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, Southern 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.
WEI Yi-Dong,MAO Xiao-Hui,HE Wei, et al. Determination of the Copy Number of Exogenous Gene in Transgenic Rice (Oryza sativa) by Multiplex qPCR[J]. , 2017, 25(12): 2072-2078.
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