Cloning and expression of wheat Rht-D1a cDNA in E.coli

Authors

  • I. T. Smekenov
  • T. I. Ayupov
  • M. K. Bakhtambayeva
  • G. T. Rakhmatullaeva
  • S. M. Taipakova
  • A. K. Bissenbaev Scientific Research Institute of Biology and Biotechnology Problems, Al-Farabi Kazakh National University, Kazakhstan, Almaty

DOI:

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

Abstract

The wheat DELLA proteins encoded by the plant height reducing (Rht-1) genes. Wheat DELLA genes have three homologous loci (Rht-A1, Rht-B1, and Rht-D1) in the 4A, 4B, and 4D chromosomes, respectively. Despite the importance of the Rht-1 proteins, little biochemical analyses have been reported, mainly due to difficulties with purification of sufficient quality of protein and absence specific antibodies to this protein. Here, we isolated the Rht-D1a cDNA gene encoding the wheat Rht-D1a using the reverse transcription - polymerase chain reaction (RT-PCR).  Rht-D1a with a 6xHis end was functionally expressed in E. coli and purified by nickel affinity chromatography. Using MALDI-TOF mass spectrometry, a complete coincidence of the amino acid sequence of the recombinant protein with the primary structure of the DELLA protein Triticum aestivum was established. It was revealed that the product of gene expression is a globular protein with a mass of 65.3  kDa, consisting of 623 amino acids (pI 4.99). Purified Rht-D1a was used as an immunogen to generate rabbit polyclonal anti-Rht-D1a antibodies. Our purified preparation of the wheat Rht-D1a and antibodies generated against the recombinant Rht-D1a is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through Rht-D1a proteins.

Key words: Triticum aestivum, Rht-D1a, DELLA-protein, aleurone, expression.

References

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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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2018-11-17

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МOLECULAR BIOLOGY AND GENETICS

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