ISSN 16076729, Doklady Biochemistry and Biophysics, 2015, Vol. 463, pp. 264–267. © Pleiades Publishing, Ltd., 2015. Original Russian Text © M.A. Filyushin, E.Z. Kochieva, K.G. Skryabin , 2015, published in Doklady Akademii Nauk, 2015, Vol. 463, No. 6, pp. 730–733.
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
5.8S rRNA Sequence and Secondary Structure in Monotropa hypopitys and Related Ericaceae Species M. A. Filyushin, E. Z. Kochieva, and Academician K. G. Skryabin Received April 10, 2015
Abstract—This is the first study to investigate the secondary structure of 5.8S rRNA in M. hypopitys and related Ericaceae species. The identified nucleotide substitutions are localized mostly at the 3' and 5' ends of the gene, in the region of the third hairpin, and do not significantly affect the topology of the secondary struc ture of the 5.8S rRNA molecule. DOI: 10.1134/S1607672915040183
Family Ericaceae includes approximately 120 gen era comprising more than 4000 species of grasses, shrubs, and trees, which are widespread around the world. The variety of plants of this family includes a number of berry (e.g., arbutus, cranberries, blueber ries, and cowberries) and ornamental (heather, rhodo dendron, labrador tea, etc.) cultures. This family is interesting because it also includes eight genera of chlorophyllfree mycoheterotrophic plants combined into the tribe Monotropeae, which are spread primarily in North America. In the Euro pean part of Russia, tribe Monotropeae is represented by the species Monotropa hypopitys L. s.l., or the pine sap. Another species, Monotropa uniflora, grows in the Far East. The pinesap is of special scientific interest because of its parasitic lifestyle. Through haustoria on the rhizome it connects to the mycorrhiza of fungi (mostly of the genus Tricholoma) [1, 2], which, in turn, is connected to the root system of trees. This allows the pinesap to get nutrients and minerals not only from fungi but also from trees. In the genomes of higher plants, ribosomal RNA genes form the tandemly repeated cistron 18SITS1 5.8SITS226S [3]. Ribosomal RNA molecules form specific secondary structures that bind to ribosomal proteins. The resulting riboprotein complexes are recruited during the assembly of ribosomes and subse quent translation. The secondary structure of the 5.8S rRNA gene consists of four loops (1a, 1b, 2, and 3) [4] and contains three highly conserved motifs [5]. Loop 1b contains the 14nt motif inherent in all higher plants, which binds to the ribosomal protein during the assembly of the large ribosomal subunit, and two other motives that are required for maintaining the
Bioengineering Center, Russian Academy of Sciences, pr. 60letiya Oktyabrya 7/1, Moscow, 117312 Russia email:
[email protected],
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correct secondary structure of the 5.8S RNA molecule in a 3D environment [5]. The aim of this work was to analyze the 5.8S rRNA gene sequence and to study the possible influence of nucleotide substitutions on the topology of the sec ondary structure of the 5.8S rRNA molecule in M. hypopitys and related Ericaceae species. As a result, we for the first time studied the 5.8S rRNA gene sequence of the members of the family Ericaceae and determined the probable secondary structure of the 5.8S rRNA molecule. To analyze the 5.8S rRNA gene sequences in the representatives of Ericaceae, accessions of chloro phyllfree parasitic species of three genera of the tribe Monotropeae Pterospora andromedea (California, United States), Monotropastrum humile (Akita Prefec ture, Japan), Monotropa uniflora (Delaware, United States), and Monotropa hypopitys were chosen from the collection of the Tsitsin Main Botanical Garden, Russian Academy of Sciences. M. hypopitys was repre sented by accessions of seven remote populations reflecting the area of distribution of this species in the European part of Russia and Ukraine: Belgorod (#1), Vologda (#2), Kaluga (#3), and Tver (#4) regions, the Republic of Bashkortostan (#5), Crimea (#6), and Transcarpathia (#7). In addition, we analyzed the chlorophyllcontaining species of four other genera of the family Ericaceae (Arbutus andrachne (Greek strawberry tree), Calluna vulgaris (heather), Pyrola rotundifolia (wintergreen), Vaccinium myrtillus (black berries), and Vaccinium oxycoccos (cranberry). From the fresh plant tissues, DNA was extracted by the CTAB method with double purification with chlo roform [6]. From the herbarium specimens, DNA was extracted using the Plant/Seed DNA MicroPrep kit (ZYMO RESEARCH, United States). The ITS1 5.8SITS2 sequences were amplified using the stan dard PCR primers ITS4 (TCCTCCGCTTAT TGATATGC) and ITS5 (GGAAGTAAAAGTCG TAACAAG) [7]. The amplicons were sequenced using
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Fig. 1. The 5.8S rRNA gene sequence in the Ericaceae samples analyzed.
the Bigdye system (Applied Biosystems, United States) and ABI PRISM 310 analyzer (Applied Bio systems). Alignment, analysis of the derived nucle otide sequences, and cluster analysis were performed using MEGA 6.0 software [8]. The probable secondary structure of the 5.8S rRNA molecule was constructed using mFold software (http://mfold.rit.albany.edu/). ITS15.8SITS2 sequences were amplified and sequenced, and the boundaries of the ITS spacers and the 5.8SrRNA gene were determined. The length of the nucleotide sequence of the 5.8S rRNA gene in the accessions of M. hypopitys, M. uniflora, M. humile, R. andromedea, and V. oxycoccos was 159 bp, whereas in the species A. andrachne, C. vulgaris, P. rotundifolia, and V. myrtillus is was 163 bp, which was due to the presence of a fournucleotide AACG insertion at the beginning of the gene (Fig. 1). The GCcomposition of the sequences ranged from 47.2 to 53.4%, which, in general, was consistent with the data for the 5.8S rRNA gene of different plant groups obtained previ ously [9]. In total, 20 variable sites in the 5.8S rRNA gene sequence were detected in the samples analyzed. In the seven accessions of M. hypopitys, representing highly remote populations, the nucleotide sequence of this gene was invariable. M. hypopitys differed from the related species M. uniflora by a number of specific nucleotide substitutions (C/T12, C/T103, C/T148). Despite the fact that both species, M. hypopitys and M. uniflora, belong to the same genus, the 5.8S rRNA sequence of M. uniflora was more similar and had common nucleotide substitutions with the species Monotropastrum humile (T/C1, G/A138, and C/T152), DOKLADY BIOCHEMISTRY AND BIOPHYSICS
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which is also included in the tribe Monotropeae and looks very similar to M. uniflora. The 5.8S rRNA sequence analysis has not revealed any specific substitutions or indels typical of chloro phyllfree parasitic species of the family Ericaceae. Moreover, for all species analyzed, except M. hypopi tys, common SNPs were identified (Fig. 1). In the heather C. vulgaris, the CA dinucleotide sub stitution was detected at the 5' end of the 5.8S rRNA gene sequence. The analysis of the NCBI database showed that CA at the 5' end of the 5.8S rRNA sequence is quite rare. The same beginning of the gene was found in Lissanthe sapida (KC197100), an Austra lian endemic species of the family Ericaceae. The tet ranucleotide AACG at the beginning of the 5.8S rRNA gene is absent in all analyzed chloropohyllfree Ericaceae and in the cranberry V. oxycoccos. However, this insertion is present in the blueberry Vaccinium myrtillus and in the majority of species of the genus Vaccinium [10]. In all Ericaceae species analyzed, three highly con served motifs were identified in the 5.8S rRNA gene sequences (the first motive—CGATGAAGAACG TAGC (16 bp), the second motive—GAATTGCA GAATCC (14 bp), and the third motive— TTTGAAYGCA (10 bp)), which are common to all higher plants [5]. The identified nucleotide substitu tions in the 5.8S rRNA gene in the analyzed samples of the family Ericaceae do not affect the conserved motifs and are located primarily in the 3' and 5' ends of the 5.8S rRNA gene. Using mFold software, the probable secondary structure of the 5.8S rRNA gene was constructed. 2015
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A. andrachne C. vulgaris P. rotundifolia V. myrtillus V. oxycoccos
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A. andrachne C. vulgaris P. rotundifolia G123 V. myrtillus C118 V. oxycoccos
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Fig. 2. The secondary structure of the 5.8S rRNA molecule in M. hypopitys.
When constructing the secondary structure of the 5.8S rRNA gene sequence, the sequence of M. hypopitys was used as a consensus sequence. Figure 2 shows the identified highly conserved motifs and the nucleotide substitutions found in other analyzed representatives of the family Ericaceae. Currently, the secondary structure of the 5.8S rRNA gene has been described only for a limited number of plant species, primarily in some gymnosperm groups [9] and in a number of flow ering plants [9, 11]. In general, the topology of the sec ondary structure of the 5.8S rRNA molecule in M. hypopitys and other Ericaceae species analyzed is similar to that described previously for the representa tives of the genus Poa (family Poaceae) [4], genus Allium (family Alliaceae) [11], and genus Cycas [12]. The greatest number of nucleotide substitutions in the studied samples was detected in the third hairpin region; the secondary structure of this hairpin on the whole remains unchanged and is maintained by four GC pairs. Such nucleotide variability of the third hair
pin is apparently due to the fact that it contains no domains or motifs that have a functional significance for the assembly of ribosomes. In M. uniflora, a single nucleotide substitution (C/U99) was found at the base of loops 1a and 1b, which has no effect on the topology of the secondary structure of the 5.8S rRNA molecule. Thus, in our work we for the first time investigated the secondary structure of 5.8S rRNA in the pinesap M. hypopitys and related Ericaceae species. The iden tified nucleotide substitutions are located primarily in the 3' and 5' ends of the gene, in the third hairpin region, and have no significant effect on the topology of the secondary structure of the 5.8S rRNA molecule. ACKNOWLEDGMENTS This study was performed using the scientific equipment of the Bioengineering Shared Access Cen ter, with the financial support by the Russian Science
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Foundation (project no. 142400175 “Genome Study of Molecular Adaptation”).
7. White, T.J., Bruns, T., Lee, S., and Taylor, J., PCR Pro tocols: A Guide to Methods and Applications, 1990, pp. 315–322.
REFERENCES
8. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S., Mol. Biol. Evol., 2013, vol. 30, no. 12, pp. 2725–2729.
1. Leake, J.R., McKendrick, S.L., Bidartondo, M., and Read, D.J., New Phytol., 2004, vol. 163, pp. 405–423. 2. Min, S., ChangQin, Z., YongPeng, M., Welti, S., Moreau, P., and Selosse, M., Symbiosis, 2012, vol. 57, pp. 1–13. 3. Sone, T., Fujisawa, M., Takenaka, M., et al., Plant Mol. Biol., 1999, vol. 41, pp. 679–685. 4. Nosov, N.N. and Rodionov, A.V., Bot. Zh., 2008, vol. 93, no. 12, pp. 1919–1936. 5. Harpke, D. and Peterson, A., Plant Res., 2008, vol. 121, pp. 261–270. 6. Puchooa, D., Int. J. Agric. Biol., 2004, vol. 6, pp. 884– 888.
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9. Won, H. and Renner, S.S., Mol. Phylogenet. Evol., 2005, vol. 36, pp. 581–597. 10. Tsutsumi, C., Bull. Nat. Mus. Nat. Sci. Ser., 2011, vol. 37, no. 2, pp. 79–86. 11. Filyushin, M.A. and Kochieva, E.Z., Russian Journal of Genetics, 2014, vol. 50, no. 10, pp. 1120–1124. 12. Xiao, L., Muller, M., and Zhu, H., Mol. Phylogenet. Evol., 2010, vol. 55, pp. 168–177.
Translated by M. Batrukova
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