Molecular genetic analysis of Rauvolfia serpentina tissue culture strain K-27M using the ITS1-5.8S-ITS2 region of 35S rRNA genes

DOI: https://doi.org/10.7124/visnyk.utgis.22.1-2.1685
Keywords: Rauvolfia serpentina Benth. ex Kurz, plant tissue culture, molecular-genetic markers, 35S ribosomal RNA genes, internal transcribed spacer

Abstract

Hormone-independent strain K-27M of Rauvolfia serpentina tissue culture, which is a valuable source of indoline alkaloids, was created in the Department of genetics of cell populations at the Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine. This strain differs significantly from R. serpentina plants in composition and content of alkaloids. Furthermore, there are no clear data on the plant material used to obtain this tissue culture strain. The aim of this study was to carry out species identification of tissue culture strain K-27M based on molecular genetic analysis of the ITS1-5.8S-ITS2 region of the 35S ribosomal RNA genes (35S rDNA). Methods. Polymerase chain reaction, cloning, sequencing, phylogenetic analysis. Results. The ITS1-5.8S-ITS2 region of the 35S rDNA of the K-27M strain was amplified with PCR using specific primers. Several clones were obtained, two of which were used for sequencing. The sequenced clones differed in length due to two deletions in one of them, as well as in nucleotide sequence. The presence of a deletion in the 18S rRNA gene region and numerous single nucleotide substitutions in the 18S and 5.8S rRNA gene regions in one of the clones may indicate that the 35S rRNA gene variant from which it was amplified is non-functional. Phylogenetic analysis using 26 sequences of the ITS1-5.8S-ITS2 region from 7 species of the genus Rauvolfia found in GenBank showed that the clones obtained from strain K-27M were grouped in a separate cluster together with other samples of R. serpentina. Conclusions. Based on molecular genetic analysis of the ITS1-5.8S-ITS2 region of the 35S rDNA, the tissue culture strain K-27M was found to belong to the species R. serpentina.

References

Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T.L. BLAST+: architecture and applications. BMC Bioinformatics. 2009. Vol. 10(1). P. 421. doi: 10.1186/1471-2105-10-421.

Deshmukh S.R., Ashrit D.S., Patil B.A. Extraction and evaluation of indole alkaloids from Rauwolfia serpentina for their antimicrobial and antiproliferative activities. International Journal of Pharmacy and Pharmaceutical Sciences. 2012. Vol. 4(Suppl. 5). P. 329–334.

Kovaleva T.A., Shamina Z.B., Butenko R.G. Cytological study of tissue culture of rauvolfia (Rauwolfia serpentina Benth.). Genetica. 1968. Vol. 4(15). P. 5–13. [In Russian].

Kumari R., Rathi B., Rani A., Tiwari S.M., Bhatnagar S. Rauvolfia serpentina L. Benth. ex Kurz.: Phytochemical, pharmacological and therapeutic aspects. International Journal of Pharmaceutical Sciences Review and Research. 2013. Vol. 23(2). P. 348–355.

Kunakh V.A. Biotechnology of medicinal plants. Genetic, physiological and biochemical basis. Kyiv: Logos, 2005. 730 p. [In Ukrainian].

Kunakh V.A., Konvalyuk I.I., Mozhylevska L.P., Bieda O.A., Twardovska M.O., Andreev I.O., Yarmolyuk S.M. Comprehensive study of hormone-independent highly productive strain of Rauvolfia serpentina tissue culture as a source of indole alkaloids. Biopolymers and Cell. 2023. Vol. 39(4). P. 283–298. doi: 10.7124/bc.000AA7.

Letsiou S., Madesis P., Vasdekis E., Montemurro C., Grigoriou M.E., Skavdis G., Moussis V., Koutelidakis A.E., Tzakos A.G. DNA Barcoding as a Plant Identification Method. Applied Sciences. 2024. Vol. 14(4). P. 1415. doi: 10.3390/app14041415.

Mishchenko A.M., Andreev I.O. Selection and optimization of the method for DNA isolation and purification from Corylus species for PCR analysis. Faktori eksperimental’noi evolucii organizmiv. 2023. Vol. 32. P. 53–58. doi: 10.7124/FEEO.v32.1535.

Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum. 1962. Vol. 15(3). P. 473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x.

Okonechnikov K., Golosova O., Fursov M., Varlamov A., Vaskin Y., Efremov I., German Grehov O.G., Kandrov D., Rasputin K., Syabro M., Tleukenov T. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics. 2012. Vol. 28(8). P. 1166–1167. doi: 10.1093/BIOINFORMATICS/BTS091.

Singh M., Kaur R., Rajput R., Mathur G. Evaluating the therapeutic efficiency and drug targeting ability of alkaloids present in Rauwolfia serpentina. International Journal of Green Pharmacy (IJGP). 2017. Vol. 11(03). P. 132. doi: 10.22377/IJGP.V11I03.1116.

Trifinopoulos J., Nguyen L.-T., von Haeseler A., Minh B.Q. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research. 2016. Vol. 44(W1). P. W232–W235. doi: 10.1093/nar/gkw256.

Vollosovich N.E., Vollosovich A.G., Kovaleva T.A., Shamina Z.B., Butenko R.G. Tissue culture strains of Rauwolfia serpentina Benth. and their productivity. Rastitel’nyye resursy. 1976. Vol. 12(4). P. 578–583. [In Russian].