应用Tail-PCR扩增蓝猪耳T-DNA侧翼序列

蓝猪耳是一种重要的具有观赏和科研价值的花卉植物.采用TAIL-PCR成功扩增了转基因蓝猪耳T-DNA插入位点的侧翼序列,扩增片断长度为200～2 000 bp,大多数片段在400 bp和800 bp左右,其中36%的序列含有植物的同源序列.通过与GenBank数据库比对,确定了部分T-DNA插入位点周边序列编码的可能蛋白,并提交序列7条;另外还对T-DNA转化植物时整合的位点进行了分析,发现断裂位点集中在距右边界15～18 bD和右边界外234 bp处,分别占总扩增片段的47.62%和38.10%.这为利用T-DNA标签进行蓝猪耳基因克隆和功能分析提供了实验技术上的保证.图2表2参24

Amplification of Tail-PCR in Amplifying Flanking Sequence of T-DNA in Torenia

Torenia (Torenia fournieri Lind.) is an important flowering plant for scientific research with ornamental values. The flanking sequence of T-DNA insertion site was successfully amplified using TAIL-PCR in torenia, and the length of the amplified products ranged about from 200 bp to 2 000 bp and most of them were about 400 bp and 800 bp. Among them, 36% had homologous plant sequences. By BLAST analysis in NCBI, the predicted protein encoded by the flanking sequence of T-DNA insertion site was determined and seven gene sequences were submitted to GenBank. In addition, the rule was analyzed for T-DNA integrated into plant genome, and it suggested that the T-DNA integration occurred in the T-DNA region 15～18 bp from right border (RB) and 234 bp from outside of the RB, and the percentages of total fragments were 47.62% and 38.1% respectively. These provide the guarantee in experimental techniques for gene cloning and functional study of torenia by T-DNA tagging. Fig 2, Tab 2, Ref 24