectively. The activity of luciferase of former was 7 times higher than the latter after transfection for 48h. The expression of reporter gene could be respectively increased up to 7 and 2.5 times by T3 after pGL3-promoter-PAL TRE with pcDNA3.1-TRβ△mut1 and pcDNA3.1-TRβ△mut2 co-transfected into cells.Conclusion:A novel thyroid hormone receptor isoforms TRβ△the same as TRβ1 had the transactivation effect on target genes through the TRE.The expression of reporter gene could be increased by T3. TRβ△was a functional transcription factor induced by T3. The DBD P-box amino acid sequence in TRβ△and the original P-box in TRβ1 had the same transactivation effect. The DBD P-box amino acid sequence in TRβ△was mutated to the sequence in TRβ1 with more than 30aa behind the P-box in TRβ1,it still had the transactivation effect on target genes through the TRE. The expression of reporter gene could be increased by T3. TRβ△Amutl protein and the dimerization complex of TRβ△mutl protein with RXR protein can all combine with labeled DR4.But two the most important amino acid in DBD P-box were changed, transactivation effect was lose. TRβ△mut2 protein itself can not combine with labeled DR4.It was important for the binding DNA and transactivation of TRβ△receptor to maintain the conservation of its P-box amino acid sequence in DBD.更多还原