|
|
|
| cDNA Cloning and Expression Characterization of WRKY69 Gene in Oilseed Rape (Brassica napus) |
| YAO Ling-Fang*, CUI Xing*, LIANG Wan-Wan, GAO Shi-Dong, ZHAO Pei-Yu, CHEN Qin-Qin, YAN Jing-Li, LI Cui, JIANG Yuan-Qing, YANG Bo** |
| College of Life Sciences, Northwest A&F University, Yangling 712100, China |
|
|
|
|
Abstract WRKY transcription factors constitute a class of proteins involved in growth, development, stress and defense responses in plants. In this study, the cDNA sequence of WRKY69 was cloned through reverse transcription PCR (RT-PCR) from oilseed rape (Brassica napus) (GenBank No. KC246583). Sequence analysis indicated the encoded protein had 279 amino acids with a single WRKY domain and a C2H2-type zinc finger motif, which belonged to GroupⅡ. Yeast transcriptional activity analysis showed that BnaWRKY69 might have transcriptional activation activity, and its activity was mainly determined by the carboxyl terminus. GFP-based subcellular localization showed that BnaWRKY69 was located not only in the nucleus but also at the cell membrane. A prediction of BnaWRKY69 protein sequence suggested it might undergo 2 types of post-translational modifications, myristoylation and farnesylation, which possibly determined its special membrane localization. Under hydroxylamine (NH2OH) treatment, the BnaWRKY69 signals at the plasma membrane decreased gradually and even disappeared. The transcriptional level of BnaWRKY69 under various abiotic stresses, hormone and Sclerotinia sclerotiorum treatments was analyzed by quantitative real-time PCR (qRT-PCR). It was found that BnaWRKY69 was specifically induced by jasmonic acid (JA) and S. sclerotiorum. Further detection by qRT-PCR showed that transient overexpression of BnaWRKY69 gene in tobacco (Nicotiana benthamiana) leaves caused significant up-regulation of Pathogenesis-related 1a (PR1a), PR2 and Non-expressor of PR1 (NPR1) and down-regulation of Plant defense 1.2 (PDF1.2). In conclusion, this study preliminarily analyzed the basic features of BnaWRKY69 in different hormones and abiotic stress treatments and molecular mechanism responding to JA and S. sclerotiorum, which could provide a reference to breed oilseed rape varieties with improved disease resistance.
|
|
Received: 31 July 2019
|
|
|
|
Corresponding Authors:
**yangwl@nwafu.edu.cn
|
|
|
|
[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] 李琪, 李翠, 王琛, 等. 2019. 甘蓝型油菜转录因子WRKY72的基因克隆、表达分析及互作蛋白鉴定[J]. 农业生物技术学报, 27(5): 761-772.
(Li Q, Li C, Wang C, et al.2019. Gene cloning, expression analysis and identification of interacting proteins of transcription factor WRKY72 in oilseed rape (Brassica napus)[J]. Journal of Agricultural Biotechnology, 27(5): 761-772.)
[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] 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.
[5] Chisholm S T, Coaker G, Day B, et al.2006. Host-microbe interactions: Shaping the evolution of the plant immune response[J]. Cell, 124(4): 803-814.
[6] Duan M, Zhang R, Zhu F, et al.2017. A lipid-anchored NAC transcription factor is translocated into the nucleus and activates glyoxalase Ⅰ expression during drought stress[J]. The Plant Cell, 29(7): 1748-1772.
[7] Eulgem T, Somssich I E.2007. Networks of WRKY transcription factors in defense signaling[J]. Current Opinion in Plant Biology, 10(4): 366-371.
[8] Eulgem T, Rushton P J, Robatzek S, et al.2000. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science, 5(5): 199-206.
[9] Jiang Y, Deyholos M K.2009. Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses[J]. Plant Molecular Biology, 69(1-2): 91-105.
[10] Jiang Y, Liang G, Yu D.2012. Activated expression of WRKY57 confers drought tolerance in Arabidopsis[J]. Molecular Plant, 5(6): 1375-1388.
[11] Jones J D, Dangl J L.2006. The plant immune system[J]. Nature, 444(7117): 323-329.
[12] Lai Z, Li Y, Wang F, et al.2011. Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense[J]. The Plant Cell, 23(10): 3824-3841.
[13] 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: 392.
[14] 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.
[15] Martin M L, Busconi L.2000. Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation[J]. The Plant Journal 24(4): 429-435.
[16] Morrison D F, O'Brien P J, Pepperberg D R.1991. Depalmitylation with hydroxylamine alters the functional properties of rhodopsin[J]. The Journal of Biological Chemistry, 266(30): 20118-20123.
[17] Nadolski M J, Linder M E.2007. Protein lipidation[J]. The FEBS Journal 274(20): 5202-5210.
[18] Neik T X, Barbetti M J, Batley J.2017. Current status and challenges in identifying disease resistance genes in Brassica napus[J]. Frontiers in Plant Science, 8: 1788.
[19] Qiao Z, Li C L, Zhang W.2016. WRKY1 regulates stomatal movement in drought-stressed Arabidopsis thaliana[J]. Plant Molecular Biology, 91(1-2): 53-65.
[20] Qiu J L, Fiil B K, Petersen K, et al.2008. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus[J]. The EMBO Journal, 27(16): 2214-2221.
[21] Rushton P J, Somssich I E, Ringler P, et al.2010. WRKY transcription factors[J]. Trends in Plant Science, 15(5): 247-258.
[22] Shen Q H, Saijo Y, Mauch S, et al.2007. Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses[J]. Science, 315(5815): 1098-1103.
[23] Wang Z, Fang H, Chen Y, et al.2014. Overexpression of BnWRKY33 in oilseed rape enhances resistance to Sclerotinia sclerotiorum[J]. Molecular Plant Pathology, 15(7): 677-689.
[24] Yang B, Jiang Y, Rahman M H, 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.
[25] 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.
[26] Zhang Y, Wang L.2005. The WRKY transcription factor superfamily: Its origin in eukaryotes and expansion in plants[J]. BMC Evolutionary Biology, 5: 1.
[27] Zheng Z, Qamar S A, Chen Z, et al.2006. Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens[J]. The Plant Journal, 48(4): 592-605. |
|
|
|
