转TGEV-S基因玉米中目的基因的PCR检测及优化

PCR detection of the target gene in transgenic maize derived spike glycoprotein gene of porcinetransmissible gastroenteritis virus

为提高转基因玉米中目的基因的检出效率,以猪传染性胃肠炎病毒纤突糖蛋白（TGEV-S）转基因玉米为材料,利用PCR方法检测样品中的目的基因.通过对PCR反应体系中4种不同DNA聚合酶和8种退火温度进行比较,建立和优化了转基因玉米中TGEV-S基因的PCR检测方法.对450株转基因玉米叶片DNA和种子DNA中的TGEV-S基因片段进行检测,并设计33对引物检测插入转基因玉米基因组DNA中的质粒pBAC9020DNA片段.结果显示,LA Taq酶对叶片DNA和种子DNA中TGEV-S片段的PCR扩增敏感性和特异性均优于其他Taq聚合酶,且退火温度为53～55℃时扩增效果较好.分别对450份转基因玉米叶片DNA和种子DNA检测结果显示阳性率分别为82.5％和76.3％.利用33对引物进行的PCR扩增及测序结果显示质粒pBAC9020基因片段已全部插入该玉米基因组DNA中.本试验建立的转TGEV-S基因玉米PCR检测方法敏感性和特异性高,为转基因玉米阳性植株的检测奠定了坚实的基础.

To improve detection efficiency of the target gene in transgenic maize,the transgenic maize derived spike glycoprotein of transmissible gastroenteritis virus （TGEV-S） was used to detect the target gene by PCR.In this study,the optimal PCR reaction system was established by comparison of 4 commonly used DNA polymerases and 8 annealing temperatures.The results showed that LA Taq polymerase had higher specificity and sensitivity by amplifying the fragment of TGEV-S than the other three polymerases.And the best annealing temperature range is 53℃-55℃.450 transgenic plants were detected,82.5％ leaves genomic DNA and 76.3％ seed genomic DNA were amplified the TGEV-S fragment.Using 33 pairs of primers to amplified the plasmid vector pBAC9020 and sequencing the PCR fragments,we found the whole plasmid pBAC9020 was inserted into the genome of the transgenic maize.The optimized PCR system provided a specific and sensitive method for detecting the TGEV-S fragment in the transgenic maize,which will play an essential role in detecting positive plants of transgenic maizes.