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"10.29235/1029-8940-2020-65-3-319-327vestib-691Research ArticleСтатьиКлонирование кДНК глюкоамилазы Aspergillus awamori в интегративный вектор для мицелиальных грибов методом кольцевой полимеразной реакцииCircular polymerase extension cloning of cDNA glucoamylase Aspergillus awamori into integrative vector for filamentous fungiКуликЕ. В.KulikA. V.

Кулик Елена Вячеславовна - кандидат биологических наук, ведущий научный сотрудник.

пр. Независимости, 4, 220030, Минск.

Alena V. Kulik - Ph. D. (Biol.), Leading Researcher, Belarusian State University.

4, Nezavisimosti Ave., 220030, Minsk.

alena.kulik31@gmail.comСелезневаЮ. В.SeleznevaYu. V.

Селезнева Юлия Валерьевна - кандидат биологических наук, заведующий лабораторией.

пр. Независимости, 4, 220030, Минск.

Yuliya V. Selezneva - Ph. D. (Biol.), Head of the Laboratory, Belarusian State University.

4, Nezavisimosti Ave., 220030, Minsk.

mamalanka@mail.ruКачанА. В.KachanA. V.

Качан Александр Вячеславович - кандидат биологических наук, доцент.

пр. Независимости, 4, 220030, Минск.

Alexandr V. Kachan - Ph. D. (Biol.), Assistant Professor, Belarusian State University.

4, Nezavisimosti Ave., 220030, Minsk.

av.kachan@mail.ruРусьО. Б.RusO. B.

Русь Ольга Борисовна - кандидат химических наук, доцент.

пр. Независимости, 4, 220030, Минск.

Olga B. Rus - Ph. D. (Chem.), Assistant Professor, Belarusian State University.

4, Nezavisimosti Ave., 220030, Minsk.

zrbio@mail.ruЕвтушенковА. Н.EvtushenkovA. N.

Евтушенков Анатолий Николаевич - доктор биологических наук, профессор, заведующий кафедрой.

пр. Независимости, 4, 220030, Минск.

Anatoliy N. Evtushenkov - D. Sc. (Biol.), Professor, Head of the Department, Belarusian State University.

4, Nezavisimosti Ave., 220030, Minsk.

evtushenkov@bsu.byБелорусский государственный университетBelarusian State University202006082020653319327Copyright © Кулик Е.В., Селезнева Ю.В., Качан А.В., Русь О.Б., Евтушенков А.Н., 20202020Кулик Е.В., Селезнева Ю.В., Качан А.В., Русь О.Б., Евтушенков А.Н.Kulik A.V., Selezneva Y.V., Kachan A.V., Rus O.B., Evtushenkov A.N.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/691

C использованием метода кольцевой полимеразной реакции (circular polymerase extension cloning, СРЕС) было проведено успешное клонирование кДНК гена глюкоамилазы Aspergillus awamori в интегративный вектор для мицелиальных грибов pH4Hyg. Вначале были проведены две полимеразные цепные реакции (ПЦР). В результате первой амплифицирована кДНК гена glaA, ранее клонированная в плазмиду на основе вектора pBluescript II SK(-), в результате второй - вектор pH4Hyg в линейной форме, содержащий промотор и терминатор целевого гена. Полученные ампликоны были очищены от компонентов смеси ПЦР. После 20 циклов реакции СРЕС была синтезирована конечная генетическая конструкция pH4HygPTgla, которая может использоваться в создании штаммов мицелиальных грибов, секретирующих глюкоамилазу в стабильной конформации, обладающую биотехнологически значимыми свойствами.

This article describes successful insertion the cDNA glucoamylase from Aspergillus awamori into the integrative vector for filamentous fungi pH4Hyg using the CPEC (circular polymerase extension cloning) strategy. The prior step was the PCR amplification of the cDNA glaA gene cloned into a vector based on the plasmid pBluescript II SK(-) and the amplification of linear pH4Hyg vector with promotor and terminator of the glaA gene. The PCR products were purified and used for CPEC reaction. The CPEC product was formed after 20 cycles of the reaction. It is proposed to use the final plasmid pH4HygPTgla in engineering of filamentous fungi strains producing stable forms of glucoamylase with biotechnological interest.

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