The influence of structural and functional characteristics of lipid transfer protein on biological resistance of pinus sylvestris l
- Autores: Nataliya Hrunyk
- Directores de la Tesis: Hryhoriy Tomkovych Krynytskyy (dir. tes.), José Luis Caballero (dir. tes.)
- Lectura: En la Universidad de Córdoba (ESP) ( España ) en 2021
- Idioma: español
- Tribunal Calificador de la Tesis: Volodymyr Zaika (presid.), Myroslava Soroka (secret.), Jesús V. Jorrin Novo (voc.)
- Programa de doctorado: Programa de Doctorado en Ingeniería Agraria, Alimentaria, Forestal y del Desarrollo Rural Sostenible por la Universidad de Córdoba y la Universidad de Sevilla
- Materias:
- Enlaces
- Tesis en acceso abierto en: Helvia
- Resumen
1. introducción o motivación de la tesis Scots pine (Pinus sylvestris L.) is a valuable tree species in the boreal forests of the northern hemisphere. In Ukraine, Scots pine occupies 23 % of the total forest area, and in Polissya, the area of pine stands increases to 64.5 %, thus making pine a good and widely used wooden species for reforestation and afforestation. Besides the economic value, pine forests are of great ecological importance and are the habitat of many plant and animal species.
Together, global climate change and the high background of pollution pose a significant threat to forest biocenoses. Therefore, there is a need to improve the resistance of woody plants to phytopathogenic organisms and other adverse environmental factors on silvicultural and molecular genetic principles.
In recent decades, much research has focused on identifying a new generation of biologically active compounds that can be used to protect plants from diseases and insect damage. The main requirements for these substances are environmental safety and minimal sensitivity of the plant to them. A wide range of compounds with antimicrobial properties has been found in plants. In particular, proteins with pronounced antimicrobial activity were extracted from different organs of different plant species. Among them, a special place belongs to lipid transfer proteins (LTP), which have a wide range of biological activities. These include the ability of these proteins to bind fatty acids, which are components of the cell membrane, the inhibitory activity of amylase and proteases of insects and mammals, and participation in defense reactions during biotic and abiotic stresses. In this regard, the study of the functional characteristics of LTP in Scots pine will significantly expand the current understanding of this group of antimicrobial peptides and their involvement in the response of trees to phytopathogenic fungi, as well as to develop molecular genetic markers to assess genetically resistant pine species, which grow, in particular, in saline conditions and high levels of osmotic stress.
2.contenido de la investigación The aim of the work is to elucidate the structure of lipid transfer proteins of Scots pine, to obtain recombinant lipid transfer protein, and to reveal its functional features related to the biological resistance of trees under the influence of various environmental factors of biotic and abiotic nature. To achieve this goal, we set the following tasks: - to detect, clone and sequence genes of Scots pine lipid transfer proteins; - to conduct a multiple comparative analysis of Scots pine LTP sequences with other members of Pinaceae family and compare the results with the well studied LTP genes in angiosperms; - to obtain a recombinant Scots pine LTP in bacterial expression system; - to investigate the functional properties of PsLTP1: antibacterial and antifungal activity; lipid-binding activity; ability to inhibit amylases and proteases; - to determine the subcellular localization of fluorescently tagged PsLTP1 in transiently transformed Nicotiana benthamiana leaves; - to investigate the Scots pine LTPs (PsLTP1, PsLTP-B, PaLTP-D) expression patterns in different organs and at different stages of development of Scots pine; - to study the influence of stressors of biotic and abiotic origin on the levels of PsLTP genes expression; - to reveal the impact of growth conditions of the maternal organism on the seed material and changes in the expression of PsLTP genes at the transcription level caused by the heavy metal ions.
The object of research - seeds, embryos, seedlings, pine trees under the influence of various environmental factors of biotic and abiotic origins.
3.conclusión In this work, we identified the genes of Scots pine lipid transfer proteins and named them PsLTP. Coding sequences and intron-containing gene sequences of Scots pine lipid transfer proteins were cloned, sequenced, and deposited in the GenBank. In the databases of the National Center for Biotechnological Information - NCBI, 10 LTP genes of Scots pine were identified and phylogenetic affinity with LTP genes of other members of the Pinaceae family was demonstrated.
Peculiarities of PsLTP1, PsLTP-B, PsLTP-D gene transcription in vegetative and generative organs of Scots pine have been studied as well as transcript accumulation at different stages of plant ontogenesis. There were also investigated changes in the expression levels of lipid transfer protein genes in juvenile plants inoculated with phytopathogenic fungi such as Alternaria alternata (Fr.) Keissl., Fusarium solani (Mart.) Sacc., Phytophthora cinnamomi Rands and Heterobasidion annosum (Fr.) Bref. The effect of plant-growth regulators, heavy metals, and abiotic stressors on the transcription of PsLTP genes in juvenile plants have been studied. The PsLTP1 gene product has been described as a lipid-binding protein that binds unsaturated fatty acid molecules and methyl jasmonate jasminic acid in vitro.
Recombinant PsLTP1 has been shown to inhibit trypsin and insect amylases from the larvae of the May beetle (Melelontha melelontha L.) and the common pine sawfly (Diprion pini L.). Fluorescently tagged PsLTP1 has been accumulated in the epithelial cell membrane of transiently transformed Nicotiana benthamiana leaves.
The practical importance of the results obtained in this work lies in the possibility of using PsLTP genes as genetic markers for the detection of Scots pine genotypes, which will confer resistance to salinity, osmotic stress, excessive moisture and plant diseases. In addition, data on the ability of PsLTP1 to inhibit insect amylase and protease activity can be used to develop approaches to generate transgenic plants resistant to insect infestation. The obtained results are also of value for basic research on the development of the theory of plant membranogenesis and can be used in the educational process of the courses "Forest Pathology" and "Plant Physiology".
4. Bibliografía Part of the result of doctoral thesis are presented in next articles and conference papers 1. Hrunyk N., Kovaleva V., Krynytskyy H., Gout I., Amil-Ruiz F., Muñoz-Blanco J., Caballero J-L., Gout R. Molecular cloning and characterization of a lipid transfer protein gene (PsLTP1) from Pinus sylvestris (L.) Journal of Forestry Research. 2019, 30. P.1149-1158. https://doi.org/10.1007/s11676-018-0648-z 2. Hrunyk N.I., Gout R.T., Kovaleva V.A. Regulation of gene expression for defensins and lipid transfer protein in Scots pine seedlings by necrotrophic pathogen Alternaria alternata (Fr.) // Folia Forestalia polonica. – 2017, 59 (2): 152–158. https://doi.org/10.1515/ffp-2017-0015 3. Hrunyk N.I., Kovaleva V.A., Gout R.T. Expression of defensins and lipid transfer protein genes in Scots pine seedlings inoculated with necrotrophic and hemibiotrophic pathogens. Scientific Bulletin of UNFU. 2017, 27 (8): 105-110. https://doi.org/10.15421/40270817 4. Hrunyk N.I., Yusypovych Y., Kovaleva V.A., Gout R.T. Heterobasidion annosum root rot infection development in Scots pine and evaluation of the expression levels of lipid transfer protein and defensins in infected tissues. Scientific Bulletin of UNFU. 2015; 25 (8): 25-32. http://nltu.edu.ua/nv/Archive/2015/25_8/5.pdf 5. Hrunyk N.I., Kovaleva V.A., Gout R.T. Features of gene expression of a lipid transfer protein in pine seedlings during infection by annosum root rot. The Bulletin of Kharkiv National Agrarian University. Series Biology. Collected papers, 2013; 3 (30): 56-60. http://nbuv.gov.ua/UJRN/Vkhnau_biol_2013_3_8 (Article in Ukrainian) 6. Hrunyk N.I., Kovaleva V.A., Gout R.T. Patterns of expression of a lipid-transfer protein gene in organs of scots pine (Pinus sylvestris L.). Studia Biologica. 2012; 6(2): 151-160. 10.30970/sbi.0602.210 (Article in Ukrainian) 7. Hrunyk N.I., Shalovylo Yu.I., Kovaleva V.A., Gout R.T. Plant lipid transfer proteins: structure, function and biotechnological prospects. Scientific Bulletin of UNFU. 2011; 21 (8): 32-35. https://nv.nltu.edu.ua/Archive/2011/21_8/32_Gru.pdf (Article in Ukrainian) Conference papers and short communcation 1. Hrunyk N.I., Kovaleva V.A., Gout R.T. The effect of heavy metals treatment of scots pine seedlings on the expression patterns of three PsLTP genes. Forest, Science, Youth: VІ National scientific conference of students, masters, graduate students and young scientists. Book of abstracts – Zhytomyr, 22nd of November 2018: 301-302.
2. Hrunyk N.I., Kovaleva V.A., Gout R.T. Efficacy of recombinant PsLTP1 towards Scots pine pests. Forest, Science, Youth: VІ National scientific conference of students, masters, graduate students and young scientists. Book of abstracts – Zhytomyr, 22nd of November 2018: 303.
3. Hrunyk N.I., Yusypovych Y., Kovaleva V.A., Gout R.T. Developing of the infection process caused by root sponge in Scots pine and evaluation of the expression levels of the antimicrobial peptides in infected tissues Problems of forest phytopathology and mycology: Materials of the IX International Conference. Minsk: BSTU, 19-24th of October 2015: 75-77.
4. Hrunyk N.I., Amil-Ruiz F., Gout R.T., Caballero J.L.GATEWAY cloning and localization of lipid transfer protein from Scots pine in tobacco leaves Young people and progress of biology: XI International Conference of Undergraduate and Postgraduate Students. Book of abstracts – Lviv, 20-23d of April 2015: 503.
5. Hrunyk N.I., Amil-Ruiz F., Gout R.T., Caballero J.L. Characterization, expression and purification of a lipid transfer protein from Scots pine (Pinus sylvestris L). IV Congreso Científico de Investigadores en Formación de la Universidad de Córdoba. III Congreso Científico de Investigadores en Formación en Agroalimentación ceiA3. Poster – Córdoba, 18-19th of November 2014.
6. Hrunyk N.I., Kovaleva V.A., Gout R.T. Molecular cloning of a lipid transfer protein from Scots pine. X International Interdisciplinary Scientific Conference of Students and Young Scientists. Shevchenkivska Vesna 2012: Life Sciences. Book of abstracts – Kyiv, 19-23d of March 2012: 89-90. (Communication in Ukrainian) 7. Hrunyk N.I., Kovaleva V.A., Gout R.T. Organ-specific expression of a lipid transfer protein at different developmental stages of Scots pine. II International Conference. Regulation of the plants growth and development. Book of abstracts – Kharkiv, 11-13th of October 2011: 21-22. (Communication in Ukrainian)
