转基因水稻B2A68事件特异性检测方法的建立

Establishment of an Event-specific Method to Detect Transgenic Rice B2A68

利用hiTAIL-PCR方法扩增获得转基因水稻B2A68中T-DNA右边界591 bp的旁侧序列,该序列位于水稻第3染色体上。参考已公布的该插入位点的水稻基因组序列和载体左边界序列分别设计上下游引物,扩增获得外源基因插入位点的左边界925 bp旁侧序列。以左右旁侧序列为基础,建立了转基因水稻B2A68事件特异性定性检测方法。采用该方法可从转基因水稻B2A68中扩增到521 bp和307 bp的2条特异性目标条带,其它水稻不会扩增到条带或者上述相同大小的目标条带。该方法特异性强,检测灵敏度达到0.1%,能满足海关、工商、农业等部门的检测需求。

The transgenic rice B2A68 contained both insect-resistant gene Cry2Aa#and herbicide-tolerant gene Bar.The right flanking sequence of T-DNA（Transfer DNA） right border was isolated using hiTAIL-PCR（High-efficiency thermal asymmetric interlaced PCR）.Blast analysis in NCBI database showed that the right flanking sequence,a 591-bp fragment,included a 120-bp vector sequence and a 471-bp rice genomic sequence on Chromosome 3.The primers used to amplify the left flanking sequence of T-DNA left border were designed according to the published sequence near the inserted site on Chromosome 3 and the T-DNA left border sequence.A 925-bp left flanking sequence was isolated,which included a 613-bp rice genomic sequence on Chromosome 3 and a 312-bp vector sequence.An event-specific detection method of transgenic rice B2A68 was established based on the right and left flanking sequences.The specific fragments of 521-bp and 307-bp were amplified from the left and right flanking sequence,respectively,in transgenic rice B2A68 using this method,but they were not amplified in any other rice variety.The qualitative detection limit was assessed to be 0.1%,which can satisfy the detection demand of the departments of customs,administration for industry and commerce,agriculture,etc.