一种启动子克隆的改良Adaptor
摘要： 真核生物通过顺式作用元件和反式作用因子相互作用实现基因的转录调控，而启动子区域含有多种顺式元件，作为基因表达调控网络的枢纽控制着基因转录的起始与效率，一直是基因表达调控研究的重点［1］。本文提供了一种改良的基于Adaptor-PCR启动子克隆方法，改进了接头序列，设计了适合两步法PCR的接头引物。选取了25种限制性内切酶对橡胶树基因组DNA进行酶切，在所有酶切产物都平端化后，与改进的接头连接，成功构建了以Adaptor-PCR为基础的橡胶树基因启动子克隆文库，并利用此文库成功克隆了橡胶树6个蔗糖转运蛋白基因、1个转化酶基因和1个海藻糖合酶基因的启动子。本文研究结果为橡胶树基因启动子克隆提供了一个高效平台，也为其他生物基因启动子克隆提供了有益的参考。
关键词: 橡胶树, 启动子克隆, Adaptor-PCR, 方法改良, 应用实例
Abstract: Transcriptional regulation of eukaryotic genes is realized by the interactions between cis-elements and trans-acting factors. Most of the cis-elements reside in the promoter regions, and form the major factor controlling the start site and efficiency of transcription. Therefore, cloning and functional characterization of the promoter of a target gene becomes an important task in unraveling the mechanisms of gene expression and regulation. Here, an improved method of promoter cloning was established based on adaptor-PCR. We modified the adaptor sequences of the adaptor by increasing the specificity and annealing temperatures for the adaptor primers, and made it more suitable for two-step PCR. The genomic DNA of Rubber tree was cleaved by 25 selected restriction enzymes, then blunted and ligated to the same modified adaptor, and formed the genome-walking library for promoter cloning. The utility of this library was verified by the successful promoter cloning for six sucrose transporter genes, one invertase gene and one trehalose synthase gene. The method established in this work is suitable for promoter cloning in Rubber tree as well as other plant species.
Key words: Hevea brasiliensis, promoter cloning, adaptor-PCR, method improvement, application
中图分类号:
Q78
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