转基因油菜W-4 T-DNA旁侧序列分析与事件特异性检测

Analysis of the flanking sequence and event-specific detection of transgenic line W-4 of Brassica napus

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。本研究应用温度不对称PCR(TAIL-PCR)技术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A/T区。依据左、右边界旁侧序列和转基因载体的T-DNA左右边界序列设计的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 bp和405 bp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,检测灵敏度达到0.1%。表明应用该技术可对W-4转基因事件进行特异性检测。

The transgenic rapeseed line W-4 is a genetically modified high oleic rapeseed （Brassica napus L. ）transformed with a binary vector harbouring an inverted repeat expression cassette of the fad2 gene by agrobacterium-media-ted transformation to the rapeseed variety Westar. Both the left and right flanking sequences to the T-DNA insertion sitefrom the genomic DNA of W-4 were amplified by temperature asymmetric PCR. The flanking sequences to the right borderwas 290 bp in length and the nucleotide composition were 31.27% for G＋C content and 68.73% for A＋T content. Theflanking sequence to the left border was 365 bp in lengthand the G＋C content was 32.6% and the A ＋ T contentwas 67.4%. These results indicated that the T-DNA wasintegrated in the A/ T-rich region. Based on the flankingsequences and the left and right boundaries of the T-DNAsequences,the specific primes TLF/ TLR and TRF/ TRRwere designed. Using the primes, the event-specific PCRdetection methods for transgenic rapeseed W-4 lines was established. The expected sizes of 485 bp and 405 bp products could be amplified from the W-4 genomic DNA by the PCR,respectively, while no products could be amplified from the genomic DNA of other transgenic oilseed rapes and nontrans-genic control and control. By the PCR, it was possible to detect the W-4 genomic DNA from a mixed sample of genomicDNA, and the detection sensibility was 0. 1%. The method developed in this study was highly specific, sensitive and suit-able for event-specific detection of the transgenic line W-4.