利用外源基因侧翼序列扩增筛选纯合转基因植株

摘要：传统的纯化外源基因的方法主要是通过多代自交，该方法耗时长，效率低。通过对转基因材料中外源基因T-DNA区域插入位点侧翼序列的分析，本文开发了筛选纯合转基因植株的新方法。首先，利用酶切连接接头的PCR方法，我们在番茄转基因植株中扩增外源基因Avr3a的两翼序列并测序；然后通过该序列设计的引物在转基因植株中验证，得到的两翼序列通过与SGN数据库比对分析，发现外源基因的插入位点有40bp的碱基缺失；最后利用侧翼序列和边界序列设计的引物，成功在转基因T1代植株中筛选出纯合单株，并在T2代进行了验证。

Screening Homozygous Transgenic Plants by Flanking Sequences Amplification of T-DNA in Tomatoes

Abstract: Traditional method of screening homozygous transgenic plants is several generations self-pollenation, this is a time consuming work and low efficiency. We created a new method by flanking sequences amplification of T-DNA insertion site to screen homozygous plants. Firstly, we using modified adaptor ligation PCR method amplified flanking sequences of T-DNA in tomato transgenic plants. Then flanking sequences were comfirmed by PCR analysis in transgenic plants. BLAST results in SGN database showed that T-DNA integration site lost 40 base pair. Finally, we screened out homozygous lines in T1 transgenic generation using flanking sequence primers and border sequence primers, and verified the results in T2 generation.