vestibИзвестия Национальной академии наук Беларуси. Серия биологических наукProceedings of the National Academy of Sciences of Belarus, Biological Series1029-89402524-230XThe Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1029-8940-2024-69-4-298-308vestib-941Research ArticleСтатьиCRISPR/Cas9-опосредованный направленный мутагенез гена PDS Nicotiana tabacum L.CRISPR/Cas9-mediated site-directed mutagenesis of the PDS gene of Nicotiana tabacum L.Шишлова-СоколовскаяА. М.Shishlova-SokolovskayaA. M.

Шишлова-Соколовская Анастасия Михайловна ‒ ст. науч. cотрудник.

Ул. Ф. Скорины, 34, 220072, Минск

Anastasia M. Shyshlova-Sokolovskaya ‒ Senior R esearcher.

34, F. Skoryna Str., 220072, Minsk

s_anastasia78@mail.ruУрбановичО. Ю.UrbanovichO. Yu.

Урбанович Оксана Юрьевна ‒ д -р б иол. н аук, п рофессор, заведующий лабораторией.

Ул. Ф. Скорины, 34, 220072, Минск

Oksana Yu. Urbanovich ‒ D. Sc. (Biol.), Professor, Head of the Laboratory.

34, F. Skoryna Str., 220072, Minsk

O.Urbanovich@igc.byИнститут генетики и цитологии НАН БеларусиInstitute of Genetics and Cytology of the National Academy of Sciences of Belarus202406112024694298308Copyright © Шишлова-Соколовская А.М., Урбанович О.Ю., 20242024Шишлова-Соколовская А.М., Урбанович О.Ю.Shishlova-Sokolovskaya A.M., Urbanovich O.Y.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestibio.belnauka.by/jour/article/view/941

Система CRISPR/Cas9 является одним из эффективных инструментов для редактирования геномов растений. С помощью этой системы нами получены трансформанты Nicotiana tabacum с различным фенотипом, несущие в своем геноме мутационные события инсерционно-делеционного типа в гене PDS, кодирующем фермент 15-цис-фитоендесатуразу. Эндогенный ген табака NtPDS был выбран в качестве мишени, так как внесение мутаций в его нуклеотидную последовательность позволяет легко визуализировать признаки. Тетрааллельный нокаут гена NtPDS приводил к полному альбинизму растений. Трансформанты-альбиносы отличались более медленным ростом, карликовостью и были нежизнеспособны при культивировании in vitro. Мутанты с пестролистностью в культуре in vitro образовывали корни и не отличались от контрольных растений по размеру листьев. Анализ нуклеотидной последовательности протоспейсера гена мишени NtPDS был выполнен на случайной выборке, включавшей 21 трансгенное растение поколения Т0 с различным фенотипом – от полностью альбиносного до дикого типа. Результаты секвенирования показали, что все трансформанты с видимыми фенотипическими проявлениями несут мутантные последовательности в гене NtPDS с частотой 51,0‒80,0 %, при этом эффективность направленного мутагенеза составила 33,33 %. У трансформантов с фенотипом дикого типа не обнаружено мутированных последовательностей в мишени.

CRISPR/Cas9 system is one of the effective tools for editing plant genomes. Using the CRISPR/Cas9 system, we have obtained different-type Nicotiana tabacum transformants carrying in their genome the mutational events of insertiondeletion type in the PDS gene, encoding15-cis-phytoene desaturase enzyme. The endogenous tobacco NtPDS gene was chosen as a target, since introducing mutations into its nucleotide sequence leads to appearing easily visualized characters. The tetraallelic knockout of the NtPDS gene led to complete albinism in plants. Albino transformants were characterized by a slower growth, dwarfism, and were not viable when cultivated in vitro. Mutants with variegated leaves in the in vitro culture formed roots and did not differ from the control in leaf size. A nucleotide sequence of the protospacer of the target NtPDSt gene was analyzed on a random sample, including 21 transgenic plants of the T0 generation with different phenotypes – from completely albino to wild type. Sequencing results showed that all transformants with visible phenotypic manifestations carried mutant sequences in the NtPDS gene with a frequency of 51.0–80.0 %, while the efficiency of site-directed mutagenesis was 33.33 %. In transformants of a wild-type phenotype, no mutated sequences were detected in the target gene.

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