普通小麦转录因子基因TaWRKY35的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2016.12.001

摘要/Abstract

摘要： 【目的】非生物逆境是制约小麦生产的主要因素。WRKY转录因子是植物特有的转录因子家族，参与对多种非生物逆境胁迫的应答。普通小麦基因组中至少含有200个WRKY，目前仅对少数成员的功能进行了鉴定。通过克隆小麦WRKY转录因子TaWRKY35并揭示其功能，为改良小麦的抗逆性提供基因资源。【方法】利用小麦全长cDNA数据信息，从强抗旱小麦品种旱选10号中克隆逆境胁迫应答转录因子基因TaWRKY35；采用实时定量PCR技术，分析目标基因在不同发育时期、不同组织器官中的表达水平，以及在PEG-6000、NaCl、ABA和低温等逆境胁迫下的表达模式；构建目标基因与GFP融合的表达载体，转化小麦原生质体，以确定目标蛋白在细胞中的作用位置；构建TaWRKY35过表达载体，通过农杆菌介导的方法转化拟南芥，揭示目标基因的功能。【结果】TaWRKY35的开放阅读框全长1 134 bp，编码377个氨基酸的蛋白。TaWRKY35的N端有一个典型的WRKY结构域，C端有一个C₂HC型的锌指结构域，属于WRKY家族第Ⅲ亚家族。测序发现TaWRKY35编码区序列非常保守，在32份多态性较高的六倍体小麦材料中未检测到DNA多态性。TaWRKY35在小麦的不同发育时期、不同组织中均有表达，其中在幼苗根基部表达量最高，约为苗期叶片中表达量的26倍；其次为幼芽根基，约为苗期叶片中表达量的21倍；接下来依次为孕穗期茎节、孕穗期穗、芽期根、孕穗期根、苗期根、胚芽、孕穗期叶片，而在幼苗叶片中表达水平最低；同时在ABA、NaCl、PEG和低温胁迫条件下，小麦幼苗TaWRKY35的表达量均显著上升，表明TaWRKY35参与对4种逆境胁迫的应答，但在不同胁迫条件下表达模式差异显著，其中对盐胁迫最敏感。亚细胞定位发现，TaWRKY35特异定位在细胞核上，是典型的核定位蛋白。在高盐胁迫条件下，TaWRKY35过表达转基因拟南芥子叶白化率显著低于对照，TaWRKY35过表达转基因拟南芥的细胞膜稳定性和幼苗存活率均显著高于野生型对照，表明TaWRKY35能增强植物的耐盐性。【结论】TaWRKY35编码蛋白含有WRKY和C₂HC结构域，为WRKY家族第Ⅲ亚家族成员。TaWRKY35在小麦发育的不同时期、不同组织中均有表达，且受ABA、PEG、NaCl及低温等逆境胁迫诱导。过表达TaWRKY35能显著提高转基因拟南芥的耐盐性。

关键词: 普通小麦, WRKY转录因子基因, 非生物胁迫, 耐盐性

Abstract: 【Objective】Abiotic stresses are major limitations to wheat production worldwide. Transcription factors play crucial roles in abiotic stress signaling in plants. It is predicted that there are at least 200 WRKY genes in common wheat genomes, yet only a few of them have been functionally characterized. The aim of this study is to decipher the roles of WRKY transcription factors in abiotic stress signaling and facilitate the utilization of WRKY genes in the improvement of abiotic stress tolerance in wheat.【Method】A wheat WRKY gene designated TaWRKY35 was cloned via the wheat full-length cDNA libraries. Quantitative real-time PCR was performed to identify the dynamic expression of target gene in different tissues at various developmental stages, and to characterize the transcriptional patterns responding to ABA, PEG, NaCl and low temperature treatments in wheat. To probe the subcellular location of TaWRKY35, the construct encoding TaWRYK35::GFP fusion protein was transferred into wheat protoplast by PEG mediated method. To characterize the function of TaWRYK35, target gene driven by the 35S promoter was delivered into Arabidopsis by Agrobacterium mediated method. 【Result】The cDNA of TaWRYK35 contains an 1 134 bp open reading frame, encoding a 377- amino acid protein. TaWRYK35 possesses a typical WRKY domain in the N-terminal and a C2HC type zinc finger domain in the C-terminal, belonging to group III of WRKY family. In 32 hexaploid wheat materials of highly polymorphic, TaWRKY35 coding region sequence is very conservative. The dynamic expression of TaWRKY35 was identified in different tissues at various developmental stages, and the highest expression occurred in the root base of seedling, while the lowest expression was observed in seedling leaf. Furthermore, its transcript was inducible by ABA, PEG, NaCl and low temperature treatments, yet the expression patterns to difference stress varied significantly. Subcellular localization indicated that TaWRKY35 specifically located in the nucleus. Overexpression of TaWRKY35 resulted in enhanced tolerance to high salinity, supported by improved cell membrane stability and survival rate relative to wild type Arabidopsis. 【Conclusion】TaWRKY35 transcription factor contains a WRKY and a C2HC type zinc finger domain, belonging to subgroup III of the WRKY family. The expression of TaWRKY35 occurs in different tissues at various developmental stages, and TaWRKY35 is an abiotic stress responsive gene. Overexpression of TaWRKY35 confers remarkably enhanced tolerance to high salinity.

Key words: common wheat, WRKY transcription factor gene, abiotic stress, salt tolerance

刘自成，苗丽丽，王景一，杨德龙，毛新国，景蕊莲. 普通小麦转录因子基因TaWRKY35的克隆及功能分析[J]. 中国农业科学, 2016, 49(12): 2245-2254.

LIU Zi-cheng, MIAO Li-li, WANG Jing-yi, YANG De-long, MAO Xin-guo, JING Rui-lian. Cloning and Characterization of Transcription Factor TaWRKY35 in Wheat (Triticum aestivum)[J]. Scientia Agricultura Sinica, 2016, 49(12): 2245-2254.

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参考文献

[1] Suzuki N, Rivero R M, Shulaev V, Blumwald E, Mittler R. Abiotic and biotic stress combinations. New Phytologist, 2014, 203: 32-43.

[2] Mickelbart M V, Hasegawa P M, Bailey-Serres J. Genetic mechanisms of abiotic stress tolerance that translate to crop yield stability. Nature Reviews Genetics, 2015, 16: 237-251.

[3] Ishiguro S, Nakamura K. 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. Molecular and General Genetics, 1994, 244: 563-571.

[4] Eulgem T, Rushton P J, Robatzek S, Somssich I E. The WRKY superfamily of plant transcription factors. Trends in Plant Science, 2000, 5: 199-206.

[5] Maeo K, Hayashi S, Kojima-Suzuki H, Morikami A, Nakamura K. Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins. Bioscience, Biotechnology, and Biochemistry, 2001, 65: 2428-2436.

[6] Wu K L, Guo Z J, Wang H H, Li J. The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Research, 2005, 12: 9-26.

[7] Ciolkowski I, Wanke D, Birkenbihl R P, Somssich I E. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function. Plant Molecular Biology, 2008, 68: 81-92.

[8] Rushton P J, Somssich I E, Ringler P, Shen Q J. WRKY transcription factors. Trends in Plant Science, 2010, 15: 247-258.

[9] Rushton D L, Tripathi P, Rabara R C, Lin J, Ringler P, Boken A K, Langum T J, Smidt L, Boomsma D D, Emme N J, Chen X, Finer J J, Shen Q J, Rushton P J. WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnology Journal, 2012, 10: 2-11.

[10] Li C, Lü J, Zhao X, Ai X, Zhu X, Wang M, Zhao S, Xia G. TaCHP: a wheat zinc finger protein gene down-regulated by abscisic acid and salinity stress plays a positive role in stress tolerance. Plant Physiology, 2010, 154: 211-221.

[11] Li H, Xu Y, Xiao Y, Zhu Z, Xie X, Zhao H, Wang Y. Expression and functional analysis of two genes encoding transcription factors, VpWRKY1 and VpWRKY2, isolated from Chinese wild Vitis pseudoreticulata. Planta, 2010, 232: 1325-1337.

[12] Jiang Y, Deyholos M K. Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Molecular Biology, 2009, 69: 91-105.

[13] Li S, Fu Q, Chen L, Huang W, Yu D. Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta, 2011, 233: 1237-1252.

[14] Li S, Fu Q, Huang W, Yu D. Functional analysis of an Arabidopsis transcription factor WRKY25 in heat stress. Plant Cell Reports, 2009, 28: 683-693.

[15] Sun J, Hu W, Zhou R, Wang L, Wang X, Wang Q, Feng Z, Li Y, Qiu D, He G, Yang G. The Brachypodium distachyon BdWRKY36 gene confers tolerance to drought stress in transgenic tobacco plants. Plant Cell Reports, 2015, 34: 23-35.

[16] Zhou Q Y, Tian A G, Zou H F, Xie Z M, Lei G, Huang J, Wang C M, Wang H W, Zhang J S, Chen S Y. Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnology Journal, 2008, 6: 486-503.

[17] Wu X, Shiroto Y, Kishitani S, Ito Y, Toriyama K. Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter. Plant Cell Reports, 2009, 28: 21-30.

[18] Raineri J, Wang S, Peleg Z, Blumwald E, Chan R L. The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress. Plant Molecular Biology, 2015, 88: 401-413.

[19] Okay S, Derelli E, Unver T. Transcriptome-wide identification of bread wheat WRKY transcription factors in response to drought stress. Molecular Genetics and Genomics, 2014, 289: 765-781.

[20] Wang X, Zeng J, Li Y, Rong X, Sun J, Sun T, Li M, Wang L, Feng Y, Chai R, Chen M, Chang J, Li K, Yang G, He G. Expression of TaWRKY44, a wheat WRKY gene, in transgenic tobacco confers multiple abiotic stress tolerances. Frontiers in Plant Science, 2015, 6: 615.

[21] Wang C, Deng P, Chen L, Wang X, Ma H, Hu W, Yao N, Feng Y, Chai R, Yang G, He G. A wheat WRKY transcription factor TaWRKY10 confers tolerance to multiple abiotic stresses in transgenic tobacco. PLoS One, 2013, 8: e65120.

[22] Niu C F, Wei W, Zhou Q Y, Tian A G, Hao Y J, Zhang W K, Ma B, Lin Q, Zhang Z B, Zhang J S, Chen S Y. Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants. Plant Cell and Environment, 2012, 35: 1156-1170.

[23] Qin Y, Tian Y, Liu X. A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana. Biochemical and Biophysical Research Communications, 2015, 464: 428-433.

[24] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25: 402-408.

[25] Pandey S P, Somssich I E. The role of WRKY transcription factors in plant immunity. Plant Physiology, 2009, 150: 1648-1655.

[26] Zou C, Jiang W, Yu D. Male gametophyte-specific WRKY34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis. Journal of Experimental Botany, 2010, 61: 3901-3914.

[27] Wang F, Hou X, Tang J, Wang Z, Wang S, Jiang F, Li Y. A novel cold-inducible gene from Pak-choi (Brassica campestris ssp. chinensis), BcWRKY46, enhances the cold, salt and dehydration stress tolerance in transgenic tobacco. Molecular Biology Reports, 2012, 39: 4553-4564.

[28] Xiong X, James V A, Zhang H, Altpeter F. Constitutive expression of the barley HvWRKY38 transcription factor enhances drought tolerance in turf and forage grass (Paspalum notatum Flugge). Molecular Breeding, 2009, 25: 419-432.

[29] Xu Y, Hu W, Liu J, Zhang J, Jia C, Miao H, Xu B, Jin Z. A banana aquaporin gene, MaPIP1;1, is involved in tolerance to drought and salt stresses. BMC Plant Biology, 2014, 14: 59.