vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-25-124vavilov-4917Research ArticleХРОМОСОМНАЯ И ГЕНЕТИЧЕСКАЯ ИНЖЕНЕРИЯCHROMOSOME AND GENE ENGINEERINGЗамалчивание гена TaAOS2 с помощью РНК-интерференции влияет на накопление фитогормонов, рост и продуктивность мягкой пшеницыEffect of RNAi-mediated silencing of the TaAOS2 gene on phytohormone accumulation, growth and productivity in bread wheatМирошниченкоД. Н.MiroshnichenkoD. N.

Пущино, Московская область

Pushchino, Moscow region

miroshnichenko@bibch.ruПиголевА. В.PigolevA. V.

Пущино, Московская область

Pushchino, Moscow region

ДегтярёвЕ. А.DegtyaryovE. A.

Пущино, Московская область

Pushchino, Moscow region

ДегтярёваВ. И.DegtyaryovaV. I.

Пущино, Московская область

Pushchino, Moscow region

АлексееваВ. В.AlekseevaV. V.

Пущино, Московская область

Pushchino, Moscow region

ПушинА. С.PushinA. S.

Пущино, Московская область

Pushchino, Moscow region

ДолговС. В.DolgovS. V.

Пущино, Московская область

Pushchino, Moscow region

СавченкоТ. В.SavchenkoT. V.

Пущино, Московская область

Pushchino, Moscow region

Институт фундаментальных проблем биологии Российской академии наук – обособленное подразделение Федерального исследовательского центра «Пущинский научный центр биологических исследований Российской академии наук»; Филиал Института биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наукРоссияInstitute of Basic Biological Problems of the Russian Academy of Sciences; Branch of Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesRussian FederationИнститут фундаментальных проблем биологии Российской академии наук – обособленное подразделение Федерального исследовательского центра «Пущинский научный центр биологических исследований Российской академии наук»РоссияInstitute of Basic Biological Problems of the Russian Academy of SciencesRussian FederationИнститут фундаментальных проблем биологии Российской академии наук – обособленное подразделение Федерального исследовательского центра «Пущинский научный центр биологических исследований Российской академии наук»; Филиал Института биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук; Пущинский филиал Российского биотехнологического университета (РОСБИОТЕХ)РоссияInstitute of Basic Biological Problems of the Russian Academy of Sciences; Branch of Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Pushchino Branch of Russian Biotechnological UniversityRussian FederationФилиал Института биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наукРоссияBranch of Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesRussian Federation20251101202629811761183Copyright © Мирошниченко Д.Н., Пиголев А.В., Дегтярёв Е.А., Дегтярёва В.И., Алексеева В.В., Пушин А.С., Долгов С.В., Савченко Т.В., 20262026Мирошниченко Д.Н., Пиголев А.В., Дегтярёв Е.А., Дегтярёва В.И., Алексеева В.В., Пушин А.С., Долгов С.В., Савченко Т.В.Miroshnichenko D.N., Pigolev A.V., Degtyaryov E.A., Degtyaryova V.I., Alekseeva V.V., Pushin A.S., Dolgov S.V., Savchenko T.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/4917

Методы обратной генетики активно используются в биологии растений для изучения функций генов, ответственных за адаптацию растений к различным стрессовым факторам окружающей среды. Данная работа посвящена получению и первичному анализу трансгенных растений мягкой пшеницы с подавленной экспрессией одного из генов алленоксидсинтазы (АОS). АОS является ключевым ферментом, участвующим в начальном этапе биосинтеза стрессовых фитогормонов, известных как жасмонаты. Для подавления экспрессии гена АОS в пшенице создан вектор для РНК-интерференционного замалчивания, содержащий инвертированную область TaAOS2 из генома яровой мягкой пшеницы сорта Chinese Spring. Путем биобаллистической генетической трансформации получен ряд трансгенных растений Chinese Spring, в которых подавление экспрессии целевого гена подтверждено с помощью количественной ПЦР в режиме реального времени, а изменение уровней накопления отдельных жасмонатов выявлено методом газохромато масс-спектрометрии. В стрессовых условиях (механическое поранение листовой пластинки молодых растений) трансгенные dsRNAi линии накапливали лишь 21–44 % мРНК TaAOS2 по сравнению с нетрансгенным пораненным контролем пшеницы. Подавление экспрессии гена TaAOS2 существенно снизило накопление жасмоновой кислоты и конъюгата жасмоноил-изолейцина, в то время как уровень содержания в листьях других фитогормонов, таких как абсцизовая кислота и салициловая кислота, не изменился. Трансгенные линии пшеницы с подавленной экспрессией гена TaAOS2 демонстрировали уменьшение длины листьев на ранних стадиях развития растений; также для них была характерна тенденция к сокращению высоты растений и снижению массы зерна. При этом замалчивание не привело к изменению сроков цветения и не повлияло на фертильность растений, так как среднее количество семян в колосе не изменилось. Полученные трансгенные линии пшеницы в сочетании с линиями, сверхэкспрессирующими АОS, служат хорошим инструментом для дальнейшего детального анализа адаптивных реакций растений, контролируемых жасмонатным сигналингом.

Reverse genetics methods are actively used in plant biology to study the functions of specific genes responsible for the adaptation of plants to various environmental stresses. The present study describes the production and primary characterization of transgenic bread wheat with silenced expression of allen oxide synthase (AOS). AOS is a key enzyme involved in the initial step of biosynthesis of stress-related phytohormones known as jasmonates. To induce silencing of AOS in wheat, we designed the RNA interference (RNAi) vector containing an inverted repeat region of the TaAOS2 gene cloned from genome DNA of cv. Chinese Spring. With the help of biolistic-mediated transformation, a number of transgenic Chinese Spring plants have been produced. Real-Time PCR analysis confirmed the suppression of target gene expression, since transgenic dsRNAi lines accumulated only 21–44 % mRNA of TaAOS2 after leaf wounding compared to the wound-induced level in non-transgenic control. Gas chromatography–mass spectrometry revealed that the silencing of TaAOS2 substantially reduced the accumulation of jasmonic acid (JA) and jasmonoyl-isoleucine conjugate (JA-Ile), while the production of other phytohormones, such as abscisic acid and salicylic acid, was not affected. TaAOS2-silenced lines were characterized by shorter leaves at the juvenile stage, demonstrated a tendency towards reduced plant height and decreased grain weight, while the average flowering time and plant fertility (number of seeds per spike) were not affected. The obtained transgenic lines in combination with AOS-overexpressing lines can be used for further detailed analysis of the adaptive responses controlled by the jasmonate hormonal system.

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