转CBF1基因烟草植株的花器官变异及其插入区侧翼序列的分析

在转CBF1基因烟草的后代中发现了花器官变异的植株，主要有花瓣裂瓣、花型变小、花丝变短花粉萌发率降低和育性降低等表型变化。采用TAIL－PCR方法，从花器官变异明显和不明显的植株中分别克隆到了各自的T－DNA插入区域的侧翼序列。将这些侧翼序列与GenBank database及pBI121质粒进行比对。结果表明表型差异明显的2个植株的T－DNA插入位点相同，属于一个转基因株系；且T－DNA插入区位于烟草基因组的非编码区。说明这些花型的变异并非由T－DNA在受体基因组中插入位点不同造成的，也不是由外源转基因对受体功能基因的破坏引起的。

Floral Aberrance of CBF1 Transgenic Tobacco and Analysis of Its Flanking Sequences

In the present work，floral aberrances appeared in some of CBF1 transformed tobacco plants. Comparisons of wild type plants, the main phenotype changes of transformed plants were aberrant petals, smaller flowers and shorter androeciums. Furthermore, the ratio of pollen germination was decreased and the capsule number was reduced. Using genomic DNA of each aberrant plant as a template, TAIL-PCR was performed with five arbitary degenerate (AD) primers pairing with 3 nested specific primers designed toward outside in pBI121 plasmid. After 3-step PCR reactions, the flanking sequence of each T-DNA inserting site was obtained. The PCR products were sequenced directly if it is longer than 300 bp. These sequences were searched against GenBank database using blast n or searched against pBI121 sequence using bl2seq. Analysis of flanking sequences demonstrated that they shared the same T-DNA inserting site. It was suggested that the aberrance of the transformed tobacco did not result from different sites of T-DNA insertion.