多重PCR快速确证外源基因在转基因小麦后代的传递

Detection of Segregation of Foreign Genes in Transgenic Wheat Lines by Multiplex Polymerase Chain Reaction

根据转入小麦0世代中的高分子谷蛋白亚基1Dx5基因和报告基因uidA、作为选择标记的除草剂抗性基因bar的序列,设计合成三对引物。以整合uidA+bar的质粒pAHC25和整合1Dx5 的质粒p1Dx5为模板寻找uidA与1Dx5及或bar多重扩增的最佳模板浓度及最适退火温度。 MPCR模板量是单对引物扩增时的两倍,引物浓度同常规PCR为0．3μmol／L,uidA与bar的适宜退火温度范围为57．1-62．3℃;uidA与1Dx5为60．0℃-60．6℃;uidA、bar、1Dx5的最适合退火温度范围为57．0℃～58．4℃。MPCR对大小相差50bp及以下的多重扩增片段可通过10％的非变性聚丙烯酰胺凝胶电泳分离。在此基础上对14株T1代转基因小麦基因组DNA进行多重PCR 扩增,筛选出基因未分离的小麦后代,并与常规PCR比较,结果一致,其中11株同时传递1Dx5和 bar基因、1株同时传递uidA、bar和1Dx5基因,3株未检测到外源基因。表明MPCR在快速确证外源基因在转基因植株后代的传递中作用显著。研究在常规PCR反应体系上,对模板浓度和多重引物退火温度进行微调,且把MPCR技术与PAGE技术结合起来,提高了研究结果的准确性,获得了较好的扩增和检测效果,简化了MPCR优化程序,使MPCR的优势更明显,为该技术的广泛应用提供了借鉴。

The method of multiplex PCR was set up to identify two or three transgenes in one reaction such as uidA and bar; uidA and 1Dx5 or uidA, bar and 1Dx5 genes. Three sets of primer pairs which was specific to each of these three genes respectivel.y were designed and synthesized. Recombinant plasmids pAHC25 and pl Dx5 harboring uidA ＋ bar and 1Dx5 gene separately were used as template DNA in the process of optimizing an multiplex PCR reaction. The optimal annealing temperature for uidA and bar MPCR is range from 57.1 ℃ ～ 62. 3℃, for uidA and 1Dx5 is range from 60℃ to 60.6℃, and for uidA,bar and 1Dx5 range from 57.0℃～ 58.4℃. The amount of template for MPCR is twice as much as that for simplex PCR, while the concentration of primers is the same with simplex one. Less than 50bp MPCR products can be separated clearly by 10% nondenaturalized polyacrylamid gel electrophoresis. Fourteen transgenic wheat lines were tested by multiplex and simplex PCR respectively, which shows the same results and hence presents that MPCR is the reliable, rapid and high-effective approach to detect foreign genes from transgenic plant.