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.
1. Stam, M., J.N. Mol and J.M. Kooter. Review article: the silence of genes in transgenic plants [J]. Annals of Botany, 1997, 79(1): p. 3-12.2. 李淑洁 and 张正英. REAL-TIME PCR 方法测定转基因小麦中外源基因拷贝数 [J]. 中国生物工程杂志, 2010, 30(3): p. 90-94.3. 王盛, 谢芝勋, 谢丽基, et al. 转基因烟草中外源基因实时荧光定量 PCR 检测方法的建立 [J]. 南方农业学报, 2015, 46(5): p. 745-749.4. 杨晓杰, 刘传亮, 张朝军, et al. 不同转化方法获得的转基因棉花外源基因拷贝数分析 [J]. 农业生物技术学报, 2011, 19(2): p. 221-229.5. Jain, M., A. Nijhawan, A.K. Tyagi, et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR [J]. Biochemical and biophysical research communications, 2006, 345(2): p. 646-651.6. 魏毅东, 陈玉, 郭海萍谢华安, et al. 水稻缺素胁迫下实时荧光定量 RT-PCR 的内参基因的选择 [J]. 农业生物技术学报, 2013, 21(11): p. 1302-1312.7. Porebski, S., L.G. Bailey and B.R. Baum. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components [J]. Plant molecular biology reporter, 1997, 15(1): p. 8-15.8. DY, J. and M. Li, Molecular Cloning-A Laboratory Manual (分子克隆实验指南). 2002, 2nd, Beijing: Science Press.9. Yoshida, S. Routine procedure for growing rice plants in culture solution [J]. Laboratory manual for physiological studies of rice, 1976: p. 61-66.10. Pinto, F.L., A. Thapper, W. Sontheim, et al. Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria [J]. BMC molecular biology, 2009, 10(1): p. 79.11. Ingham, D.J., S. Beer, S. Money, et al. Quantitative real-time PCR assay for determining transgene copy number in transformed plants [J]. Biotechniques, 2001, 31(1): p. 132-141.12. Mason, G., P. Provero, A.M. Vaira, et al. Estimating the number of integrations in transformed plants by quantitative real-time PCR [J]. BMC biotechnology, 2002, 2(1): p. 20.13. Englerblum, G., M. Meier, J. Frank, et al. Reduction of background problems in nonradioactive Northern and Southern blot analyses enables higher sensitivity than 32P-based hybridizations [J]. Analytical biochemistry, 1993, 210(2): p. 235-244.14. McCabe, M.S., J.B. Power, A.M. de Laat, et al. Detection of single-copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis [J]. Molecular biotechnology, 1997, 7(1): p. 79-84.15. Jeon, J.S., S. Lee, K.H. Jung, et al. T‐DNA insertional mutagenesis for functional genomics in rice [J]. The Plant Journal, 2000, 22(6): p. 561-570.16. Udvardi, M.K., T. Czechowski and W.-R. Scheible. Eleven golden rules of quantitative RT-PCR [J]. The Plant Cell, 2008, 20(7): p. 1736-1737.17. Bustin, S.A., V. Benes, J.A. Garson, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments [J]. Clinical chemistry, 2009, 55(4): p. 611-622.18. Livak, K.J. and T.D. Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2? ΔΔCT method [J]. methods, 2001, 25(4): p. 402-408.
