Homologs of the rolC gene of naturally transgenic toadflaxes Linaria vulgaris and Linaria creticola are expressed in vitro

Agrobacterium mediated transformation is the most common way for obtaining transgenic plants in laboratory conditions. At the same time, there are species inside the genera Nicotiana, Linaria and Ipomoea that contain homologs of agrobacterial T-DNA genes as a result of genetic transformation of their ancestral forms in natural conditions. Such plants are called naturally transgenic plants, and T-DNA in their genomes is called cellular (cT-DNA). It is proposed that in the evolution of these genera, the introduced sequences played an important role. This idea is confirmed by the data on the expression of some T-DNA genes in Nicotiana and Ipomoea. Until the last moment, the expression of cT-DNA genes in Linaria has not been documented. However, the analysis of the nucleotide sequence indicates the functionality of rolC gene in L. vulgaris Mill., L. acutiloba Fisch. ex Rchb., L. genistifolia (L.) Mill. In this research work, we have sequenced the rolC homolog in one more toadflax species (Linaria creticola Kuprian). The in silico analysis of this gene has shown that it can encode a full-length peptide. Using the real time RT-PCR method, we have demonstrated that the rolC homolog is expressed in vitro in shoots, roots and calli of L. vulgaris Mill., as well as in shoots of L. creticola Kuprian. The results obtained are an important argument in favor of the fact that cT-DNA is functional and that its fixation in genomes played a certain role in the evolutionary process. However, the level of expression of the gene studied is quite low. A similar trend was observed in other naturally transgenic species. This can explain the absence of explicit morphological differences of species containing cT-DNA from their non-transgenic relatives.

Linaria, cT-DNA, rolC, transgene expression, in vitro

1. Chen K., Dorlhac de Borne F., Szegedi E., Otten L. Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T-DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana. Plant J. 2014;80(4):669-682.

2. Chen K., Otten L. Natural agrobacterium transformants, recent results and some theoretical considerations. Front. Plant Sci. 2017. DOI 10.3389/fpls.2017.01600.

3. Draper J., Scott R., Armitage P., Walden R. Plant Genetic Transformation and Gene Expression: A Laboratory Manual. Oxford: Blackwell Sci., 1988. (Russ. ed.: Dreyper Dzh., Skott R., Armitidzh F., Uolden R. Gennaya inzheneriya rasteniy. Laboratornoe rukovodstvo. Moscow: Mir Publ., 1991).

4. Furner I.J., Huffman G.A., Amasino R.M., Garfinkel D.J., Gordon M.P., Nester E.W. An Agrobacterium transformation in the evolution of the genus Nicotiana. Nature. 1986;319(6052):422-427.

5. Intrieri M.C., Buiatti M. The horizontal transfer of Agrobacterium rhizogenes genes and the evolution of the genus Nicotiana. Mol. Phylogenet. Evol. 2001;20:100-110. DOI 10.1006/mpev.2001.0927.

6. Kumar S., Stecher G., Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016;33:1870-1874. DOI 10.1093/molbev/msw054.

7. Kyndt T., Quispe D., Zhai H., Jarret R., Ghislain M., Liu Q., Gheysen G., Kreuze J.F. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop. Proc. Natl. Acad. Sci. USA. 2015; 112(18):5844-5849. DOI 10.1073/pnas.1419685112.

8. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). method. Methods. 2001;25(4):402-408.

9. Lutova L.A., Bondarenko L.V., Buzovkina I.S., Levashina E.A., Tikhodeyev O.N., Khodjayova L.T., Sharova N.V., Shishkova S.O. Role of the genotype in plant regeneration processes. Genetika = Genetics (Moscow). 1994;30(8):1065-1074. (in Russian)

10. Matveeva T.V., Bogomaz D.I., Pavlova O.A., Nester E.W., Lutova L.A., Horizontal gene transfer from genus Agrobacterium to the plant Linaria in nature. Mol. Plant Microbe Interact. 2012;25:1542-1551. DOI 10.1094/MPMI-07-12-0169-R.

11. Matveeva T.V., Lutova L.A. Horizontal gene transfer from Agrobacterium to plants. Front. Plant. Sci. 2014;5:326. DOI 10.3389/fpls. 2014.00326.

12. Matveeva T.V., Sokornova S.V. Biological traits of naturally transgenic plants and their evolutional roles. Russ. J. Plant Physiol. 2017;64: 635-648. DOI 10.1134/S1021443717050089.

13. Meyer A.D., Ichikawa T., Meins F. Horizontal gene transfer: regulated expression of tobacco homologue of the Agrobacterium rhizogenes rolC gene. Mol. Gen. Genet. 1995;249:265-273. DOI 10.1007/ BF00290526.

14. Murashige T., Skoog F. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 1962;15:165-170.

15. Nagata N., Kosono S., Sekine M., Shinmyo A., Syono K. Different expression patterns of the promoters of the NgrolB and NgrolC genes during the development of tobacco genetic tumors. Plant Cell Phys. 1996;37:489-498. DOI 10.1093/oxfordjournals.pcp.a028971.

16. Terentyev P.V., Rostova N.S. Praktikum po biometrii [Workshop on Biometrics]. Leningrad: LGU Publ., 1977. (in Russian)

17. Vain P. Thirty years of plant transformation technology development. Plant Biotechnol. J. 2007;5:221-229. DOI 10.1111/j.1467-7652.2006. 00225.x.

18. White F.F., Garfinkel D.J., Huffman G.A., Gordon M.P., Nester E.W. Sequence homologous to Agrobacterium rhizogenes T-DNA in the genomes of uninfected plants. Nature. 1983;301:348-350.

19. www.plantarium.ru. Accessed on 1.09.2017.

20. www.isaaa.org. Accessed on 3.09.2017.