Purification and Function Exploration of Transcription Factor MYB54 in Arabidopsis thaliana

doi:10.7668/hbnxb.20192158

Abstract

Abstract: In order to make a preliminary exploration of the function of MYB54 gene, we have analyzed the evolutionary conservation of MYB54, the secondary and tertiary structure of the protein, and tissue specific expression, high temperature stress response, in vitro protein purification and Western Blot, detection and transcription activation activity. The results showed that the MYB54 protein in Arabidopsis was similar to Brassica oleracea, Brassica rapa and Camelina sativa in the cruciferous family, reaching 87%, 87%, 81%, respectively.It had low homology with grasses such as Brassica rapa, Setaria italica and Oryza sativa, which were 54%, 57%, 47% respectively, the homology with Triticum aestivum and Solanum lycopersicum was 42%.The MYB54 protein in wheat and Arabidopsis were very similar in conserved domains, hydrophilicity, secondary structure and tertiary structure. RT-PCR results showed that the MYB54 gene in Arabidopsis was expressed the most in pods, suggesting that MYB54 might play a certain role in the process of reproductive development, and the expression of MYB54 in Arabidopsis and wheat under high temperature stress was significantly reduced, indicating that this gene also played an important function in response to heat stress. MYB54 protein could be successfully induced under the conditions of 18, 30, 37℃. The amount of purified protein induced at 37℃ was the highest. Western Blot results showed that MYB54 protein could be correctly translated and expressed.The yeast GAL4 system was used to investigate the transcription activation activity of MYB54 gene, and it was found that MYB54 protein could still activate the expression of downstream reporter genes without binding to the transcription activation domain, indicating that MYB54 gene did have transcription activation activity. MYB54 protein structure, tissue expression, protein induction and purification, transcription activity and stress expression were preliminarily analyzed, which provided certain theoretical basis for the study of MYB54 function in the growth and development process of Arabidopsis thaliana and wheat.

Key words: Arabidopsis thaliana, MYB54, Tissue expression, Induction purification, Transcription activation, Abiotic stress

CLC Number:

Q943.2

ZHENG Yichao, ZHANG Hao, ZHAO Dan, WANG Fengru, LIU Xigang, GUO Lin. Purification and Function Exploration of Transcription Factor MYB54 in Arabidopsis thaliana[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(4): 47-55. doi: 10.7668/hbnxb.20192158.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20192158

http://www.hbnxb.net/EN/Y2021/V36/I4/47

References

[1] Marocco A, Wissenbach M, Becker D, Paz-Ares J, Saedler H, Salamini F, Rohde W. Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA binding domain of the myb oncoproteins[J]. Molecular and General Genetics, 1989, 216(2/3):183-187.doi:10.1007/BF00334354.
[2] Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana[J]. Current Opinion in Plant Biology, 2001, 4(5):447-456.doi:10.1016/s1369-5266(00) 00199-0.
[3] Xiao R X, Zhang C, Guo X R, Li H, Lu H. MYB transcription factors and its regulation in secondary cell wall formation and lignin biosynthesis during xylem development[J]. International Journal of Molecular Sciences, 2021, 22(7):3560.doi:10.3390/ijms22073560.
[4] Gates D J, Strickler S R, Mueller L A, Olson B J S C, Smith S D. Diversification of R2R3-MYB transcription factors in the tomato family solanaceae[J]. Journal of Molecular Evolution, 2016, 83(1/2):26-37.doi:10.1007/s00239-016-9750-z.
[5] Jiang C K, Rao G Y. Insights into the diversification and evolution of R2R3-MYB transcription factors in plants[J]. Plant Physiology, 2020, 183(2):637-655.doi:10.1104/pp.19.01082.
[6] Ito M.Conservation and diversification of three-repeat Myb transcription factors in plants[J]. Journal of Plant Research, 2005, 118(1):61-69.doi:10.1007/s10265-005-0192-8.
[7] Haga N, Kato K, Murase M, Araki S, Kubo M, Demura T, Suzuki K, Müller I, Voss U, Jürgens G, Ito M. R1R2R3-Myb proteins positively regulate cytokinesis through activation of KNOLLE transcription in Arabidopsis thaliana[J]. Development, 2007, 134(6):1101-1110.doi:10.1242/dev.02801.
[8] Wang X D, Angelis N, Thein S L. MYB-A regulatory factor in hematopoiesis[J]. Gene, 2018, 665:6-17.doi:10.1016/j.gene.2018.04.065.
[9] Kaspar P, Prochazka J, Efenberkova M, Juhasz A, Novosadova V, Sedlacek R. c-Myb regulates tumorigenic potential of embryonal rhabdomyosarcoma cells[J]. Scientific Reports, 2019, 9(1):6342.doi:10.1038/s41598-019-42684-y.
[10] Bender J, Fink G R. A Myb homologue, ATR1, activates tryptophan gene expression in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(10):5655-5660.doi:10.1073/pnas.95.10.5655.
[11] Allen R S, Li J Y, Stahle M I, Dubroué A, Gubler F, Millar A A.Genetic analysis reveals functional redundancy and the major target genes of the Arabidopsis miR159 family[J]. PNAS, 2007, 104(41):16371-16376.doi:10.1073/pnas.0707653104.
[12] Wang B H, Luo Q, Li Y P, Yin L F, Zhou N N, Li X N, Gan J H, Dong A W.Structural insights into target DNA recognition by R2R3-MYB transcription factors[J]. Nucleic Acids Research, 2020, 48(1):460-471.doi:10.1093/nar/gkz1081.
[13] Yan H L, Pei X N, Zhang H, Li X, Zhang X X, Zhao M H, Chiang V L, Sederoff R R, Zhao X Y. MYB-Mediated regulation of anthocyanin biosynthesis[J]. Int J Mol Sci, 2021, 22(6):3103.doi:10.3390/ijms22063103.
[14] Ma D W, Constabel C P.MYB repressors as regulators of phenylpropanoid metabolism in plants[J]. Trends in Plant Science, 2019, 24(3):275-289.doi:10.1016/j.tplants.2018.12.003.
[15] Chen L H, Hu B, Qin Y H, Hu G B, Zhao J T. Advance of the negative regulation of anthocyanin biosynthesis by MYB transcription factors[J]. Plant Physiology and Biochemistry, 2019, 136:178-187.doi:10.1016/j.plaphy.2019.01.024.
[16] Li L, Yu X F, Thompson A, Guo M, Yoshida S, Asami T, Chory J, Yin Y H. Arabidopsis MYB30 is a direct target of BES1 and cooperates with BES1 to regulate brassinosteroid-induced gene expression[J]. The Plant Journal, 2009, 58(2):275-286.doi:10.1111/j.1365-313X.2008.03778.x.
[17] Zhang J, Zhou H, Zhou M J, Ge Z L, Zhang F, Foyer C H, Yuan X X, Xie Y J.The coordination of guard-cell autonomous ABA synthesis and DES1 function in situ regulates plant water deficit responses[J]. Journal of Advanced Research, 2021, 27:191-197.doi:10.1016/j.jare.2020.07.013.
[18] Seo P J, Park C M.MYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis[J]. New Phytologist, 2010, 186(2):471-483.doi:10.1111/j.1469-8137.2010.03183.x.
[19] Kirik V, Lee M M, Wester K, Herrmann U, Zheng G, Oppenheimer D, Schiefelbein J, Hulskamp M. Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation[J]. Development, 2005, 132(7):1477-1485.doi:10.1242/dev.01708.
[20] Li S F, Milliken O N, Pham H, Seyit R, Napoli R, Preston J, Koltunow A M, Parish R W.The Arabidopsis MYB5 transcription factor regulates mucilage synthesis, seed coat development, and trichome morphogenesis[J]. The Plant Cell, 2009, 21(1):72-89.doi:10.1105/tpc.108.063503.
[21] Gonzalez A, Mendenhall J, Huo Y J, Lloyd A.TTG1 complex MYBs, MYB5 and TT2, control outer seed coat differentiation[J]. Developmental Biology, 2009, 325(2):412-421.doi:10.1016/j.ydbio.2008.10.005.
[22] Baumann K, Perez-Rodriguez M, Bradley D, Venail J, Bailey P, Jin H, Koes R, Roberts K, Martin C.Control of cell and petal morphogenesis by R2R3 MYB transcription factors[J]. Development, 2007, 134(9):1691-1701.doi:10.1242/dev.02836.
[23] Zhang Y F, Cao G Y, Qu L J, Gu H Y.Characterization of Arabidopsis MYB transcription factor gene AtMYB17 and its possible regulation by LEAFY and AGL15[J]. Journal of Genetics and Genomics, 2009, 36(2):99-107.doi:10.1016/S1673-8527(08) 60096-X.
[24] Coleto I, Bejarano I, Marín-Peña A J, Medina J, Rioja C, Burow M, Marino D. Arabidopsis thaliana transcription factors MYB28 and MYB29 shape ammonium stress responses by regulating Fe homeostasis[J]. New Phytologist, 2021, 229(2):1021-1035.doi:10.1111/nph.16918.
[25] MacMillan C P, Birke H, Chuah A, Brill E, Tsuji Y, Ralph J, Dennis E S, Llewellyn D, Pettolino F A. Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls[J]. BMC Genomics, 2017, 18(1):539.doi:10.1186/s12864-017-3902-4.
[26] Matías-Hernández L, Jiang W M, Yang K, Tang K X, Brodelius P E, Pelaz S.AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana[J]. The Plant Journal, 2017, 90(3):520-534.doi:10.1111/tpj.13509.
[27] Penfield S, Meissner R C, Shoue D A, Carpita N C, Bevan M W. MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat[J]. The Plant Cell, 2001, 13(12):2777-2791.doi:10.1105/tpc.010265.
[28] Liang Y K, Dubos C, Dodd I C, Holroyd G H, Hetherington A M, Campbell M M. AtMYB61, an R2R3-MYB transcription factor controlling stomatal aperture in Arabidopsis thaliana[J]. Current Biology, 2005, 15(13):1201-1206.doi:10.1016/j.cub.2005.06.041.
[29] Bucholc K, Skrajna A, Adamska K, Yang X C, Krajewski K, Poznański J, Dadlez M, Domiński Z, Zhukov I. Structural analysis of the SANT/Myb domain of FLASH and YARP proteins and their complex with the C-Terminal fragment of NPAT by NMR spectroscopy and computer simulations[J]. International Journal of Molecular Sciences, 2020, 21(15):5268.doi:10.3390/ijms21155268.
[30] Ogata K, Hojo H, Aimoto S, Nakai T, Nakamura H, Sarai A, Ishii S, Nishimura Y. Solution structure of a DNA-binding unit of Myb:a helix-turn-helix-related motif with conserved tryptophans forming a hydrophobic core[J]. PNAS, 1992, 89(14):6428-6432.doi:10.1073/pnas.89.14.6428.

Please choose a citation manager

Content to export