摘要
为解决转基因农产品检测中阳性标准物质匮乏的问题,利用重叠PCR技术将检测过程中常用的调控元件Ca MV35S启动子、FMV35S启动子、PAT29启动子、Ubiquitin启动子、NOS启动子、35S终止子、NOS终止子和E9终止子依次连接获得融合片段,再利用分子克隆技术将融合片段转化到载体p MD-19T中,获得含有调控元件的标准质粒p MD-RG。PCR和测序结果验证了标准分子构建的正确性,并且标准质粒的均匀性和稳定性比较好,符合标准物质候选物的要求,可代替阳性标准物质用于转基因成分的检测和研究。该质粒可用于大多数已经批准的转基因作物的检测和监管工作中。
In order to solve the problem of the lack of positive standard material for PCR detection of genetically modified(GM)crops,common regulatory elements during the detection process,such as CaMV35S promoter,FMV35S promoter,PAT29 promoter,Ubiquitin promoter,NOS promoter,35S terminator,NOS terminator and E9 terminator,were selected and connected successively by overlapping PCR technology to obtain the fusion gene fragments.The fragments were fused to pMD-19T vector to get the standard plasmid pMD-RG by the molecular cloning technology.The accuracy of standard molecular construction was verified by PCR and sequencing.The uniformity and stability of reference molecules were very well,and the reference molecule could be used as the candidate of reference material.The constructed plasmids can be used in the detection and supervision of most approved GM crops.
作者
邢宇俊
陆丹丹
吴季荣
徐剑宏
王园园
梁杰
史建荣
XING Yu-jun;LU Dan-dan;WU Ji-rong;XU Jian-hong;WANG Yuan-yuan;LIANG Jie;SHI Jian-rong(Institute of Food Safety and Nutrition,Jiangsu Academy of Agricultural Sciences/Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology/Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality,Ministry of Agriculture/Key Laboratory of Agro-product Safety Risk Evaluation,Ministry of Agriculture/Jiangsu Center for GMO Evaluation and Detection/Collaborative Innovation Center for Modern Grain Circulation and Safety/The Sino-German R&D Center for Agro-product Safety and Quality Control,Nanjing 210014,China)
出处
《江苏农业学报》
CSCD
北大核心
2018年第6期1392-1400,共9页
Jiangsu Journal of Agricultural Sciences
基金
国家转基因生物新品种培育重大专项(2016ZX08011-003)
国家自然科学基金项目(31301488)
江苏省农业科技自主创新基金项目[CX(14)5065]
关键词
转基因作物
质粒标准分子
调控元件
genetically modified crop
standard plasmid molecules
regulatory element
