| 摘要: |
| 【目的】体外转录并纯化酵母端粒酶RNA，明确缓冲液中组分对其构型的影响，初步研究四膜虫端粒酶相关蛋白p65与酵母端粒酶RNA的结合情况，为酵母端粒酶活性鉴定及构型功能研究奠定基础。【方法】利用核酶的自剪切功能，通过在3′端引入HDV核酶序列获得完整的酵母端粒酶RNA；并用分子筛SuperDeX-200/16/60凝胶过滤纯化酵母端粒酶RNA；琼脂糖凝胶电泳检测RNA Buffer组分中NaCl和MgCl2浓度对酵母端粒酶RNA构型的影响；非变性聚丙烯酰胺凝胶电泳（PAGE）检测四膜虫端粒酶相关蛋白p65与酵母端粒酶RNA的结合情况。【结果】通过基因序列合成，结合PCR扩增方法获得了酵母端粒酶RNA基因，并将其构建到体外转录载体上；体外转录获得大量的酵母端粒酶RNA（Yeast-RNA-239）；使用SuperDeX-200/16/60成功分离得到了纯净的Yeast-RNA-239。NaCl、MgCl2浓度对酵母端粒酶RNA构型有一定的影响，表现为没有NaCl或MgCl2时，RNA的构型均一且致密；随NaCl、MgCl2浓度的增大，RNA构型逐渐变得松散；而四膜虫端粒酶相关蛋白p65能与酵母端粒酶RNA有效结合。【结论】在体外成功制备了高纯度的酵母端粒酶RNA，NaCl、MgCl2浓度对其构型有影响，四膜虫端粒酶相关蛋白p65能够帮助端粒酶RNA正确折叠。 |
| 关键词: 体外转录 端粒酶RNA 端粒 端粒酶相关蛋白p65 |
| DOI： |
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| 基金项目:教育部回国人员科研启动基金项目 |
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| Preparation of the yeast telomerase RNA and analysis of the factors influencing its configuration |
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XIE Yan,FU Jing,XI Xu-guang,et al
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| Abstract: |
| 【Objective】This study purified yeast telomerase RNA (TER) from in vitro transcription reactions to confirm effects of components in different buffers on the structure of transcription products,and to investigate the combination of Tetrahymena thermophila telomerase-associated protein p65 and yeast TER.【Method】This study used ribozyme self-cleavage function to obtain complete RHA sequence of yeast TER by introducing HDV to 3′ location,which overcome the bases errors in the end of transcription products in the process of in vitro transcription.A molecule sieve SuperDeX-200/16/60 was used to purify the target RNA product.Agarose gel electrophoresis was used to detect the effects of NaCl and MgCl2 contents in RNA buffers on configuration of yeast TER RNA.PAGE was applied to determine the combination of T.thermophila telomerase associated protein p65 and yeast TER.【Result】The yeast TER gene was synthesized with PCR amplification and sub-cloned to the in vitro transcription vector.Large amount of yeast TER Yeast-RNA-239 was obtained by in vitro transcription.Pure Yeast-RNA-239 was separated using SuperDeX-200/16/60.NaCl and MgCl2 concentrations had effects on yeast telomerase RNA configuration.RNA configuration was uniform and dense when there was no NaCl or MgCl2.As the increase of NaCl or MgCl2 concentrations,RNA configuration became loose.Combination of T.thermophila telomerase-associated proteinp65 with yeast TER was verified.【Conclusion】The study successfully prepared yeast TER in vitro with high purity,and demonstrated the effects of NaCl and MgCl2 on yeast TER configuration and the combination of T.thermophila telomerase-associated protein p65 and yeast TER. |
| Key words: in vitro transcription telomerase RNA telomere telomerase-associated protein p65 |
