agronaukaАграрная наука Евро-Северо-ВостокаAgricultural Science Euro-North-East2072-90812500-1396FARC North-East10.30766/2072-9081.2024.25.3.368-378agronauka-1665Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИ: РАСТЕНИЕВОДСТВООRIGINAL SCIENTIFIC ARTICLES: PLANT GROWINGИзучение экспрессии генов целлюлозосинтаз у льна в процессе ростаStudy of the expression of cellulose synthase genes during plant growth of flaxhttps://orcid.org/0000-0001-9544-5528БазановТ. А.BazanovT. A.

Базанов Тарас Александрович, кандидат хим. наук, заведующий лабораторией молекулярно-генетических исследований и клеточной селекции, ведущий научный сотрудник

Комсомольский проспект, д. 17/56, г. Тверь, 170041

Taras A. Bazanov, PhD in Chemical Science, Head of the Laboratory of the molecular-genetic researches and cell selection, leading researcher

Komsomolsky prospect, 17/56, Tver, 170041

info@fnclk.ruhttps://orcid.org/0000-0002-0602-1211УщаповскийИ. В.UshapovskyI. V.

Ущаповский Игорь Валентинович, кандидат биол. наук, ведущий научный сотрудник лаборатории молекулярно-генетических исследований и клеточной селекции, заместитель директора по науке

Комсомольский проспект, д. 17/56, г. Тверь, , 170041

Igor V. Ushapovsky, PhD in Biological Science, leading  researcher, the Laboratory of the molecular-genetic  researches and cell selection, Deputy Director for Science

Komsomolsky prospect, 17/56, Tver, 170041

info@fnclk.ruhttps://orcid.org/0000-0003-4633-392XЛогиноваН. Н.LoginovaN. N.

Логинова Наталья Николаевна, научный сотрудник лаборатории молекулярно-генетических исследований и клеточной селекции

Комсомольский проспект, д. 17/56, г. Тверь, 170041

Natalya N. Loginova, researcher, the Laboratory of the molecular-genetic researches and cell selection

Komsomolsky prospect, 17/56, Tver, 170041

info@fnclk.ruhttps://orcid.org/0000-0001-6559-9577МининаЕ. В.MininaE. V.

Минина Екатерина Витальевна, аспирант, младший научный сотрудник лаборатории молекулярно-генетических исследований и клеточной селекции

Комсомольский проспект, д. 17/56, г. Тверь, 170041

Ekaterina V. Minina, postgraduate, junior researcher, the Laboratory of the molecular-genetic researches and cell selection

Komsomolsky prospect, 17/56, Tver, 170041

info@fnclk.ruhttps://orcid.org/0000-0001-7797-2578ВересоваП. Д.VeresovaP. D.

Вересова Полина Дмитриевна, младший научный сотрудник лаборатории молекулярно-генетических исследований клеточной селекции

Комсомольский проспект, д. 17/56, г. Тверь, 170041

Polina D. Veresova, junior researcher, the Laboratory of the molecular-genetic researches and cell selection

Komsomolsky prospect, 17/56, Tver, 170041

info@fnclk.ruФГБНУ «Федеральный научный центр лубяных культур»РоссияFederal Research Center for Bast Fiber CropsRussian Federation202426062024253368378Copyright © Базанов Т.А., Ущаповский И.В., Логинова Н.Н., Минина Е.В., Вересова П.Д., 20242024Базанов Т.А., Ущаповский И.В., Логинова Н.Н., Минина Е.В., Вересова П.Д.Bazanov T.A., Ushapovsky I.V., Loginova N.N., Minina E.V., Veresova P.D.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.agronauka-sv.ru/jour/article/view/1665

В селекции лубяных культур, направленной на создание высоковолокнистых сортов, важным является выявление закономерностей формирования волокна на молекулярном уровне, определение генотипспецифичности процессов синтеза целлюлозы, изучение эффективности работы генов, отвечающих за синтез целлюлозы на различных этапах онтогенеза растений. Целью работы являлось изучение экспрессии генов целлюлозосинтаз льна. Объектом исследования являлись 8 образцов льна (род Linum), отличающихся по содержанию волокна: 7 сортов Linum usitatissimum L., из которых 5 сортов льна-долгунца, 2 сорта льна масличного, один образец льна крупноцветкового (L. grandiflorum Desf.). Исследуемые сорта выращивали в растильной установке. Пробы растительного материала отбирали на 10-й, 20-й, 30-й, 40-й и 50-й дни после появления всходов. Проводили извлечение РНК, синтез кДНК. Были получены последовательности РНК CesA1, CesA4, CesA6, CesA7 и разработаны ген-специфические праймеры для ПЦР. Экспрессию генов определяли методом полуколичественной RT-PCR, продукты реакции разделяли по размеру с помощью 1,2%-го агарозного геля. На основании полученных данных было установлено наличие фазо- и генотипспецифичности в процессе экспрессии целлюлозосинтаз семейства CesA в стебле льна. Целлюлозосинтазы первичной клеточной стенки льна (CesA1, CesA6) проявляют динамику роста экспрессии до стадии «елочка», а затем экспрессия постепенно снижается к фазе наступления быстрого роста, тогда как целлюлозосинтазы вторичной клеточной стенки (CesA4, CesA7) характеризуются постоянным увеличением экспрессии от первых дней развития проростков до фазы быстрого роста.

During the selection of crops aimed at creating high-fiber varieties, it is important to determine the patterns of fiber formation at the molecular level and the genotype specificity of cellulose synthesis processes, to study the efficiency of the genes responsible for cellulose synthesis at various stages of plant ontogenesis. The aim of the work was to study the expression of flax cellulose synthase genes. The objects of the study were 8 samples of flax (genus Linum), differing in fiber content: 7 cultivarеs of Linum usitatissimum L., of which 5 cultivarеs of fiber flax, 2 cultivarеs of oilseed flax, as well as one sample of large-flowered flax (L. grandiflorum Desf.). The studied cultivar were grown in a growth chamber. Samples of plant material were taken on the 10th, 20th, 30th, 40th and 50th day after germination. RNA extraction and cDNA synthesis were carried out. RNA sequences of CesA1, CesA4, CesA6, CesA7 were obtained and gene-specific primers for PCR were developed. Gene expression was determined by semi-quantitative RT-PCR, and reaction products were separated by size using a 1.2% agarose gel. Based on the data obtained, the presence of phase- and genotype-specificity in the process of expression of cellulose synthases of the CesA family in the flax stem was established. Cellulose synthases of the primary cell wall of flax (CesA1, CesA6) show a dynamic increase in expression up to the «herringbone» stage, and then the expression gradually decreases towards the onset of rapid growth phase, while cellulose synthases of the secondary cell wall (CESA4, CESA7) are characterized by a constant increase in expression from the first days of the development of seedlings to the phase of rapid growth.

лён-долгунецлен масличныйлен крупноцветковыйпервичная клеточная стенкавторичная клеточная стенкаCesAРНКкДНКПЦРгель-электрофорезflaxlinseedlarge-flowered flaxprimary cell wallsecondary cell wallCesARNAcDNAPCRgel electrophoresisработа выполнена при поддержке Минобрнауки РФ в рамках государственного задания ФГБНУ «Федеральный научный центр лубяных культур» (тема № FGSS-2024-0002).the research was carried out under the support of the Ministry of Science and Higher Education of the Russian Federation within the state assignment of the Federal Research Center for Bast Fiber Crops (theme No. FGSS-2024-0002).ReferencesDhaliwal J. S. Natural fibers: applications. Generation, development and modifications of natural fibers. London: IntechOpen, 2020. Pp. 1–23. DOI: https://doi.org/10.5772/intechopen.86884Dhaliwal J. S. Natural fibers: applications. Generation, development and modifications of natural fibers. London: IntechOpen, 2020. Pp. 1–23. DOI: https://doi.org/10.5772/intechopen.86884Mohite S., Patil C. K. Linseed Biodiesel – A Review. FLAME 2022: Advances in Fluid and Thermal Engineering. Lecture Notes in Mechanical Engineering. Singapore: Springer, 2023. Pp. 131–141. DOI: https://doi.org/10.1007/978-981-99-2382-3_11Mohite S., Patil C. K. Linseed Biodiesel – A Review. FLAME 2022: Advances in Fluid and Thermal Engineering. Lecture Notes in Mechanical Engineering. Singapore: Springer, 2023. Pp. 131–141. DOI: https://doi.org/10.1007/978-981-99-2382-3_11Haag K., Padovani J., Fita S., Trouvé J., Pineau C., Hawkins S., De Jong H., Deyholos M., Chabbert B., Müssig J., Beaugrand J. Influence of flax fibre variety and year-to-year variability on composite properties. Industrial Crops and Products. 2017;98:1–9. DOI: https://doi.org/10.1016/j.indcrop.2016.12.028Haag K., Padovani J., Fita S., Trouvé J., Pineau C., Hawkins S., De Jong H., Deyholos M., Chabbert B., Müssig J., Beaugrand J. Influence of flax fibre variety and year-to-year variability on composite properties. Industrial Crops and Products. 2017;98:1–9. DOI: https://doi.org/10.1016/j.indcrop.2016.12.028Pisupati A., Willaert L., Goethals F., Uyttendaele W., Park C. H. Variety and growing condition effect on the yield and tensile strength of flax fibers. Industrial crops and products. 2021;170:113736. DOI: https://doi.org/10.1016/j.indcrop.2021.113736Pisupati A., Willaert L., Goethals F., Uyttendaele W., Park C. H. Variety and growing condition effect on the yield and tensile strength of flax fibers. Industrial crops and products. 2021;170:113736. DOI: https://doi.org/10.1016/j.indcrop.2021.113736Kvavadze E., Bar-Yosef O., Belfer-Cohen A., Boaretto E., Jakeli N., Matskevich Z., Meshveliani T. 30,000-year-old wild flax fibers. Science. 2009;325(5946):1359. DOI: https://doi.org/10.1126/science.1175404Kvavadze E., Bar-Yosef O., Belfer-Cohen A., Boaretto E., Jakeli N., Matskevich Z., Meshveliani T. 30,000-year-old wild flax fibers. Science. 2009;325(5946):1359. DOI: https://doi.org/10.1126/science.1175404Gorshkova T., Chernova T., Mokshina N., Ageeva M., Mikshina P. Plant ‘muscles’: fibers with a tertiary cell wall. New Phytologist. 2018;218(1):66–72. DOI: https://doi.org/10.1111/nph.14997Gorshkova T., Chernova T., Mokshina N., Ageeva M., Mikshina P. Plant ‘muscles’: fibers with a tertiary cell wall. New Phytologist. 2018;218(1):66–72. DOI: https://doi.org/10.1111/nph.14997Ageeva M., Petrovská B., Kieft H., Sal’nikov V., Snegireva A., Van Dam J., Van Veenendaal W., Emons A., Gorshkova T., Van Lammeren A. Intrusive growth of flax phloem fibers is of intercalary type. Planta. 2005;222:565–574. DOI: https://doi.org/10.1007/s00425-005-1536-2Ageeva M., Petrovská B., Kieft H., Sal’nikov V., Snegireva A., Van Dam J., Van Veenendaal W., Emons A., Gorshkova T., Van Lammeren A. Intrusive growth of flax phloem fibers is of intercalary type. Planta. 2005;222:565–574. DOI: https://doi.org/10.1007/s00425-005-1536-2Snegireva A., Chernova T., Ageeva M., Lev-Yadun S., Gorshkova T. Intrusive growth of primary and secondary phloem fibres in hemp stem determines fibre-bundle formation and structure. AoB Plants. 2015;7:plv061. DOI: https://doi.org/10.1093/aobpla/plv061Snegireva A., Chernova T., Ageeva M., Lev-Yadun S., Gorshkova T. Intrusive growth of primary and secondary phloem fibres in hemp stem determines fibre-bundle formation and structure. AoB Plants. 2015;7:plv061. DOI: https://doi.org/10.1093/aobpla/plv061Mokshina N., Chernova T., Galinousky D., Gorshkov O., Gorshkova T. Key stages of fiber development as determinants of bast fiber yield and quality. Fibers. 2018;6(2):20. DOI: https://doi.org/10.3390/fib6020020Mokshina N., Chernova T., Galinousky D., Gorshkov O., Gorshkova T. Key stages of fiber development as determinants of bast fiber yield and quality. Fibers. 2018;6(2):20. DOI: https://doi.org/10.3390/fib6020020Gorshkova T., Mokshina N., Chernova T., Ibragimova N., Salnikov V., Mikshina P., Tryfona T., Banasiak A., Immerzeel P., Dupree P., Mellerowicz E. Aspen tension wood fibers contain β-(1→ 4)-galactans and acidic arabinogalactans retained by cellulose microfibrils in gelatinous walls. Plant Physiology. 2015;169(3):2048–2063. DOI: https://doi.org/10.1104/pp.15.00690Gorshkova T., Mokshina N., Chernova T., Ibragimova N., Salnikov V., Mikshina P., Tryfona T., Banasiak A., Immerzeel P., Dupree P., Mellerowicz E. Aspen tension wood fibers contain β-(1→ 4)-galactans and acidic arabinogalactans retained by cellulose microfibrils in gelatinous walls. Plant Physiology. 2015;169(3):2048–2063. DOI: https://doi.org/10.1104/pp.15.00690Lampugnani E. R., Flores-Sandoval E., Tan Q. W., Mutwil M., Bowman J. L., Persson S. Cellulose synthesis – central components and their evolutionary relationships. Trends in Plant Science. 2019;24(5):402–412. DOI: https://doi.org/10.1016/j.tplants.2019.02.011Lampugnani E. R., Flores-Sandoval E., Tan Q. W., Mutwil M., Bowman J. L., Persson S. Cellulose synthesis – central components and their evolutionary relationships. Trends in Plant Science. 2019;24(5):402–412. DOI: https://doi.org/10.1016/j.tplants.2019.02.011Daras G., Templalexis D., Avgeri F., Tsitsekian D., Karamanou K., Rigas S. Updating insights into the catalytic domain properties of plant cellulose synthase (CesA) and cellulose synthase-like (Csl) proteins. Molecules. 2021;26(14):4335. DOI: https://doi.org/10.3390/molecules26144335Daras G., Templalexis D., Avgeri F., Tsitsekian D., Karamanou K., Rigas S. Updating insights into the catalytic domain properties of plant cellulose synthase (CesA) and cellulose synthase-like (Csl) proteins. Molecules. 2021;26(14):4335. DOI: https://doi.org/10.3390/molecules26144335Wilson T. H., Kumar M., Turner S. R. The molecular basis of plant cellulose synthase complex organisation and assembly. Biochemical Society Transactions. 2021;49(1):379–391. DOI: https://doi.org/10.1042/BST20200697Wilson T. H., Kumar M., Turner S. R. The molecular basis of plant cellulose synthase complex organisation and assembly. Biochemical Society Transactions. 2021;49(1):379–391. DOI: https://doi.org/10.1042/BST20200697Haigler C. H., Roberts A. W. Structure/function relationships in the rosette cellulose synthesis complex illuminated by an evolutionary perspective. Cellulose. 2019;26:227–247. DOI: https://doi.org/10.1007/s10570-018-2157-9Haigler C. H., Roberts A. W. Structure/function relationships in the rosette cellulose synthesis complex illuminated by an evolutionary perspective. Cellulose. 2019;26:227–247. DOI: https://doi.org/10.1007/s10570-018-2157-9Purushotham P., Ho R., Zimmer J. Architecture of a catalytically active homotrimeric plant cellulose synthase complex. Science. 2020;369(6507):1089–1094. DOI: https://doi.org/10.1126/science.abb2978Purushotham P., Ho R., Zimmer J. Architecture of a catalytically active homotrimeric plant cellulose synthase complex. Science. 2020;369(6507):1089–1094. DOI: https://doi.org/10.1126/science.abb2978Roach M. J., Deyholos M. K. Microarray analysis of flax (Linum usitatissimum L.) stems identifies transcripts enriched in fibre-bearing phloem tissues. Molecular Genetics and Genomics. 2007;278:149–165. DOI: https://doi.org/10.1007/s00438-007-0241-1Roach M. J., Deyholos M. K. Microarray analysis of flax (Linum usitatissimum L.) stems identifies transcripts enriched in fibre-bearing phloem tissues. Molecular Genetics and Genomics. 2007;278:149–165. DOI: https://doi.org/10.1007/s00438-007-0241-1Mokshina N., Gorshkov O., Galinousky D., Gorshkova T. Genes with bast fiber-specific expression in flax plants – Molecular keys for targeted fiber crop improvement. Industrial crops and products. 2020;152:112549. DOI: https://doi.org/10.1016/j.indcrop.2020.112549Mokshina N., Gorshkov O., Galinousky D., Gorshkova T. Genes with bast fiber-specific expression in flax plants – Molecular keys for targeted fiber crop improvement. Industrial crops and products. 2020;152:112549. DOI: https://doi.org/10.1016/j.indcrop.2020.112549Горбова М. А., Мансапова А. И. Изучение сортов льна-долгунца томской селекции в подтаёжной зоне Омской области. Актуальные вопросы биологии, селекции, технологии возделывания и переработки сельскохозяйственных культур: сб. мат-лов 11-й Всеросс. конф. молодых учёных и специалистов. Краснодар: ФГБНУ ФНЦ ВНИИМК имени В. С. Пустовойта, 2021. С. 44–49. DOI: https://doi.org/10.25230/conf11-2021-44-49Горбова М. А., Мансапова А. И. Изучение сортов льна-долгунца томской селекции в подтаёжной зоне Омской области. Актуальные вопросы биологии, селекции, технологии возделывания и переработки сельскохозяйственных культур: сб. мат-лов 11-й Всеросс. конф. молодых учёных и специалистов. Краснодар: ФГБНУ ФНЦ ВНИИМК имени В. С. Пустовойта, 2021. С. 44–49. DOI: https://doi.org/10.25230/conf11-2021-44-49Gorshkova T. A., Sal'nikov V. V., Chemikosova S. B., Ageeva M. V., Pavlencheva N. V., Van Dam J. E. The snap point: a transition point in Linum usitatissimum bast fiber development. Industrial Crops and Products. 2003;18(3):213–221. DOI: https://doi.org/10.1016/S0926-6690(03)00043-8Gorshkova T. A., Sal'nikov V. V., Chemikosova S. B., Ageeva M. V., Pavlencheva N. V., Van Dam J. E. The snap point: a transition point in Linum usitatissimum bast fiber development. Industrial Crops and Products. 2003;18(3):213–221. DOI: https://doi.org/10.1016/S0926-6690(03)00043-8Guenin S., Mauriat M., Pelloux J., Van Wuytswinkel O., Bellini C., Gutierrez L. Normalization of qRT-PCR data: the necessity of adopting a sysematic, experimental conditions-specific, validation of references. Journal of experimental botany. 2009;60(2):487–493. DOI: https://doi.org/10.1093/jxb/ern305Guenin S., Mauriat M., Pelloux J., Van Wuytswinkel O., Bellini C., Gutierrez L. Normalization of qRT-PCR data: the necessity of adopting a sysematic, experimental conditions-specific, validation of references. Journal of experimental botany. 2009;60(2):487–493. DOI: https://doi.org/10.1093/jxb/ern305Kumar S., Jordan M. C., Datla R., Cloutier S. The LuWD40-1 gene encoding WD repeat protein regulates growth and pollen viability in flax (Linum usitatissimum L.). PloS One. 2013;8(7):e69124. DOI: https://doi.org/10.1371/journal.pone.0069124Kumar S., Jordan M. C., Datla R., Cloutier S. The LuWD40-1 gene encoding WD repeat protein regulates growth and pollen viability in flax (Linum usitatissimum L.). PloS One. 2013;8(7):e69124. DOI: https://doi.org/10.1371/journal.pone.0069124Thambugala D., Cloutier S. Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.). Journal of Applied Genetics. 2014;55:423–432. DOI: https://doi.org/10.1007/s13353-014-0222-0Thambugala D., Cloutier S. Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.). Journal of Applied Genetics. 2014;55:423–432. DOI: https://doi.org/10.1007/s13353-014-0222-0Huis R., Hawkins S., Neutelings G. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC plant biology. 2010;10:71. DOI: https://doi.org/10.1186/1471-2229-10-71Huis R., Hawkins S., Neutelings G. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC plant biology. 2010;10:71. DOI: https://doi.org/10.1186/1471-2229-10-71Galinousky D., Padvitski T., Bayer G., Pirko Y., Pydiura N., Anisimova N., Nikitinskaya T., Khotyleva L., Yemets A., Kilchevsky A., Blume Ya. Expression analysis of cellulose synthase and main cytoskeletal protein genes in flax (Linum usitatissimum L.). Cell Biology International. 2019;43(9):1065–1071. DOI: https://doi.org/10.1002/cbin.10837Galinousky D., Padvitski T., Bayer G., Pirko Y., Pydiura N., Anisimova N., Nikitinskaya T., Khotyleva L., Yemets A., Kilchevsky A., Blume Ya. Expression analysis of cellulose synthase and main cytoskeletal protein genes in flax (Linum usitatissimum L.). Cell Biology International. 2019;43(9):1065–1071. DOI: https://doi.org/10.1002/cbin.10837Chantreau M., Chabbert B., Billiard S., Hawkins S., Neutelings G. Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus‐induced gene silencing. Plant biotechnology journal. 2015;13(9):1312–1324. DOI: https://doi.org/10.1111/pbi.12350Chantreau M., Chabbert B., Billiard S., Hawkins S., Neutelings G. Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus‐induced gene silencing. Plant biotechnology journal. 2015;13(9):1312–1324. DOI: https://doi.org/10.1111/pbi.12350Mokshina N., Gorshkova T., Deyholos M. K. Chitinase-like (CTL) and cellulose synthase (CESA) gene expression in gelatinous-type cellulosic walls of flax (Linum usitatissimum L.) bast fibers. PLoS One. 2014;9(6):e97949. DOI: https://doi.org/10.1371/journal.pone.0097949Mokshina N., Gorshkova T., Deyholos M. K. Chitinase-like (CTL) and cellulose synthase (CESA) gene expression in gelatinous-type cellulosic walls of flax (Linum usitatissimum L.) bast fibers. PLoS One. 2014;9(6):e97949. DOI: https://doi.org/10.1371/journal.pone.0097949

The authors declare that there are no conflicts of interest present.