抗虫转基因欧洲黑杨多重PCR技术检测体系构建

摘要

In order to establish a multiplex PCR detection method of genetically modified ingredient in Populus nigra, primers were designed according to CaMV 35s promoter, NOS terminator, NPT I1 marker gene and Bt target gene sequences 9f insect-resistant transgenic P. nigra. The promoter, terminator, marker gene and target gene were detected by simplex PCR and multiplex PCR. The PCR system such as annealing temperature and content of primers were optimized. The results showed that the high efficiency PCR system was obtained when the final ratio of the primer is 1.0: 0. 5: 0. 5 for Bt, NPT 1I and NOS respectively, and the annealing temperature is 59 ℃. Base on these, a technical system was set up to detect the Bt, NPT II, NOS gene of insect-resistant transgenic P. nigra with triple PCR, and the quick, efficient and accurate identification way of insect-resistant transgenic P. nigra was achieved.%根据抗虫转基因欧洲黑杨中的CaMV 35S启动子、NOS终止子、NPTⅡ标记基因以及Bt目的基因序列,设计合成了相应引物,采用单重PCR和多重PCR技术对启动子、终止子、选择标记基因以及目的基因等多个外源基因进行检测,并对各引物的退火温度及引物之间的浓度配比进行优化,建立了适用于抗虫转基因欧洲黑杨检测的Bt、NPTⅡ和NOS基因的三重PCR分析的技术体系.结果表明:当各组引物的终浓度配比为1.0:0.5:0.5,退火温度为59℃时,所建立的三重PCR检测体系能够有效地检测出抗虫转基因欧洲黑杨中的转基因成分,实现了对抗虫转基因欧洲黑杨的快速、高效、准确的鉴定.