盐藻cbr基因启动子及其3'-非翻译区序列的克隆

目的：克隆盐藻cbr基因启动子和3‘-非翻译9(3‘-UTR)片段。方法：设计2对引物，通过2轮巢式PCR反应，扩增cbr基因启动子，其中首轮PCR反应使用改良的降落PCR程序，第二轮巢式PCR反应的模板是第一轮PCR产物，使用普通PCR程序；cbr基因3‘-UTR片段的克隆采用一对引物，通过一般PCR程序得到。结果：通过巢式PCR得到长约1．1kb的cbr基因启动子片段，DNA测序结果表明碱基序列与文献记载完全相同，无任何碱基突变；克隆的长0．3kh的chr基因3‘-UTR片段经人工比较，与收录的序列吻合，无碱基突变。结论：利用普通Taq酶对用PCR方法克隆得到了盐藻胡萝卜素生物合成相天基因cbr的2个基因表达渊控序列；结合使用巢式PCR和改良降落PCR，可以非常有效地克隆具有复杂二级结构的DNA片段；通过两端人工添加的限制序列．将cbr基因启动子和3‘-UTR2个调控片段构建到同一个载体上，形成cbr基因表达盒，为构建转基因盐藻表达载体打下了基础。

Cloning of promoter and 3'-untranslated region of the cbr gene from Dunaliella bardawil

Aim: To clone the promoter and 3'-untranslated region (3'-UTR) of the cbr (carotene biosynthesis related) gene from Dunaliella bardawil. Methods: Succeeded cloning of the cbr promoter was carried out by a nested PCR in which a modified touch-down PCR (MTD-PCR) program was taken in the first cycle reactions. The cloning of 3'-UTR of cbr was accomplished with a simple traditional PCR method. Results: A fragment (1.1 kb) of the cbr promoter was cloned into the intermediate vector pGPD1, and the fragment (0.3 kb) of 3'-UTR of cbr was inserted into the plasmid pGP3D2. Base sequencing results displayed that no base mutation was brought in the two fragments of gene expression. Conclusion: A combination of nested PCR and modified touch-down PCR could efficiently be used to clone fragments of DNA that may contain some complicated secondary structure. Construction of the vector pGP3D2, which bore both the promoter and the 3'-UTR of cbr gene, makes it a good preparation to create a light-inducible expression box for the transgenic study of 3'-UTR of cbr.