农业生物技术学报2017，Vol.25Issue(12)：2072-2078,7.DOI:10.3969/j.issn.1674-7968.2017.12.018
利用多重荧光定量PCR检测转基因水稻外源基因拷贝数
Determination of the Copy Number of Exogenous Gene in Transgenic Rice (Oryza sativa) by Multiplex qPCR
摘要
Abstract
The copy number of exogenous gene affects the genetic stability and gene expression level in transgenic plants,and the identification and screening of single copy homozygous plants are necessary for the functional analysis of progeny.To fast and accurately identify gene copy of transgenic rice materials harboring hygromycin resistant gene (hygromycin phosphotransferase II (hptII),Taqman based Multiplex qRT-PCR was developed using constructive gene Oryza sativa splicing factor u2af (OsSFu2a) as reference gene.By comparison of amplification efficiency of different primer sets,the primer set of hptII-1 and OsFU2a-2 was the best one.The copy number of 10 transgenic clones was determinated using this system,and the results shown that 7 of 10 were single copy and 3 were double copy.Furthermore,Southem blot and segregation ratio analysis were performed to validate this system,and when compared with traditional singlex qRT-PCR,this system improved the stability and accuracy of detection result.Therefore,this system could be great help for plant biotechnologists to fast,accurately and high-throughput identify endogenous gene copy number and homozygous plant.关键词
转基因水稻/拷贝数/多重荧光定量PCRKey words
Transgenic rice/Copy number/Multiplex real-time quantitative PCR (Multiplex qRT-PCR)分类
农业科技引用本文复制引用
魏毅东,毛小辉,何炜,王进兰,连玲,许秀宝,林悦龙,谢华安,张建福..利用多重荧光定量PCR检测转基因水稻外源基因拷贝数[J].农业生物技术学报,2017,25(12):2072-2078,7.基金项目
国家转基因农作物新品种培育重大专项(No.2016ZX08001-006和No.2016ZX08001-004)和福建省财政专项-福建省农业科学院创新团队PI项目(No.2016PI-15) （No.2016ZX08001-006和No.2016ZX08001-004）
