Abstract WRKY transcription factors are a class of plant-specific proteins involved in the regulation of growth and development, secondary metabolism, senescence and response to biotic and abiotic stresses.The present study firstly cloned the cDNA sequence of WRKY72 (GenBank No.KC246585) from oilseed rape (Brassica napus) by the method of reverse transcription PCR (RT-PCR).The encoded protein of BnaWRKY72 included a conserved WRKY domain and a C2H2-type zinc finger motif.Subcellular localization study using green fluorescence protein revealed that BnaWRKY72 transcription factor localized in the nucleus.Dual luciferase reporter assay detected that BnaWRKY72 had trans-activation activity towards firefly luciferase gene driven by tandem repeat of W-box element.The expression of BnaWRKY72 was examined by qRT-PCR under various hormones, biotic and abiotic stresses.It was found that BnaWRKY72 responded to cold stress and Sclerotinia sclerotiorum infection.Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays were used to screen and confirm the interacting proteins of WRKY72 which included homologous BnaWRKY31 and BnaWRKY42 in the same subclade and itself.It was also found that BnaWRKY72 could interact with small ubiquitin-like modifier (SUMO) 3, suggesting that SUMO modification might participate in regulating of BnaWRKY72 activity.The present study explored the molecular characteristics of BnaWRKY72 for the first time, which provides a reference for further study of its stress resistance function and related molecular mechanism.
LI Qi,LI Cui,WANG, et al. Gene Cloning, Expression Analysis and Identification of Interacting Proteins of Transcription Factor WRKY72 in Oilseed Rape (Brassica napus)[J]. 农业生物技术学报, 2019, 27(5): 761-772.
[1] 陈思雀, 翁群清, 曹红瑞, 等.2017.WRKY转录因子在生物和非生物胁迫中的功能和调控机理的研究进展[J].农业生物技术学报, 25(4): 668-682. (Chen S Q, Weng Q Q, Cao H R, et al.2017.The functions and regulation mechanisms of WRKY transcription factors in response to biotic and abiotic stresses[J].Journal of Agricultural Biotechnology, 25(4): 668-682) [2] Asai T, Tena G, Plotnikova J, et al.2002.MAP kinase signalling cascade in Arabidopsis innate immunity[J].Nature, 415(6875): 977-983. [3] Andreasson E, Jenkins T, Brodersen P, et al.2005.The MAP kinase substrate MKS1 is a regulator of plant defense responses[J].The EMBO Journal, 24(14): 2579-2589. [4] Augustine R C, Vierstra R D.2018.SUMOylation: Re-wiring the plant nucleus during stress and development[J].Current Opinion in Plant Biology, 45(Pt A): 143-154. [5] Banerjee A, Roychoudhury A.2015.WRKY proteins: Signaling and regulation of expression during abiotic stress responses[J].Scientific World Journal.DOI: 10.1155/2015/807560 [6] Buscaill P, Rivas S.2014.Transcriptional control of plant defence responses[J].Current Opinion in Plant Biology, 8-20: 35-46. [7] Chen C, Chen Z.2002.Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor[J].Plant Physiology, 129(2): 706-716. [8] Chen X, Liu J, Lin G, et al.2013.Overexpression of AtWRKY28 and AtWRKY75 in Arabidopsis enhances resistance to oxalic acid and Sclerotinia sclerotiorum[J].Plant Cell Reports, 32(10): 1589-1599. [9] Chi Y, Yang Y, Zhou Y, et al.2013.Protein-protein interactions in the regulation of WRKY transcription factors[J].Molecular Plant, 6(2): 287-300. [10] Chujo T, Kato T, Yamada K, et al.2008.Characterization of an elicitor-induced rice WRKY gene, OsWRKY71[J].Bioscience Biotechnology and Biochemistry, 72(1): 240-245. [11] Elrouby N.2014.Extent and significance of non-covalent SUMO interactions in plant development[J].Plant Signaling & Behavior, 9(2): e27948. [12] Eulgem T, Rushton P, Robatzek S, et al.2000.The WRKY superfamily of plant transcription factors[J].Trends in Plant Science, 5(5): 199-206. [13] He Y, Mao S, Gao Y, et al.2016.Genome-wide identification and expression analysis of WRKY transcription factors under multiple stresses in Brassica napus[J].PLoS ONE, 11(6): e0157558. [14] Hellens R P, Allan A C, Friel E N, et al.2005.Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants[J].Plant Methods, 1: 13. [15] Hendriks I A, Vertegaal A C.2016.A comprehensive compilation of SUMO proteomics[J].Nature Reviews Molecular Cell Biology, 17(9): 581-595. [16] Ishiguro S, Nakamura K.1994.Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5' upstream regions of genes coding for sporamin and beta-amylase from sweet potato[J].Molecular & General Genetics, 244(6): 563-571. [17] Ishihama N, Yoshioka H.2012.Post-translational regulation of WRKY transcription factors in plant immunity[J].Current Opinion in Plant Biology, 15(4): 431-437. [18] Jiang J, Ma S, Ye N, et al.2017.WRKY transcription factors in plant responses to stresses[J].Journal of Integrative Plant Biology, 59(2): 86-101. [19] Lahaye T.2002.The Arabidopsis RRS1-R disease resistance gene-uncovering the plant's nucleus as the new battlefield of plant defense[J].Trends in Plant Science, 7(10): 425-427. [20] Li J, Brader G, Palva E.2004.The WRKY70 transcription factor: A node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense[J].Plant Cell, 16(2): 319-331. [21] Liang W, Yang B, Yu B J, et al.2013.Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.)[J].BMC Genomics, 14(1): 392. [22] Liu F, Li X, Wang M, et al.2018.Interactions of WRKY15 and WRKY33 transcription factors and their roles in the resistance of oilseed rape to Sclerotinia infection[J].Plant Biotechnology Journal, 16(4): 911-925. [23] Liu X Q, Bai X Q, Qian Q, et al.2005.OsWRKY03, a rice transcriptional activator that functions in defense signaling pathway upstream of OsNPR1[J].Cell Research, 15(8): 593-603. [24] Mao G, Meng X, Liu Y, et al.2011.Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis[J].Plant Cell, 23(4): 1639-1653. [25] Miura K, Jin J B, Hasegawa P M.2007.Sumoylation, a post-translational regulatory process in plants[J].Current Opinion in Plant Biology, 10(5): 495-502. [26] Neik T X, Barbetti M J, Batley J.2017.Current status and challenges in identifying disease resistance genes in Brassica napus[J].Frontiers Plant Science, 8: 1788. [27] Pfaffl M W.2001.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research, 29(9): e45. [28] Qiu D, Xiao J, Ding X, et al.2007.OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling[J].Molecular Plant-Microbe Interactions, 20(5): 492-499. [29] Qiu D, Xiao J, Xie W, et al.2008.Rice gene network inferred from expression profiling of plants overexpressing OsWRKY13, a positive regulator of disease resistance[J].Molecular Plant, 1(3): 538-551. [30] Sun Y, Yu D.2015.Activated expression of AtWRKY53 negatively regulates drought tolerance by mediating stomatal movement[J].Plant Cell Reports, 34(8): 1295-1306. [31] Ulker B, Somssich I.2004.WRKY transcription factors: From DNA binding towards biological function[J].Current Opinion in Plant Biology, 7(5): 491-498. [32] Van Den Burg H, Takken F.2010.SUMO-, MAPK-, and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity[J].Plant Signaling & Behavior, 5(12): 1597-1601. [33] Wei K, Chen J, Chen Y, et al.2012.Molecular phylogenetic and expression analysis of the complete WRKY transcription factor family in maize[J].DNA Research, 19(2): 153-164. [34] Wu K, Guo Z, Wang H, et al.2005.The WRKY family of transcription factors in rice and Arabidopsis and their origins[J].DNA Research, 12(1): 9-26. [35] Xie Z, Zhang Z L, Zou X, et al.2006.Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells[J].Plant Journal, 46(2): 231-242. [36] Xu L, Jin L, Long L, et al.2012.Overexpression of GbWRKY1 positively regulates the Pi starvation response by alteration of auxin sensitivity in Arabidopsis[J].Plant Cell Reports, 31(12): 2177-2188. [37] Yamasaki K, Kigawa T, Seki M, et al.2013.DNA-binding domains of plant-specific transcription factors: Structure, function, and evolution[J].Trends in Plant Science, 18(5): 267-276. [38] Yamasaki K, Kigawa T, Watanabe S, et al.2012.Structural basis for sequence-specific DNA recognition by an Arabidopsis WRKY transcription factor[J].Journal of Biology Chemistry, 287(10): 7683-7691. [39] Yang B, Jiang Y, Rahman M, et al.2009.Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments[J].BMC Plant Biology, 9: 68. [40] Yang L, Ye C, Zhao Y, et al.2018.An oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF[J].Planta, 247(6): 1323-1338. [41] Zhang Y, Wang L.2005.The WRKY transcription factor superfamily: Its origin in eukaryotes and expansion in plants[J].BMC Evolutionary Biology.DOI: 10.1186/1471-2148-5-1 [42] Zheng Z, Qamar S A, Chen Z, et al.2006.Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens[J].Plant Journal, 48(4): 592-605.
