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W-4是通过农杆菌介导法将fad2基因的反向重复序列表达框转入甘蓝型油菜Westar后获得的转基因高油酸油菜品系。为建立W-4的转基因事件特异性PCR检测技术,应用温度不对称PCR(TAIL-PCR)扩增获得转基因油菜W-4的T-DNA插入位点的左、右旁侧序列。其中右边界旁侧序列长度为290 bp,其碱基组成G+C含量为31.27%、A+T含量为68.73%;左边界旁侧序列长度为365 bp,其碱基组成G+C含量为32.6%、A+T含量为67.4%,表明该T-DNA整合在富含AT区。序列比对结果发现,该转基因事件中,T-DNA左边界序列完全整合到油菜基因组中,仅有1个碱基由G转换成了A。而右边界则缺失了包括RB border在内的62个碱基。结果表明:转基因高油酸油菜T-DNA的整合是一次无载体序列的整合。依据左、右边界旁侧序列和转基因载体的T-DNA左右边界序列设计了2对特异性引物TLF/TLR和TRF/TRR,能从W-4基因组DNA中扩增出大小分别为485和405 bp的预期产物,而在其他转基因油菜、非转基因油菜的基因组DNA和空白对照中均无特异性扩增产物,据此建立了W-4的转基因事件特异性PCR检测技术。应用该检测技术可以从含有0.1%W-4基因组DNA的混合样品中扩增出特异产物,检测灵敏度达0.1%。可对W-4的转基因事件进行特异性检测。
W-4 is a transgenic high-oleic rapeseed line obtained by transferring the inverted repeat sequence of the fad2 gene into Westar of Brassica napus via Agrobacterium-mediated transformation. To establish a W-4 transgenic PCR-specific PCR assay, the left and right flanking sequences of the T-DNA insertion site of transgenic rape W-4 were amplified by temperature-asymmetric PCR (TAIL-PCR). The length of the flanking sequence on the right border was 290 bp, the base composition G + C content was 31.27% and the A + T content was 68.73%. The length of the flanking sequence on the left border was 365 bp and the base composition G + C content was 32.6%, A + T content of 67.4%, indicating that the T-DNA integration in the AT-rich area. Sequence alignment revealed that the left border sequence of T-DNA was completely integrated into the genome of rapeseed and only 1 base was changed from G to A in this transgenic event. The right border is missing, including RB border, including 62 bases. The results showed that the integration of T-DNA in transgenic high oleic rape was a non-vector integration. Two pairs of specific primers TLF / TLR and TRF / TRR were designed according to the flanking sequence of the left and right borders and the transmembrane T-DNA border sequences of the transgene vector. The primers were designed to amplify the genomic DNAs of 485 and 405 bp, and no specific amplification product was found in other transgenic rapeseed and non-transgenic canola genomic DNA and blank control. Therefore, a transgenic PCR assay specific to W-4 was established. Using this detection technique, a specific product can be amplified from a mixed sample containing 0.1% W-4 genomic DNA with a detection sensitivity of 0.1%. Specific detection of W-4 transgenic events can be performed.