Клонирование и экспрессия кДНК Rht-D1a пшеницы в E.coli
DOI:
https://doi.org/10.26577/eb-2018-3-1343Аннотация
DELLA белки пшеницы кодируются генами Rht-1 и имеют три гомологичных локуса (Rht-A1, Rht-B1 и Rht-D1) в 4A, 4B и 4D хромосомах, соответственно. Несмотря на важность Rht-1 белков, практически не проводились биохимические исследования, в основном из-за трудностей с очисткой достаточного количества белка и отсутствия специфичных к этому белку антител. В представленной работе выделена кДНК гена Rht-D1а с применением реакции обратной транскрипции и полимеразной цепной реакции. Осуществлена функциональная экспрессия Rht-D1а с гистидиновым концом в E. cоli и очищена никель-аффинной хроматографией до гомогенного состояния. С помощью MALDI-TОF масс-спектрометрии установлено полное совпадение аминокислотной последовательности рекомбинантного белка с первичной структурой Rht-D1а белка Triticum aestivum. Выявлено, что продуктом экспрессии гена является глобулярный белок массой 65,3 kDa, состоящий из 623 аминокислот (pI=4,99). С применением очищенных рекомбинантных белков RHT-D1а были получены поликлональные антитела и выявлена их специфичность к Rht-D1а белку пшеницы. Полученные нами очищенный Rht-D1а белок и специфичные к нему поликлональные антитела подходят для дальнейших структурных и функциональных исследований, которые будут способствовать четкому пониманию механизма регуляции роста растений через Rht-D1a белки.
Ключевые слова: Triticum aestivum, Rht-D1а, DELLA, экспрессия.
Библиографические ссылки
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References
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