抗草甘膦转基因玉米外源基因ddPCR拷贝数分析

Estimation of Exogenous Genes Copy Number of Genetically Modified Glyphosate-Resistant Maize by Droplet Digital PCR

【目的】本研究是从前期获得的100余份转基因抗除草剂草甘膦玉米品系中,挑选T抗-1等14个转基因玉米品系进行拷贝数检测。【方法】采用一种新型的拷贝数分析方法——微滴数字PCR技术(droplet digital PCR,简称dd PCR),设计特异引物对外源抗草甘膦基因2m G2-epsps进行绝对定量分析。【结果】供试的14份转基因材料中10份是单拷贝品系。同时,通过引物探针特异性试验、叶片基因组DNA的PCR抑制和浓度检测、转基因玉米的外源基因2m G2-epsps的实时PCR检测和基因组DNA的酶切等系列研究,建立稳定的转基因玉米拷贝数分析的dd PCR检测体系。【结论】微滴数字PCR技术为这批转基因玉米的下一步转基因生物安全评价提供了重要参数,同时建立了转基因玉米(genetically modified maize)外源基因拷贝数dd PCR分析方法。

[ Objective ] The exogenous gene copy number of Tkang-1 etc. 14 was estimated genetically modified maize based on the early stage of the genetically modified more than 100 maize （Zea mays L. ） materials with higher resistance and better comprehensive agronomic charac- ters, in order to pass through the transgenic biosafety evaluation and fit in with the needs of breeding new transgenic maize varieties. [ Method ] A new kind of analysis method was adopted for the exogenous gene copy number the droplet digital PCR technology （ droplet digital PCR, ddPCR）. And specific primer of exogenous gene 2mG2-epsps was designed to analyze the copy number by absolutely quantitative method. [ Result] The analysis showed that 10 of ld transgenic materials were single copies. At the same time, a stable test system was established for analyzing the exogenous gene copy number of genetically modified maize by the studying of designing specific primer of exogenous gene 2mG2-epsps, testing the PCR inhibitor and concentration of leaf genome, real time PCR-testing for the exogenous gene of the transgenic maize, restricting genome DNA and so on. [ Conclusion ] This paper provided important parameters for the further transgenie biosafety evaluation of transgenic maize, laid a strong foundation for breeding new transgenic maize varieties and established the genetic copy number analysis method of genetically modified maize.