Клонирование и экспрессия кДНК Rht-D1a пшеницы в E.coli

Авторы

  • I. T. Smekenov
  • T. I. Ayupov
  • M. K. Bakhtambayeva
  • G. T. Rakhmatullaeva
  • S. M. Taipakova
  • A. K. Bissenbaev Научно-исследовательский институт проблем биологии и биотехнологии, Казахский национальный университет имени аль-Фараби, Казахстан, г. Алматы

DOI:

https://doi.org/10.26577/eb-2018-3-1343

Аннотация

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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23. Lou X., Li X., Li A., Pu M., Shoaib M., Liu D., Sun J., Zhang A., Yang W. Molecular Characterization of Three gibberellin-insensitive DWARF2 Homologous Genes in Common Wheat // PLoS ONE. – 2011. – Vol. 11, № 6. – P.e0157642. DOI:10.1371/journal.pone.0157642.
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References

1. Alvey L. and Harberd N.P. (2005) DELLA proteins: integrators of multiple plant growth regulatory inputs. Physiologia Plantarum, vol. 123, pp. 153-160. DOI: 10.1111/j.1399-3054.2004. 00412.x
2. Boss P.K., Thomas M.R. (2002) Association of dwarfism and floral induction with a grape «green revolution» mutation. Nature, vol. 416, no 6883, pp. 847-850. DOI: 10.1038/416847a
3. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., vol. 72, pp. 248-254.
4. Chandler P.M., Marion-Poll A., Ellis M., Gubler F. (2002) Mutants at the Slenderl locus of 'Himalaya' barley: molecular and physiological characterization. Plant Physiol., vol. 129, no 1, pp. 181-190. DOI: 10.1104/pp.010917
5. Chandler P.M., Harding C.A., Ashton A.R., Mulcair M.D., Dixon N.E., Mander L.N. (2008) Characterization of gibberellin receptor mutants of barley (Hordeum vulgare L.). Mol Plant, vol. 1, no 2, pp. 285-94. DOI: 10.1093/mp/ssn002. Epub 2008 Feb 11.
6. Cheng H., Qin L., Lee S., Fu X., Richards D.E., Cao D., Luo D., Harberd N.P., Peng J. (2004) Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function. Development, vol. 131, no 5, pp.1055–1064. DOI:10.1242/dev.00992
7. Dill A., Sun T.P. (2001) Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana. Genetics, vol. 159, pp. 777-785.
8. Dai C., Xue H.W. (2010) Rice early flowering1, a CKI, phosphorylates DELLA protein SLR1 to negatively regulate gibberellin signalling. EMBO J., vol. 29, no 11, pp. 1916-1927. DOI:10.1038/emboj.2010.75
9. Feng S., Martinez C., Gusmaroli G., Wang Y., Zhou J., Wang F., Chen L.Y., Yu L., Iglesias-Pedraz J.M., Kircher S., Schafer E., Fu X.D., Fan L.M., Deng X.W. (2008) Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature, vol. 451, no 7177, pp. 475-479. DOI: 10.1038/nature06448
10. Fu X., Richards D.E., Ait-Ali T., Hynes L.W., Ougham H., Peng J., Harberd N.P. (2002) Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor. Plant Cell, vol. 14, no 12, pp. 3191-3200.
11. Gilroy S., Jones R.L. (1992) Gibberellic acid and abcisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts. Proc. Natl. Acad.Sci. USA, vol. 89, pp. 3591-3595.
12. Gomi K., Sasaki A., Itoh H., Ueguchi-Tanaka M., Ashikari M., Kitano H., Matsuoka M. (2004) GID2, an F-box subunit of the SCF E3 complex, specifically interacts with phosphorylated SLR1 protein and regulates the gibberellin-dependent degradation of SLR1 in rice. Plant J., vol. 37, no 4, pp. 626-634.
13. Gubler, F., Chandler, P.M., White, R.G., Llewellyn, D.J., and Jacobsen, J.V. (2002) Gibberellin signaling in barley aleurone cells. Control of SLN1 and GAMYB expression. Plant Physiol., vol. 129, pp. 191–200. DOI: 10.1104/pp.010918
14. Ikeda A., Ueguchi-Tanaka M., Sonoda Y., Kitano H., Koshioka M., Futsuhara Y., Matsuoka M., Yamaguchi J. (2001) Slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8. Plant Cell, vol. 13, no 5, pp. 999-1010.
15. Itoh H., Ueguchi-Tanaka M., Sato Y., Ashikari M., Matsuoka M. (2002) The gibberellin signaling pathway is regulated by appearance and disappearance of SLENDER RICE1 in nuclei. Plant Cell, vol. 14, no 1, pp. 57-70. DOI: 10.1105/tpc.010319.
16. Itoh H., Shimada A., Ueguchi-Tanaka M., Kamiya N., Hasegawa Y., Ashikari M., Matsuoka M. (2005) Overexpression of a GRAS protein lacking the DELLA domain confers altered gibberellin responses in rice. Plant J., vol. 44, no 4, pp. 669-679. DOI: 10.1111/j.1365-313X.2005.02562.x
17. Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, vol. 227, no 5259, pp. 680-685.
18. Lou X., Li X., Li A., Pu M., Shoaib M., Liu D., Sun J., Zhang A., Yang W. (2011) Molecular Characterization of Three gibberellin-insensitive DWARF2 Homologous Genes in Common Wheat. PLoS ONE, vol. 11, no 6, pp.e0157642. DOI:10.1371/journal.pone.0157642.
19. Pearce S., Saville R.,, Vaughan S.P., Chandler P.M., Wilhelm E.P., Sparks C.A., Al-Kaff N., Korolev A., Boulton M.I., Phillips A.L., Hedden P., Nicholson P., Thomas S.G. (2011) Molecular Characterization of Rht-1 Dwarfing Genes in Hexaploid Wheat. Plant Physiology, vol. 157, pp. 1820-1831.
20. Peng J., Richards D.E., Hartley N.M., Murphy G.P., Devos K.M., Flintham J.E., Beales J., Fish L.J., Worland A.J., Pelica F., Sudhakar D., Christou P., Spape J.W., Gale M.D., Harberd N.P. (1999) «Green Revolution» genes encode mutant gibberellin response modulators. Nature, vol. 400, no 6741, pp. 256-261. DOI: 10.1038/22307
21. Phillips S. and Norton R. (2012) Global Wheat Production and Fertilizer Use. Better Crops, vol. 96, no 3, pp. 4-6.
22. Silverstone A.L., Ciampaglio C.N., Sun T. (1998) The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell, vol. 10, no 3, pp. 155-169. DOI: 10.1105/tpc.10.2.155
23. Ueguchi-Tanaka M., Nakajima M., Motoyuki A., Matsuoka M. (2007) Gibberellin receptor and its role in gibberellin signaling in plants. Annu. Rev. Plant Biol., vol. 58, no 1, pp. 183-198. DOI: 10.1146/annurev.arplant.58.032806.103830
24. Wu J., Kong X.,Wan J., Liu X., Zhang X., Guo X., Zhou R., Zhao G., Jing R., Fu X., Jia J. (2011) Dominant and pleiotropic effects of a GAI gene in wheat results from lack of interaction between DELLA and GID1. Plant Physiol., vol. 157, pp. 2120-2130. DOI: 10.1104/pp.111.185272.
25. Yamaguchi S. (2008) Gibberellin metabolism and its regulation. Annu Rev Plant Biol., vol. 59, pp. 225-251. DOI: 10.1146/annurev.arplant.59.032607.092804.

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Опубликован

2018-11-17

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МОЛЕКУЛЯРНАЯ БИОЛОГИЯ И ГЕНЕТИКА

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