Ecological geneticsEcological geneticsЭкологическая генетика1811-09322411-9202Eco-Vector62567010.17816/ecogen625670Methodology in ecological geneticsМетодология экологической генетикиResearch ArticleOptimization of transformation conditions of the yeast Saccharomyces cerevisiae to determine coregulators of the transcription factor NIN in a yeast two-hybrid systemОптимизация условий трансформации Saccharomyces cerevisiae для повышения разрешающей способности скрининга белок-синтезирующих библиотек в дрожжевой двугибридной системеhttps://orcid.org/0000-0002-5919-2487AAF-3244-2021DymoAlina M.ДымоАлина Михайловна
Russian Federation
dymoalina@yandex.ruhttps://orcid.org/0000-0002-3081-98804752-1910KantsurovaElizaveta S.КанцуроваЕлизавета Степановна
Russian Federation
rudaya.s.e@gmail.comhttps://orcid.org/0000-0003-1845-97015719038282ABC-2930-20202602-1514DolgikhAlexandra V.ДолгихАлександра Вячеславовна
Russian Federation
sqshadol@gmail.comhttps://orcid.org/0000-0002-5375-09436603496335G-6363-20174453-2060DolgikhElena A.ДолгихЕлена Анатольевна
Russian Federation

Dr. Sci. (Biology)

д-р биол. наук

dol2helen@yahoo.comAll-Russia Research Institute for Agricultural MicrobiologyВсероссийский научно-исследовательский институт сельскохозяйственной микробиологии29052024290920242232933061501202401052024Copyright ©; 2024, Eco-VectorCopyright ©; 2024, Эко-Вектор2024Eco-VectorЭко-Векторhttps://journals.eco-vector.com/ecolgenet/article/view/625670

BACKGROUND: The method of protein-protein interaction analysis using the yeast two-hybrid system in Saccharomyces cerevisiae cells is used to search for protein coregulators. This method is also used for mass screening of libraries of cloned fragments of complementary DNA (cDNA) that are translated in the cell. The key factor in the success of such screening is the level of efficiency of yeast cell transformation, since the resolution of the analysis is based on this.

AIM: The aim of this study is to search for optimal parameters for chemical transformation of yeast cells to increase the resolution of cDNA library screening.

MATERIALS AND METHODS: Plasmids pDEST22 and pDEST32 were used for chemical transformation of the yeast strain S. cerevisiae pJ69-4A. For screening, a cDNA library was used, obtained on the basis of mRNA isolated from the roots of pea Pisum sativum cultivar Finale, inoculated with rhizobia.

RESULTS: Factors influencing the efficiency of transformation were identified. Among them are the molecular weight of polyethyleneglycol used for transformation, as well as the number of cell division cycles that the culture undergoes. The effect of the number and size of plasmids used in transformation was also shown. Using the optimized protocol, a cDNA library was successfully screened to find coregulators of the NIN transcription factor.

CONCLUSIONS: Based on the data obtained, optimal parameters were determined that allow achieving a high level of competence in yeast cells. The use of the described protocol allowed for successful screening of the library to identify coregulators of the NIN transcription factor.

Актуальность. Метод анализа белок-белковых взаимодействий с помощью дрожжевой двугибридной системы в клетках Saccharomyces cerevisiae используют для поиска корегуляторов белков. С помощью этого метода также проводят массовые скрининги библиотек клонированных фрагментов комплементарной ДНК (кДНК), которые транслируются в клетке. Ключевым фактором успешности такого скрининга становится уровень эффективности трансформации дрожжевых клеток, так как на этом основывается разрешающая способность анализа.

Цель — провести поиск оптимальных параметров химической трансформации дрожжевых клеток для повышения разрешающей способности скрининга библиотек кДНК.

Материалы и методы. Для трансформации штамма дрожжей S. cerevisiae pJ69-4A химическим способом использовали плазмиды pDEST22 и pDEST32. Для скрининга применяли библиотеку кДНК, полученную на основе мРНК, выделенной из корней гороха Pisum sativum сорта Finale, инокулированных ризобией.

Результаты. Выявлены факторы, влияющие на эффективность трансформации. Среди них молекулярная масса используемого для трансформации полиэтиленгликоля, а также количество циклов деления клеток, которые претерпевает культура. Показано влияние количества и размера плазмид, используемых при трансформации. С помощью оптимизированного протокола успешно проведен скрининг библиотеки кДНК для поиска корегуляторов транскрипционного фактора NIN.

Заключение. На основе полученных данных определены оптимальные параметры, позволяющие добиться высокого уровня компетентности дрожжевых клеток. Применение описанного протокола позволило успешно провести скрининг библиотеки для выявления корегуляторов транскрипционного фактора NIN.

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