Fiziol. rast. genet. 2020, vol. 52, no. 3, 196-207, doi:

Detection of dna polymorphism of transgenic wheat plants with proline metabolism heterologous genes

Dubrovna O.V.1, Velikozhon L.G.1, Slivka L.V.1, Kondratskaya I.P.2, Reshetnikov V.N.2, Makai S.3

  1. Institute of Plant Physiology and Genetics, National Academy of Sciences of Ukraine 31/17 Vasylkivska St., Kyiv, 03022, Ukraine
  2. Central Botanical Garden, National Academy of Sciences of Belarus 2v Surganov St., Minsk, 220012, Republic of Belarus
  3. University of West-Hungary,  2 Var St., Mosonmagyarovar, H-9200, Republic of Hungary

The polymorphism level of DNA regions flanked by inverted LTR-retrotransposon repeats has been analyzed by the IRAP-PCR method in genetically modified wheat plants obtained by Agrobacterium-mediated transformation in an in vitro culture. Some plants contain the Medicago truncatula ornithine-d-aminotransferase gene, and the other contain a double-stranded RNA suppressor of the Arabidopsis thaliana proline dehydrogenase gene. In analysis of plants with the heterologous ornithine-d-aminotransferase gene, the application of the primer to the Sukkula retrotransposon was the most effective, where in the nine tested plants four new amplicons were obtained in the spectrum of DNA amplification products. The findings suggest that it is the foreign DNA insertion capable of inducing transposition of retrotransposons Sukkula/Nikita and Wham/Sabrina, because in control plants derived from in vitro culture their activity has not been established. The analysis of transgenic plants with a double-stranded RNA suppressor of the proline dehydrogenase gene using highly efficient primers for the retrotransposons Sukkula, Sabrina, Wham, Nikita, and Wilma1 no DNA polymorphism was revealed. In the course of the experiment, we did not register the disappearance of amplicons in the DNA profiles of PCR and this may be index of the rearrangements absence in the primer annealing sites and in the loci studied. The emergence of new amplicons was not observed in the spectra of DNA amplification products, what indicate the absence of activation of mobile genetic elements transposon activity in transgenic plants with a double-stranded RNA suppressor of the proline dehydrogenase gene. IRAP primer pairs were selected experimentally, but the use of this method did not reveal the disappearance or emergence of polymorphic fragments. The absence of DNA polymorphism in transgenic plants with a double-stranded RNA suppressor of the proline dehydrogenase gene may be due to the phenomenon of RNA interference that suppresses retrotransposon activity.

Keywords: Agrobacterium-mediated transformation, genes of proline metabolism, retrotransposons, IRAP-PCR

Fiziol. rast. genet.
2020, vol. 52, no. 3, 196-207

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1. Jones, H., Doherty, A. & Wu, H. (2005). Review of methodologies and a protocol for the Agrobacterium mediated transformation of wheat. Plant Methods, 1, pp. 1-5.

2. Choi, H.W., Lemaux, P.G. & Cho, M.-J. (2000). Increased chromosomal variation in transgenic versus nontransgenic barley (Hordeum vulgare L.) plants. Crop Sci., 40, pp. 524-533.

3. Choi, H.W., Lemaux, P.G. & Cho, M.-J. (2001). High frequency of cytogenetic aberration in transgenic oat (Avena sativa L.) plants. Plant Sci., 160, pp. 763-772.

4. Labra, M., Savini, C., Bracale, M., Pelucchi, N., Colombo, L., Bardini, M. & Sala, F. Genomic changes in transgenic rice (Oryza sativa L.) plants produced by infecting calli with Agrobacterium tumefaciens. Plant Cell Rep., 20, pp. 325-330.

5. Enikeev, A.G., Kopytina, T.V., Semenova, L.A., Natyaganova, A.V., Gamanetz, L.V. & Volkova, O.D. (2008). Agrobacterium transformation as complex biotical stressing factor. J. Stress Physiol. Biochem., 4, no. 1, pp. 11-19.

6. Flugge, U.I. & Klosgen, R.B. (2004). Characterization of a T-DNA insertion mutant for the protein import receptor at Toc33 from chloroplasts. Mol. Genet. Genom., 272, no. 4, pp. 379-396.

7. Gaspar, Y., Nam, J., Schultz, C., Lee, L., Gilson, P., Gelvin, S. & Bacic, A. (2004). Characterization of the Arabidopsis lysine-rich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation. Plant Physiol., 135, no. 4, pp. 2162-2171.

8. Muller, K., Heller, H. & Doerfier, W. (2001). Foreign DNA integration. Genome-wide perturbations of methylation and transcription in the recipient genomes. J. Biol. Chem., 276, pp. 14271-14278.

9. Matzke, A.J.M. & Matzke, M.A. (1998). Position effects and epigenetic silencing of plant transgenes. Curr. Opin. Plant Biol., 1, pp. 142-148.

10. Matzke, M.A., Mette, M.F. & Matzke, A.J.M. (2000). Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol. Biol., 43, pp. 401-415.

11. Kidwell, M.G. & Lisch, D.R. (2000). Transposable elements and host genome evolution, Trends Ecol. Evol., 15, pp. 95-99.

12. Todorovska, E. (2007). Retrotransposons and their role in plant-genome evolution, Biotechnol. Equip., 21, pp. 294-305.

13. Kalendar, R. & Schulman, A. (2006). IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nat. Protoc., 1, no. 5, pp. 2478-2484.

14. Leigh, F., Kalendar, R., Lea, V., Lee, D., Donini, P. & Schulman, A. (2003). Comparison of the utility of barley retrotransposon families for genetic analysis by molecular marker techniques. Mol. Gen. Genom., 269, pp. 464-474.

15. Schnell, J., Steele, M., Bean, J., Neuspiel, M., Girard, C., Dormann, N., Pearson, C., Savoie, A., Bourbonniere, L. & Macdonald, P. (2015). A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments. Transgen. Res., 24, no. 1, pp. 1-17.

16. Kaya, Y., Yilmaz, S., Gozukirmizi, N. & Huyop, F. (2013). Evaluation of transgenic Nicotiana tabacum with dehE gene using transposon based IRAP markers. Am. J. Plant Sci., 4, no. 8A, pp. 41-44.

17. Rao, J., Yang, L., Guo, J., Quan, S., Chen, G., Zhao, X., Zhang, D. & Shi, J. (2016). Development of event-specific qualitative and quantitative PCR detection methods for the transgenic maize BVLA430101. Eur. Food Res.Technol., 242, no. 8, pp. 1277-1284.

18. Bavol, A.V., Dubrovna, O.V. & Morgun, B.V. (2013). Genetic transformation and analysis of wheat transgenic cell lines by IRAP-PCR. Biotechnol. Acta, 6, no. 6, pp. 113-119 [in Ukrainian].

19. Wu, R., Guo, W., Wang, X., Wang, X., Zhuang, T., Clarke, J. & Liu, B. (2009). Unintended consequence of plant transformation: biolistic transformation caused transpositional activation of an endogenous retrotransposon Tos17 in rice ssp. japonica cv. Matsumae. Plant Cell Rep., 28, no. 7, pp. 1043-1051. https//

20. Yuzbasioglu, G., Marakli, S. & Gozukirmizi, N. (2017). Screening of Oryza sativa L. for hpt gene and evaluation of hpt positive samples using Houba retransposonbased IRAP markers. Turk. J. Agric. Res., 4, no. 1, pp. 59-64.

21. Bavol, A.V., Dubrovna, O.V., Goncharuk, O.M. & Voronova, S.S. (2014). Agrobacterium-mediated transformation of wheat using calli culture, Fakt. Eksp. Evol. Organism., 15, pp. 16-19 [in Ukrainian].

22. Trebichalsky, A., Kalendar, R., Schulman, A., Stratula, O., Galova, Z., Balazova, Z. & Chnapek, M. (2013). Detection of genetic relationships among spring and winter triticale (Triticosecale Witt.) and rye cultivars (Secale cereale L.) by using retrotransposon-based markers. Czech J. Genet. Plant Breed., 49, pp. 171-174.

23. Bavol, A.V., Velikozhon, L.G., Pykalo, S.V. & Dubrovna, O.V. (2016). IRAP-analysis of triticale plants regenerants, resistant to water deficit. Fakt. Eksp. Evol. Organism., 19, pp. 73-78 [in Ukrainian].

24. Bayram, E., Yilmaz, S. & Hamat-Mecbur H. (2012). Nikita retrotransposon movements in callus cultures of barley (Hordeum vulgare L.). Plant OMICS: Journal of Plant Molecular Biology and Omics., 5, no. 3, pp. 211-217.

25. Bavol, A.V., Lyalko, I.I., Voronova, S.S., Goncharuk, O.M. & Dubrovna, O.V. (2015). The course of meiosis in genetically modified wheat plants obtained by Agrobacterium - mediated transformation. Fiziol. rast. genet., 47, no. 6, pp. 536-544 [in Ukrainian].

26. Bhattm, A.M., Lister, C., Crawford, N. & Dean, C. (1998). The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines. Plant Cell, no. 10, pp. 427-434.

27. Casacuberta, J.M. & Santiago, N. (2003). Plant LTR-retrotransposons and MITEs: control of transposition and impact on the evolution of plant genes and genomes. Gene, 311, pp. 1-11.

28. Lister, R., O'Malley R., Tonti-Filippini, J., Gregory, B., Berry, C., Miller A. & Ecker, J. (2008). Highly integrated singlebase resolution maps of the epigenome in Arabidopsis. Cell, 133, pp. 523-536.

29. Choulet, F., Wicker, T., Rustenholz, C., Paux, E., Salse, J., Leroy, P., Schlub, S., Le Paslier, M., Magdelenat, G., Gonthier, C., Couloux, A., Budak, H., Breen, J., Pumphrey, M., Liu, S., Kong, X., Jia, J., Gut, M., Brunel, D., Anderson, J., Gill, B., Appels, R., Keller, B. & Feuillet, C. (2010). Megabase level sequencing reveals contrasted organization and evolution patterns of the wheat gene and transposable element spaces. Plant Cell, 22, no. 6, pp. 1686-1701.

30. Vicient, C.M. (2010). Transcriptional activity of transposable elements in maize, BMC. Genomics, 11, no. 601, pp. 1-10.

31. Martienssen, R.A. & Colot, V. (2001). DNA methylation and epigenetic inheritance in plants and filamentous fungi. Science, 293, pp. 1070-1074.

32. Mello, C.C. & Conte, D., Jr. (2004). Revealing the world of RNA interference. Nature, 431, pp. 338-342.

33. Meister, G. & Tuschl, T. (2004). Mechanisms of gene silencing by double-stranded RNA. Nature, 431, pp. 343-349.

34. Verdel, A., Jia, S., Gerber, S., Sugiyama, T., Gygi, S., Grewal, S.I. & Moazed, D., (2004). RNAi-mediated targeting of heterochromatin by the RITS complex. Science, 303, no. 5658, pp. 672-676.

35. Mette, M.F., Aufsatz, W., van der Winden, J., Matzke, M.A. & Matzke, A.J., (2000). Transcriptional silencing and promoter methylation triggered by double stranded RNA. EMBO J., pp. 5194-5201.

36. Gvozdev, V.A. (2003). Mobile genes and RNA interference. Genetics, 39, pp. 151-156. [in Russian].

37. Makarova, Yu.A. & Cramers, D.A. (2007). Noncoding RNA. Biochemistry, 72, no. 11, pp. 1427-1448 [in Russian].

38. Alder, M.N., Dames, S., Gaudet, J. & Mango, S.E. (2003). Gene silencing in Caenorhabditis elegans by transitive RNA interference. RNA, 9, pp. 25-32.

39. Sijen, T., Fleenor, J., Simmer, F., Thijssen, K.L., Parrish, S., Timmons, L., Plasterk, R.H. & Fire, A. (2001). On the role of RNA amplification in dsRNA-triggered gene silencing. Cell, 107, pp. 465-476.