转基因水稻BarKasalath-01事件特异性检测

Event-specific Detection of Genetically Modified Rice BarKasalath-01

利用hi TAIL-PCR方法研究了转基因水稻Bar Kasalath-01中外源基因在基因组上插入位点的序列特征,获得了外源基因T-DNA右边界旁侧序列511 bp,与水稻基因组数据库比对发现,外源基因插入位点位于水稻基因组第8号染色体的第3 326 720处。根据整合位点水稻基因组序列和外源基因T-DNA左边界序列设计引物,扩增得到了左旁侧序列783 bp。基于左右旁侧序列,建立了转基因水稻Bar Kasalath-01的事件特异性定性PCR检测方法,扩增片段分别为783 bp和411 bp。该方法特异性强、灵敏度高,能够在Bar Kasalath-01基因组DNA含量为0.1%的模板中检测出转基因成份。依据旁侧序列,还建立了快速鉴定转基因后代植株基因型的3引物PCR检测方法。这些方法的建立,实现了对转基因水稻Bar Kasalath-01转化事件的特异性检测。

The hi Tail-PCR（high-efficiency thermal asymmetric interlaced PCR） was adopted to study the characteristic of insertion site in genetically modified rice Bar Kasalath-01. As a result, a 511 bp fragment of right flanking sequence was discovered. By comparison with rice genome database, we found that the insertion site of enthetic gene located on rice genome 3 326 720 of chromosome 8. A 783 bp fragment of left flanking sequence was amplified using the primers that were designed according to sequence of integration site on rice genome and T-DNA left border sequence of enthetic gene. The event-specific PCR detection method was developed based on the left and right flanking sequences, which produced 783 bp and 411 bp fragment respectively. The event-specific PCR detection method, with high specificity and sensitivity, could detect the genetically modified ingredients in samples containing 0.1% genomicDNA of Bar Kasalath-01. Based on the flanking sequence, a tri-primer PCR method was developed to identify homozygous genotype of enthetic gene in segregation generation quickly and accurately. The establishment of above methods implemented the specific detection of transformant event Bar Kasalath-01.