vstispСадоводство и виноградарствоHorticulture and viticulture0235-25912618-9003Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»10.31676/0235-2591-2022-3-5-14vstisp-871Research ArticleГЕНЕТИКА, СЕЛЕКЦИЯ, СЕМЕНОВОДСТВОGENETICS, BREEDING, SEED PRODUCTIONСоздание трансгенной формы подвоя косточковых культур ((Prunus pumila L.×P. salicina Lindl.)×(P. cerasifera Ehrh.)) с использованием конструкции РНК-шпильки (Hairpin-RNA) для устойчивости к потивирусу шарки сливыGeneration of transgenic rootstock plum ((Prunus pumila L.×P. salicina Lindl.)×(P. cerasifera Ehrh.)) using hairpin-RNA construct for resistance to the Plum pox virusСидороваТ. Н.SidorovaT. N.

Сидорова Т. Н. — младший научный сотрудник

проспект Науки, 6, г. Пущино, Московская область, 142290

Sidorova T. N., Junior Researcher, Chief Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

ПушинА. С.PushinA. S.

Пушин А. С. — младший научный сотрудник

проспект Науки, 6, г. Пущино, Московская область, 142290

Pushin A. S., Junior Researcher, Chief Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

МирошниченкоД. Н.MiroshnichenkoD. N.

Мирошниченко Д. Н. — кандидат биологических наук, старший научный струдник

проспект Науки, 6, г. Пущино, Московская область, 142290

Miroshnichenko  D.  N., PhD (Biol.), Senior Redearcher, Chief Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

ДолговС. В.DolgovS. V.

Долгов Сергей Владимирович — доктор биологических наук, главный научный сотрудник

проспект Науки, 6, г. Пущино, Московская область, 142290

Dolgov  S.  V., Dr. Sci. (Biol.), Chief Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

dolgov@bibch.ruФилиал Института биоорганической химии им. академиков М. М. Шемякина и Ю. А. Овчинникова Российской академии наук; Никитский ботанический сад — Национальный научный центр РАНРоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Nikita Botanical Garden — National Scientific Center of the Russian Academy of SciencesRussian FederationФилиал Института биоорганической химии им. академиков М. М. Шемякина и Ю. А. Овчинникова Российской академии наукРоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesRussian FederationФилиал Института биоорганической химии им. академиков М. М. Шемякина и Ю. А. Овчинникова Российской академии наук; Никитский ботанический сад — Национальный научный центр РАН; Федеральный научный селекционно- технологический центр садоводства и питомниководстваРоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Nikita Botanical Garden — National Scientific Center of the Russian Academy of Sciences; Federal Horticultural Research Center for Breeding, Agrotechnology and NurseryRussian Federation20221406202203514Copyright © Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture», 20222022Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»https://www.sadivin.com/jour/about/submissions#copyrightNoticehttps://www.sadivin.com/jour/article/view/871

Использование подвоев сливы, устойчивых к потивирусу шарки сливы (PPV), является важной стратегией борьбы с распространением заболевания в питомниках и садах. Несмотря на значительный прогресс в разработки подвоев косточковых плодовых культур для адаптации к различным стрессам, долговременная устойчивость к вирусам традиционными методами селекции до сих пор безуспешна. По этой причине создание устойчивых к PPV растений биоинженерными методами остается актуальной в противодействии распространению этого заболевания. Целью настоящего исследования является получение трансгенных растений клонового подвоя косточковых культур `Элита`, устойчивого к PPV, с использованием технологии интерференции рибонуклеиновых кислот (RNAi). Для индуцирования механизма посттранскрипционного замалчивания экспресии генов, обеспечивающий устойчивость к вирусу, была использована генетическая конструкция, содержащая самокомплементарные фрагменты последовательности гена белка оболочки вируса шарки сливы (PPV-CP). Трансгенные растения были получены после опосредованной агробактериями трансформации листьев in vitro. Результаты полимеразной цепной реакции (ПЦР) и Саузерн-блоттинга подтвердили стабильную геномную интеграцию последовательности PPV-CP смысловой и антисмысловой интрон-РНК-шпильки последовательности. Функциональность введенной экспрессионной кассеты была подтверждена активностью включения гена uidA в переносящую Т-ДНК. Насколько нам известно, это первый межвидовой подвой сливы, полученный с помощью генной инженерии для достижения устойчивости к PPV.

The use of Prunus rootstocks that are resistant to plum pox virus (PPV) is an important agronomic strategy to combat the spread of the Sharka disease in nurseries and orchards. Despite remarkable progress in developing stone fruit rootstocks to adapt to various stresses, breeding that ensures durable virus resistance has not yet been achieved. For this reason, the engineering of PPV resistant plants through genetic transformation is a very promising approach to control sharka disease. The aim of the present study is to produce transgenic plants of the clonal rootstock `Elita`, which is resistant to PPV using ribonucleic acid interference (RNAi) technology. The genetic construct containing the self-complementary fragments of the Plum pox virus coat protein (PPV-CP) gene sequence were used to induce the mechanism of post-transcriptional gene silencing to ensure virus resistance. Transgenic plants have been produced after agrobacterium-mediated transformation of in vitro explanted leaves. The results of polymerase chain reaction (PCR) and Southern blotting analyses confirmed the stable genomic integration of the PPV-CP sense and antisense intronhairpin-RNA sequence. Th e functionality of the introduced expression cassette was confirmed by the activity of including the uidA gene into the transferring T-DNA. To our knowledge, this is the first interspecific plum rootstock produced by genetic engineering to achieve PPV resistance.

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