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Ⅰ类内含子(group I intron)是研究RNAs结构与功能关系的理想元件,在解释RNA折叠理论、催化机制等方面起着重要作用;对其结构与功能关系的研究也因此成为一个非常重要的课题.本研究建立了一个基于卡那霉素抗性进行Ⅰ类内含子结构与功能关系研究系统,将源于海洋蓝细菌Nostoc punctiforme(Npu)核糖核酸还原酶基因(ribonucleotide reductase,Rir)中的1个Ⅰ类内含子插入到pDrive质粒的卡那霉素抗性基因(kanamycin resistance gene,KanR)内构建得pKR12质粒并转化大肠杆菌(E.coli).只有内含子剪接的阳性克隆才能生成KanR蛋白并在Kan抗性平板上生长.结果显示,pKR12转入E.coli后不能在Kan抗性平板上生长,RT-PCR检测仅可见前体带,表明插入到KanR中的Npu Rir内含子没有发生剪接.随后通过易错PCR建立内含子的随机突变库并用Kan抗性筛选进行定向演化,产生有剪接活性的内含子突变体,RT-PCR检测显示剪接发生.由于内含子剪接活性的改变可通过Kan抗性变化在LB平板上得以反映,因此该系统有望成为简单快速地研究Ⅰ类内含子结构与功能关系的有利工具.
Group I intron is an ideal element to study the relationship between structure and function of RNAs and plays an important role in explaining RNA folding theory and catalytic mechanism. Therefore, the study on the relationship between structure and function has become very important The present study established a system based on kanamycin resistance class I intron structure and function of the system will be derived from the marine cyanobacteria Nostoc punctiforme (Npu) ribonucleotide reductase gene (riir) 1 intron was inserted into the kanamycin resistance gene (KanR) of pDrive plasmid to construct pKR12 plasmid and transformed into E. coli. Only intron-splicing positive The results showed that pKR12 could not grow on Kan-resistant plates after transfection into E.coli, and only the precursor bands could be detected by RT-PCR, indicating that Npu inserted into KanR Rir introns were not spliced.A random intron library was then created by error-prone PCR and directed mutagenesis with Kan resistance screening to generate splicing intron mutants, RT-PCR detection showed that splicing Since the alteration of intron splicing activity is reflected in LB plates by Kan resistance changes, the system is expected to be an advantageous tool for the simple and rapid study of the relationship between structure and function of class I intron.