采用TAIL-PCR方法研究转基因大豆外源基因LEC1插入位点序列特征,并根据此特征建立了LEC1转化事件特异性检测方法.依据正义表达载体上T-DNA区段侧翼序列设计特异性引物和简并引物,获得4个同源序列.对获得的序列进行Blastn分析发现,p69、p148、p225均为载体T-DNA区段一部分,未见大豆基因组序列；P217插入片段的序列分析表明,该序列长863bp,其中T-DNA左边界序列长143 bp,720bp为大豆基因组片段,与大豆光系统Ⅱ类囊体膜蛋白(Thylakoid membrane proteins)的206-926区段同源性为99％,这表明,外源DNA插入了大豆基因组中类囊体膜蛋白编码区的206位.根据分离的序列建立事件特异性定性检测方法,扩增片段大小为277 bp.该事件特异性检测方法具有高度的特异性和良好的灵敏性,为转基因大豆品种LEC1的身份识别提供了有效的方法.%This paper was designated to discover the integration site of the transgene in the soybean genome and to establish event specific methods for qualitative detection of LEC based on the left border junction fragment which was isolated using the TAIL-PCR. Designing the degenerate primers and specific primers based on T-DNA flanking sequence of over-expression vector, PCR to obtain sequences of four homologous. To get Blastn the sequences, analysis three of them: pl48, p225, p69, are both as the carrier T-DNA segment part, without the soybean genome sequence; the rest P217 shows that the sequence is length of 863 bp, Which contains 720 bp fragment of the soybean genome, of which was homologous 99% with soybean photosystem II thylakoid membrane protein of the 206 -926 sections. This suggests that exogenous DNA into the 206 of genome of soybean thylakoid membrane protein coding region. During the integration of the construct LEC event- specific, qualitative PCR method was established with the primers(LECl-s)targeting the junction regions to produce a 277 bp product The method developed in this study proved to be highly specific, sensitive, and suitable for LEC1 sample detection.
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