vstispСадоводство и виноградарствоHorticulture and viticulture0235-25912618-9003Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»10.31676/0235-2591-2022-2-32-41vstisp-862Research ArticleГЕНЕТИКА, СЕЛЕКЦИЯ, СЕМЕНОВОДСТВОGENETICS, BREEDING, SEED PRODUCTIONИспользование последовательности гена белка оболочки вируса шарки для создания устойчивых форм сливы (Prunus domestica L.)Use of the Plum pox virus coat protein gene sequence to create resistant forms of plum (Prunus domestica L.)ДолговС. В.DolgovS. V.

Долгов Сергей Владимирович — доктор биологических наук, главный научный сотрудник

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

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

6, Prospekt Nauki, Pushchino, Moscow region, 142290

dolgov@bibch.ruМихайловР. В.MikhailovR. V.

Михайлов Р. В. — ведущий сотрудник

г. Пущино

Mikhaylov R. V., Key specialist

Pushchino

СероваТ. А.SerovaT. А.

Серова Т. А. — аспирант

г. Пущино

Serova T. A., PhD student

Pushchino

ШульгаО. А.ShulgaO. A.

Шульга О. А. — кандидат химических наук, ведущий научный сотрудник

г. Москва

Shulga O. A., PhD (Chem.), Leading Research

Moscow

 

ФирсовА. П.FirsovA. Р.

Фирсов А. П. — кандидат биологический наук, старший научный сотрудник

г. Пущино

Firsov A. P., PhD (Biol.), senior scientist

Pushchino

Филиал Института биоорганической химии им. М. М. Шемякина и Ю. А. Овчинникова РАН; Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии; Федеральный научный селекционно-технологический центр садоводства и питомниководстваРоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; All-Russia Research Institute of Agricultural Biotechnology; Federal Horticultural Research Center for Breeding, Agrotechnology and NurseryRussian FederationФилиал Института биоорганической химии им. М. М. Шемякина и Ю. А. Овчинникова РАН; ООО «Антерикс»РоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesRussian FederationФилиал Института биоорганической химии им. М. М. Шемякина и Ю. А. Овчинникова РАНРоссияBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesRussian FederationВсероссийский научно-исследовательский институт сельскохозяйственной биотехнологииРоссияAll-Russia Research Institute of Agricultural BiotechnologyRussian Federation202213052022023241Copyright © 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/862

В настоящее время наиболее опасным патогеном абрикосов, слив и персиков считается вирус оспы сливы (PPV), возбудитель болезни шарки сливы. Трансформация сливы вирусными генами, такими как белок оболочки, может обеспечить получение устойчивых к вирусам сортов и исходных форм для использования в селекционном процессе. Для повышения устойчивости растений к вирусу шарки сливы (PPV) использовали две технологии. Одна основана на совместном подавлении, а другая — на РНК-сайленсинге (RNAi). Бинарный вектор pCamPPVcp, который содержал селективный ген hpt и ген белка оболочки вируса шарки сливы ppvcp в смысловой ориентации (управляемый двойным промотором 35S), был использован для реализации посттранскрипционного сайленсинга гена. Вектор pCamPPVRNAi содержал самокомплементарные фрагменты гена ppv-cp (698 пар оснований), управляемые двойным промотором 35S и генами hpt и gus. Фрагменты гена ppv-cp были разделены pdk-интроном для получения структуры РНК «шпильки» в антисмысловой ориентации. Были получены семь независимых трансгенных линий с геном ppv-cp и пять трансгенных линий с двумя инвертированными повторами фрагмента гена ppv-cp. Стабильная интеграция генов в геном растений подтверждена ПЦР-анализами. Накопление белка оболочки оценивали методом вестерн-блоттинга в пяти из шести проанализированных линий. Трансгенные побеги были укоренены и акклиматизированы в теплице. После прививки инфицированными PPV почками у всех контрольных и трансформированных ppv-cp растений с помощью вестерн-блоттинга обнаруживались полосы, соответствующие белку оболочки PPV, тогда как в образцах от растений, трансформированных конструкцией «шпилька», не наблюдалось никаких пятен, соответствующих белку оболочки PPV. Эти предварительные результаты подтвердили эффективность стратегии RNAi для защиты растений от вирусной атаки в целом и плодов косточковых культур и от PPV, в частности.

Plum pox virus (PPV), the causative agent of plum Sharka disease, is currently considered the most dangerous pathogen of apricots, plums and peaches. The transformation of plum with viral genes, such as coat protein, can provide novel virus resistant forms or gene resources for breeding new resistant varieties. For improving the plants resistance to Plum pox virus (PPV) two technologies were used. One based on co-suppression and another on RNA-silencing. Binary vector pCamPPVcp that contained the selective hpt gene and ppv-cp gene in sense-orientation (driven by double 35S promoter) was used for realization post-transcriptional gene silencing. Vector pCamPPVRNAi contained self-complementary fragments of gene ppv-cp (698bp) driven by double 35S promoter and the hpt and gus genes.

The fragments of ppv-cp gene were separated by pdk-intron to produce a “hairpin” RNA structure in antisense-sense orientation. Seven independent transgenic lines with ppv-cp gene and five transgenic lines with a two inverted repeats of ppv-cp gene fragment were produced. Stable integration of genes into genome of plants was confirmed by PCR analyses. The accumulation of coat protein was evaluated by Western blot assay in five from six analyzed lines. The transgenic shoots were rooted and acclimatized to the greenhouse. After grafting by PPV infected buds in all control and ppvcp transformed plants were detected by Western blot analysis lines corresponding PPV coat protein, whereas no any spots corresponding PPV coat protein were observed in samples from plants transformed “hairpin” construct. These preliminary results confirmed the efficiency of RNAi strategy for protection plants from virus attack in general, and for stone fruits from PPV in particular.

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