基于卡那霉素抗性建立的研究Ⅰ类内含子结构与功能关系的系统  

Establishment of Kanamycin Resistance-based Construct System for Structural and Functional Analyses of Group Ⅰ Intron

作　　者：殷伟[1] 林瑛[1] 孟清[1] 

机构地区：[1]东华大学生物科学与技术研究所,上海201620

出　　处：《中国生物化学与分子生物学报》2011年第4期384-389,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目资助(No.30770463)~~

摘　　要：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 molecule for studying the relationship between structure and function of RNAs.They play important roles in interpretation of RNA folding and catalytic mechanism,thus investigation of relationship between structure and function of group Ⅰ intron has become one important subject in this field.This study constructed a system based on kanamycin resistance selection for structural and functional analyses of group Ⅰ intron.Npu Rir group Ⅰ intron from the cyanobacterial ribonucleotide reductase gene of Nostoc punctiforme,was inserted into the kanamycin resistance gene of pDrive to construct pKR12 and then transformed into E.coli cells.Only positive clones in which KanR pre-mRNA splicing and KanR protein expressed can grow on the KanR plates.The results showed that E.coli cells containing pKR12 cannot grow and only precursor band can be detected in RT-PCR products,which means that intron in KanR has no splicing activity.Then Npu Rir intron was subjected to error-prone PCR to generate a random mutations library,followed by screening of kanamycin resistance.RT-PCR detection showed that splicing were occurred in some mutant introns.As the splicing activity of introns is reflected by kanamycin resistance,this system is hopefully to become a simple,fast and efficient method to study group Ⅰ intron and its splicing.

关 键 词：Ⅰ类内含子 卡那霉素抗性 RNA剪接活性 定向演化 

分 类 号：Q71[生物学—分子生物学]