烟草T-DNA插入位点侧翼序列扩增方法的筛选与优化

摘要

As the mutant pool of T-DNA insertion has been constructed, a quantity of T-DNA flanking sequences are in great need to be cloned. TAIL-PCR, PCR-walking and FPNI-PCR have been used to clone the sequences flanked by known sequences. For this reason, it will be great help to choose an efficient method applied to the analysis of the tobacco T-DNA insertion flanking sequences. This study focus on the comparison among the efficiency, T-DNA insertion localization and the researched homologous protein of the three methods, also we optimized the PCR procedures and primers to select an effective method which is suitable for cloning of tobacco T-DNA flanking sequences. By comparing the efficiency, T-DNA insertion localization and the researched homologous protein, the result of TAIL-PCR was the best. This result lays a foundation for the study on tobacco gene function by using mutant.%烟草T-DNA激活标签插入突变体库的创制过程中，需要进行大量的T-DNA插入位点的侧翼序列扩增，因此选择一种适用于烟草T-DNA侧翼序列扩增的高效方法尤为必要。本研究比较了TAIL-PCR、PCR-walking、FPNI-PCR三种方法对T-DNA插入位点侧翼序列的扩增效率、插入位点确定的比例和同源蛋白比对结果，并对3种方法的PCR程序、体系和引物设计等方面进行了优化，确定TAIL-PCR更适合烟草的侧翼序列扩增，通过产率、条带长度、插入位点和同源蛋白比对结果的比较，得出TAIL-PCR更适合烟草的侧翼序列扩增。这一结果为应用烟草突变体开展烟草基因功能的研究奠定了基础。