Abstract
Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.
This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.
[1] De Preter, K., Speleman, F., Combaret, V., Lunec, J., Laureys, G., Eusson, B.H., Francotte, N., Pearson, A.D., De Paepe, A, van Roy, N. and Vandersompele J. Quantification of MYC-N, DDX I and NAG gene copy number in neuroblastoma using real time quantitative PCR assay. Mod. Pathol. 15 (2002) 150–166. Search in Google Scholar
[2] Mason, G., Provero, P., Vaira, A.M. and Accotto, G.P. Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotech. 24 (2002) 2–20. Search in Google Scholar
[3] Ingham, D.J., Beer, S., Money, S. and Hansen, G. Quantitative realtime PCR assay for determining transgene copy number in transformed plants. Biotechniques 31 (2001) 132–140. Search in Google Scholar
[4] Li, Z., Hansen, J.L., Liu, Y., Zemetra, R.S. and Berger, P.H. Using real-time PCR to Determine transgene copy number in wheat. Plant Mol. Biol. Rep. 22 (2004) 179–188. http://dx.doi.org/10.1007/BF0277272510.1007/BF02772725Search in Google Scholar
[5] Bubner, B. and Baldwin, I.T. Use of real-time PCR for determining copy number and zygosity in transgenic plant. Plant Cell Rep. 23 (2004) 263–271. http://dx.doi.org/10.1007/s00299-004-0859-y10.1007/s00299-004-0859-ySearch in Google Scholar
[6] Yang, L., Ding, J., Zhang, C., Jia, J., Weng, H., Liu, W. and Zhang, D. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep. 23 (2005) 759–763. http://dx.doi.org/10.1007/s00299-004-0881-010.1007/s00299-004-0881-0Search in Google Scholar
[7] Schmidt, M.A. and Parrot, W.A. Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction. Plant Cell Rep. 20 (2001) 422–428. http://dx.doi.org/10.1007/s00299010032610.1007/s002990100326Search in Google Scholar
[8] Bubner, B., Gase, K. and Baldwin, I.T. Two-fold differences are the detection limit for determining transgene copy numbers in plants by realtime PCR. BMC Biotech. 4:14 (2004) 1–11. Search in Google Scholar
[9] Mackay, I.M., Arden, K.E. and Nitshe, A. Real-time PCR in virology. Nucleic Acid Res. 30 (2002) 1292–1305. http://dx.doi.org/10.1093/nar/30.6.129210.1093/nar/30.6.1292Search in Google Scholar
[10] Mackay, I.M. Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infect. 10 (2004) 190–212. http://dx.doi.org/10.1111/j.1198-743X.2004.00722.x10.1111/j.1198-743X.2004.00722.xSearch in Google Scholar
[11] Mayer, Z., Bagnara, A., Farber, P. and Geisen, R. Quantification of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods. I. J. Food Microb. 82 (2003) 143–151. http://dx.doi.org/10.1016/S0168-1605(02)00250-710.1016/S0168-1605(02)00250-7Search in Google Scholar
[12] Schnerr, H., Niessen, L. and Vogel, R.F. Real-time detection of the tri5 gene in Fusarium species by LightCycler™-PCR using SYBR-Green I for continuous fluorescence minitoring. I. J. Food Microb. 71 (2001) 53–61. http://dx.doi.org/10.1016/S0168-1605(01)00579-710.1016/S0168-1605(01)00579-7Search in Google Scholar
[13] Vaitilingom, M., Pijnenburg, H., Gendre, F. and Brignon, P. Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods. J. Agric. Food Chem. 47 (1999) 5261–5266. http://dx.doi.org/10.1021/jf981208v10.1021/jf981208vSearch in Google Scholar
[14] Shewry, P.R., Halford, N.G., Tatham, A.S., Popineau, Y., Lafiandra D. and Belton, P.S. The high molecular weight subunits of wheat glutenin and their role in determining wheat processing properties. Adv. Food Nutr. Res. 45 (2003) 219–302. http://dx.doi.org/10.1016/S1043-4526(03)45006-710.1016/S1043-4526(03)45006-7Search in Google Scholar
[15] Jones, H.D. Wheat transformation current technology and applications to grain development and composition. J. Cereal Sci. 41 (2005) 137–147. http://dx.doi.org/10.1016/j.jcs.2004.08.00910.1016/j.jcs.2004.08.009Search in Google Scholar
[16] Blechl, A., Lin, J., Nguyen, S., Chan, R., Anderson, O.D. and Dupont, F.M. Transgenic wheats with elevated levels of Dx5 and/or Dy10 high-molecular-weight glutenin subunits yield doughs with increased mixing strength and tolerance. J. Cereal Sci. 45 (2007) 172–183. http://dx.doi.org/10.1016/j.jcs.2006.07.00910.1016/j.jcs.2006.07.009Search in Google Scholar
[17] Barro, F., Rooke, L., Bekes, F., Gras, P., Tatham, A.S., Fido, R., Lazzeri, P.A., Shewry, P.R. and Barcelo, P. Transformation of wheat with high molecular weight subunit genes results in improved functional properties. Nat. Biotech. 15 (1997) 1295–1299. http://dx.doi.org/10.1038/nbt1197-129510.1038/nbt1197-1295Search in Google Scholar PubMed
[18] Rooke, I., Barro, F., Tatham, A.S., Fido, R., Steele, S., Békés, F., Gras, P., Martin, A., Lazzeri, P.A., Shewry, P.R. and Barcelo, P. Altered functional properties of tritordeum by transformation with HMW glutenin subunit genes. Theor. Appl. Genet. 99 (1999) 851–858. http://dx.doi.org/10.1007/s00122005130510.1007/s001220051305Search in Google Scholar
[19] Sangtong, V., Moran, D.L., Chikwamba, R., Wang, K.W., Woodman-Clikeman, M.J., Long, M., Lee, M. and Scott, P. Expression and inheritance of the wheat Glu-1Dx5 gene in transgenic maize. Theor. Appl. Genet. 105 (2002) 937–945. http://dx.doi.org/10.1007/s00122-002-1036-810.1007/s00122-002-1036-8Search in Google Scholar PubMed
[20] Altpeter, F., Popelka, J.C. and Wieser, H. Stable expression of 1Dx5 and 1Dy10 high molecular weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutenin fraction in rye flour. Plant Mol. Biol. 54 (2004) 783–792. http://dx.doi.org/10.1007/s11103-004-0122-510.1007/s11103-004-0122-5Search in Google Scholar PubMed
[21] Blechl, A.E. and Anderson, O.D. Expression of a novel high-molecularweight glutenin subunit gene in transgenic wheat. Nat. Biotech. 14 (1996) 875–879. http://dx.doi.org/10.1038/nbt0796-87510.1038/nbt0796-875Search in Google Scholar PubMed
[22] Leon, E., Marín, S., Gimenez, M.G., Piston, F., Rodriguez-Quijano, M., Shewry, P.R. and Barro, F. Mixing properties and dough functionality of transgenic lines of a commercial wheat cultivar expressing the 1Ax1, 1Dx5 and 1Dy10 HMW glutenin subunit genes. J. Cereal Sci. 49 (2009) 148–156. http://dx.doi.org/10.1016/j.jcs.2008.08.00210.1016/j.jcs.2008.08.002Search in Google Scholar
[23] Gadaleta, A., Blechl, A.E., Nguyen, S., Cardone, M.F., Ventura, M. and Blanco, A. Stable inheritance and expression of D-genome derived HMW glutenin subunit genes transformed into different durum wheat genotypes. Mol. Breed. 22 (2008) 267–279. http://dx.doi.org/10.1007/s11032-008-9172-810.1007/s11032-008-9172-8Search in Google Scholar
[24] Iglesias, V.A., Moscone, E.A., Papp, I., Neuhuber, F., Michalowski, S., Phelan, T., Spiker, S., Matzke, M. and Matzke, A.J.M. Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco. Plant Cell 9 (1997) 1251–1264. http://dx.doi.org/10.1105/tpc.9.8.125110.1105/tpc.9.8.1251Search in Google Scholar PubMed PubMed Central
[25] Srivastava, V., Vasil, V. and Vasil, I.K. Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.). Theor. Appl. Genet. 92 (1996) 1031–1037. http://dx.doi.org/10.1007/BF0022404510.1007/BF00224045Search in Google Scholar
[26] Anderson, O.D., Greene, F.C., Yip, R.E., Halford, N.G., Shewry, P.R. and Malpica-Romero, J-M. Nucleotide sequences of the two high-molecular weight glutenin genes form the D-genome of a hexaploid bread wheat, Triticum aestivum cv. Cheyenne. Nucleic Acids Res. 17 (1989) 461–462. http://dx.doi.org/10.1093/nar/17.1.46110.1093/nar/17.1.461Search in Google Scholar
[27] Sharp, P.J., Kreis, M., Shewry, P.R. and Gale, M.D. Location of α-amylase sequences in wheat and its relatives. Theor. Appl. Genet. 75 (1988) 286–290. http://dx.doi.org/10.1007/BF0030396610.1007/BF00303966Search in Google Scholar
[28] Kumpatla, S.P., Teng, W., Buchholz, W.G. and Hall, T.C. Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice. Plant Physiol. 115 (1997) 361–373. http://dx.doi.org/10.1104/pp.115.2.36110.1104/pp.115.2.361Search in Google Scholar
[29] Pawlowski, W.P. and Somers, D.A. Transgene inheritance in plants genetically engineered by microprojectile bombardment. Mol. Biotech. 6 (1996) 17–30. http://dx.doi.org/10.1007/BF0276232010.1007/BF02762320Search in Google Scholar
[30] Matzke, A.J.M., and Matzke, M.A. Position effect and epigenetic silencing of plant transgenes. Curr. Op. Plant Biol. 1 (1998) 142–148. http://dx.doi.org/10.1016/S1369-5266(98)80016-210.1016/S1369-5266(98)80016-2Search in Google Scholar
[31] Meyer, P. and Heidmann, I. Epigenetic variants of a transgenic petunia line show hypermetilation in transgene DNA, an indication for specific recognition of foreign DNA in transgenic plants. Mol. Gene Genet. 243 (1994) 390–399. Search in Google Scholar
[32] Altpeter, F., Vasil, V., Srivastava, V. and Vasil, I.K. Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat. Nat. Biotech. 14 (1996) 1155–1159. http://dx.doi.org/10.1038/nbt0996-115510.1038/nbt0996-1155Search in Google Scholar PubMed
[33] Blechl, A.E., Le, H.Q. and Anderson, O.D. Engineering changes in wheat flour by genetic transformation. Plant Phys. J. 152 (1998) 703–707. Search in Google Scholar
[34] Alvarez, M.L., Guelman, S., Halford, N.G., Lustig, S., Reggiardo, M.I., Ryabushkina, N., Schewry, P., Stein, J. and Vallejos, R.H. Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits. Theor. Appl. Genet. 100 (2000) 319–327. http://dx.doi.org/10.1007/s00122005004210.1007/s001220050042Search in Google Scholar
[35] Uthayakumaran, S., Lukow, O.M., Jordan, M.C. and Cloutier, S. Development of genetically modified wheat to assess its dough functional properties. Mol. Breed. 11 (2003) 249–258. http://dx.doi.org/10.1023/A:102346130584810.1023/A:1023461305848Search in Google Scholar
[36] He, G.Y., Jones, H.D., D’Ovidio, R., Masci, S., Chen, M., West, J., Butow, B., Anderson, O.D., Lazzeri, P., Fido, R. and Shewry, P.R. Expression of an extended HMW subunit in transgenic wheat and the effect on dough mixing properties. J. Cereal Sci. 42 (2005) 225–231. http://dx.doi.org/10.1016/j.jcs.2005.04.00410.1016/j.jcs.2005.04.004Search in Google Scholar
[37] Meyer, P. Understanding and controlling transgene expression. Trends Biotech. 13 (1995) 332–337. http://dx.doi.org/10.1016/S0167-7799(00)88977-510.1016/S0167-7799(00)88977-5Search in Google Scholar
[38] Callaway, A.S., Abranches, R., Scroggs, J. and Allen, G.C. and Thompson, W.F. High throughput transgene copy number estimation by competitive PCR. Plant Mol. Biol. Rep. 20 (2002) 265–277. http://dx.doi.org/10.1007/BF0278246210.1007/BF02782462Search in Google Scholar
[39] Song, P., Cai, C.Q., Skokut, M., Kosegi, B.D. and Petolino, J.F. Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKER-derived transgenic maize. Plant Cell Rep. 20 (2002) 948–954. http://dx.doi.org/10.1007/s00299-001-0432-x10.1007/s00299-001-0432-xSearch in Google Scholar
[40] Pedersen, C., Zimny, J., Becker, D., Janhne-Gartner, A. and Lorz, H. Localization of introduced genes on the chrmomosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization. Theor. Appl. Genet. 94 (1997) 749–757. http://dx.doi.org/10.1007/s00122005047410.1007/s001220050474Search in Google Scholar
[41] Rooke, L., Steele, S.H., Barcelo, P., Shewry, P.R. and Lazzeri, P. Transgene inheritance, segregation and expression in bread wheat. Euphytica 129 (2003) 301–309. http://dx.doi.org/10.1023/A:102229601780110.1023/A:1022296017801Search in Google Scholar
[42] Gautier, M.F., Cosson, P., Guirao, A., Alary, R. and Joudrier, P. Pouroindoline genes are highly conserved in diploid ancestor wheat and related species but absent in teraploid Triticum species. Plant Sci. 153 (2000) 81–91. http://dx.doi.org/10.1016/S0168-9452(99)00258-710.1016/S0168-9452(99)00258-7Search in Google Scholar
[43] Matzke, M.A., Aufsatz, W., Kanno, T., Mette, M.F. and Matzke, A.J. Homology-dependent gene silencing and host defence in plants. Adv. Gen. 46 (2002) 235–275. http://dx.doi.org/10.1016/S0065-2660(02)46009-910.1016/S0065-2660(02)46009-9Search in Google Scholar
© 2011 University of Wrocław, Poland
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.