vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-26-61vavilov-5194Research ArticleГЕНЕТИКА РАСТЕНИЙPLANT GENETICSСоздание растений картофеля (Solanum tuberosum L.) с инактивированным геном StPain-1 с помощью CRISPR/Cas9Development of potato (Solanum tuberosum L.) plants with inactivated StPain-1 gene using CRISPR/Cas9https://orcid.org/0000-0002-7468-9483КарловВ. Д.KarlovV. D.

Москва

Moscow

v4slvk@yandex.ruhttps://orcid.org/0009-0006-7985-822XВолковМ. К.VolkovM. K.

Москва

Moscow

https://orcid.org/0000-0003-3522-1674АнтиповА. Д.AntipovA. D.

Москва

Moscow

https://orcid.org/0000-0002-6772-6098МонаховаЮ. С.MonahovaYu. S.

Москва

Moscow

ТрофимовА. С.TrofimovA. S.

Москва

Moscow

https://orcid.org/0000-0001-8976-7774КоноваловаЛ. Н.KonovalovaL. N.

Москва

Moscow

БабаковА. В.BabakovA. V.

Москва

Moscow

https://orcid.org/0000-0001-5963-8111КомахинР. А.KomakhinR. A.

Москва

Moscow

https://orcid.org/0000-0002-0728-0346ТарановВ. В.TaranovV. V.

Москва

Moscow

Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологииРоссияAll-Russia Research Institute of Agricultural BiotechnologyRussian Federation202624072026304601611Copyright © Карлов В.Д., Волков М.К., Антипов А.Д., Монахова Ю.С., Трофимов А.С., Коновалова Л.Н., Бабаков А.В., Комахин Р.А., Таранов В.В., 20262026Карлов В.Д., Волков М.К., Антипов А.Д., Монахова Ю.С., Трофимов А.С., Коновалова Л.Н., Бабаков А.В., Комахин Р.А., Таранов В.В.Karlov V.D., Volkov M.K., Antipov A.D., Monahova Y.S., Trofimov A.S., Konovalova L.N., Babakov A.V., Komakhin R.A., Taranov V.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/5194

Холодовое хранение клубней картофеля для последующей переработки осложнено развитием холодового осахаривания. При пониженной температуре происходит реакция ферментативного гидролиза сахарозы на глюкозу и фруктозу. Как следствие, высокое содержание гексоз негативно сказывается на качестве продуктов переработки картофеля – чипсов и фри, а также способствует образованию нейротоксина и канцерогена акриламида. Гидролиз сахарозы в процессе холодового осахаривания катализирует вакуолярная инвертаза, кодируемая геном StPain-1. Ранее было показано, что подавление активности фермента придает картофелю устойчивость к холодовому осахариванию, не снижая при этом питательных качеств клубней. В настоящей работе с помощью CRISPR/Cas9 были созданы растения культурного картофеля Solanum tuberosum L. сорта Фрителла с нокаутом гена StPain-1. Для редактирования целевого гена были собраны две генетические конструкции на основе вектора pKSE401 (Вектор A и Вектор B), каждая из которых несла две гидовые РНК: на участки в первом (sgRNA-P1.A и sgRNA-P1.B) и третьем (sgRNA-P3.A и sgRNA-P3.B) экзонах. Эффективность редактирования каждой gRNA была охарактеризована с применением высокопроизводительного секвенирования. С конструкцией Вектор A получено 48 трансформантов, среди которых 22 содержали нокаут всех аллелей гена StPain-1; с конструкцией Вектор B – 26 трансформантов, из них 10 были с нокаутом. Путем обжарки чипсов из клубней девяти отредактированных растений картофеля сорта Фрителла было наглядно продемонстрировано снижение активности вакуолярной инвертазы. Чипсы из образцов с нокаутом гена StPain-1 имели более светлую окраску, чем чипсы из контрольного образца без редактирования. По результатам проведенной работы отобрано четыре растения с нокаутом гена StPain-1, по два для каждой конструкции, для которых количественная оценка глюкозы, фруктозы и сахарозы, а также измерение уровня мРНК подтвердили инактивацию фермента. Полученные растения обладают повышенной устойчивостью к холодовому осахариванию и являются перспективными источниками нефункциональных аллелей гена StPain-1 для селекции новых сортов картофеля.

Storage of potato tubers intended for further processing is complicated by their cold-induced sweetening (CIS). Enzymatic hydrolysis of sucrose into glucose and fructose occurs at low temperatures. The resulting high hexose content adversely affects the quality of processed potato products such as chips and fries and promotes the formation of acrylamide, which is a neurotoxin and carcinogen. During CIS, sucrose hydrolysis is catalyzed by vacuolar invertase encoded by the StPain-1 gene. Previous studies have shown that suppression of the enzyme activity confers potato resistance to CIS without reducing the nutritional value of tubers. In this study, CRISPR/Cas9 technology was used to generate Solanum tuberosum L. cv. Fritella plants with a knockout of StPain-1. Two binary vectors based on pKSE401 were constructed (Vector A and Vector B), each carrying two gRNAs targeting exon 1 (sgRNA-P1.A or sgRNA-P1.B) and exon 3 (sgRNA-P3.A or sgRNA-P3.B). Editing efficiency with each gRNA was evaluated through next-generation sequencing (NGS). Transformation with Vector A produced 48 transformants, 22 of which carried knockouts in all StPain-1 alleles. Transformation with Vector B yielded 26 transformants, including 10 plants with complete StPain-1 knockout. Chips made from tubers of nine edited Fritella plants demonstrated reduced vacuolar invertase activity: chips from StPain-1 knockout lines were lighter compared to the non-edited control sample. Quantitative assessment of glucose, fructose, and sucrose levels, as well as StPain-1 mRNA expression in tubers of four selected transformants (two per vector), confirmed enzyme inactivation. The resulting plants exhibit increased resistance to cold-induced sweetening and can be used as a promising source of nonfunctional StPain-1 alleles for breeding new potato varieties.

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