烟草T-DNA插入位点侧翼序列扩增方法的筛选与优化  

The Screening and Optimization of Three Methods to Clone the Flanking Sequences of Transgenic Tobacco

作　　者：刘慧[1,3] 刘贯山[1] 刘峰[2,3] 龚达平[1] 张雪峰[1,3] 崔萌萌[1] 路铁刚[2] 王元英[1] 

机构地区：[1]中国农业科学院烟草研究所,中国农业科学院烟草遗传改良与生物技术重点开放实验室,青岛266101 [2]中国农业科学院生物技术研究所,北京100081 [3]中国农业科学院研究生院,北京100081

出　　处：《中国烟草科学》2014年第1期96-101,107,共7页Chinese Tobacco Science

基　　金：中国烟草总公司烟草基因组计划重大专项项目“烟草突变体库创制、筛选与分析”[110201101009(JY-03)];“烟草突变体筛选与鉴定”[110201201004(JY-04)];“烟草全基因组过表达和RNA干涉体系构建”(110200902036)

摘　　要：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。As the mutant pool of T-DNA insertion has been constructed, a quantity of T-DNA flanking sequences are in great need tobe cloned. TAIL-PCR, PCR-walking and FPNI-PCR have been used to clone the sequences flanked by known sequences. For thisreason, 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 thethree methods, also we optimized the PCR procedures and primers to select an effective method which is suitable for cloning oftobacco T-DNA flanking sequences. By comparing the efficiency, T-DNA insertion localization and the researched homologousprotein, the result of TAIL-PCR was the best. This result lays a foundation for the study on tobacco gene function by using mutant.

关 键 词：烟草 T-DNA 插入位点 侧翼序列 筛选 

分 类 号：S572[农业科学—烟草工业]