转录因子GhWRKY21调控棉花耐高温机制研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-1061

摘要/Abstract

摘要：

目的探究棉花WRKY转录因子GhWRKY21在耐高温胁迫中的分子机制，揭示其在棉花抗逆中发挥的功能。方法综合运用生物信息学、分子生物学和生物化学的方法，通过病毒介导的基因沉默技术在棉花中构建GhWRKY21沉默的植株材料，并对其开展初步功能验证；利用农杆菌介导的叶盘转化法，在烟草中获得GhWRKY21过表达转基因株系，通过种子萌发实验、幼苗表型分析、叶片保卫细胞显微观察及高温胁迫下活性氧积累检测等试验，从表型、细胞和生理水平系统评估GhWRKY21在植物热应激响应中的功能。结果 GhWRKY21属于棉花WRKY转录因子家族中的IId亚族，其表达可受温度、H₂O₂及信号分子的诱导。功能研究表明，在棉花中沉默该基因可显著增强植株的耐热性；相反，在烟草中过表达GhWRKY21则导致转基因植株对高温更为敏感，具体表现为种子萌发率降低、根伸长减缓以及叶片保卫细胞气孔关闭能力减弱等现象。进一步的生理与分子证据表明，GhWRKY21可能通过调控活性氧（ROS）的积累，参与植物对高温胁迫的应答过程。结论 GhWRKY21在植物高温胁迫响应中起负调控作用，其通过调节ROS稳态影响植物抗逆性。

关键词: 棉花, WRKY转录因子, GhWRKY21, 烟草, 高温, ROS, 气孔

Abstract:

Objective To investigate the molecular mechanism of the cotton WRKY transcription factor GhWRKY21 under high-temperature stress and elucidate its functional role in cotton stress resistance. Method GhWRKY21-silenced cotton plants were constructed using virus-induced gene silencing (VIGS) and preliminarily functionally characterized. Concurrently, GhWRKY21-overexpressing transgenic tobacco lines were generated via Agrobacterium-mediated leaf disc transformation. Functional assessment of GhWRKY21 in heat-stress response was then conducted through a series of experiments spanning seed germination assays, seedling phenotypic analysis, microscopic examination of leaf guard cells, and measurement of reactive oxygen species (ROS) accumulation under high-temperature stress, thereby evaluating its role at phenotypic, cellular, and physiological levels. Result GhWRKY21 is a member of the IId subfamily of WRKY transcription factors in cotton. Its expression can be induced by temperature, H₂O₂, and signaling molecules. Functional analysis revealed that silencing GhWRKY21 in cotton significantly enhanced thermotolerance. Conversely, its overexpression in tobacco increased sensitivity to heat stress, as evidenced by reduced seed germination rates, suppressed root elongation, and impaired stomatal closure in leaf guard cells. Further physiological and molecular evidence suggests that GhWRKY21 may participate in the plant response to high-temperature stress by modulating the accumulation of ROS. Conclusion GhWRKY21 functions as a negative regulator in plant high-temperature stress responses by modulating ROS homeostasis, thereby influencing plant stress resistance.

Key words: Gossypium hirsutum, WRKY transcription factor, GhWRKY21, tobacco, heat, ROS, stomata

王佳玉, 王琛. 转录因子GhWRKY21调控棉花耐高温机制研究[J]. 生物技术通报, 2026, 42(10): 134-141.

WANG Jia-yu, WANG Chen. Mechanism of GhWRKY21 Transcription Factor in Regulating Cotton High Temperature Resistance[J]. Biotechnology Bulletin, 2026, 42(10): 134-141.

图/表 5

图1 不同物种WRKY21的氨基酸序列比对（A）和进化树分析（B）A：陆地棉GhWRKY21与拟南芥AtWRKY21、松树PmWRKY21、杨树PtWRKY21、红豆杉TcWRKY21的氨基酸序列同源性比对；黑色部分表示相同的氨基酸，WRKY结构域（60个氨基酸）用双向箭头标记，方框标注高度保守的WRKYGQK基序及C端保守序列；假定的核定位信号KKRK用****标记；B：显示WRKY家族的7个分组，用黑色矩形框标注GhWRKY21的位置，该进化树使用MEGA5.0构建

Fig. 1 Amino acid sequence alignment (A) and phylogenetic analysis (B) of WRKY21 from different speciesA: Amino acid sequence homology comparison of GhWRKY21 from Gossypium hirsutum, AtWRKY21 from Arabidopsis thaliana, PmWRKY21 from pine, PtWRKY21 from poplar, and TcWRKY21 from Taxus chinensis. The black regions indicate the same amino acids, where the 60-amino-acid WRKY domain is marked by a double-headed arrow. The boxed sequences are the highly conserved amino-acid sequence WRKYGQK and the conserved C-terminal motif. The putative nuclear localization signal, KKRK, is marked by asterisks (****). B: The phylogenetic tree shows the seven subgroups of the WRKY family. The position of GhWRKY21 is marked with a black rectangular box. The tree was constructed using MEGA 5.0

图2 棉花中GhWRKY21的表达模式分析数据为3次独立实验的平均值±标准误差（SE）；经Tukey’s HSD检验，误差线上不同字母表示组间差异具有统计学意义（P<0.05）。下同

Fig. 2 Expression pattern analysis of GhWRKY21 in cottonData are presented as the mean±standard error (SE) of three independent experiments. Different letters above the error bars indicate statistically significant differences among groups (P<0.05) based on Tukey’s HSD test. The same below

图3 GhWRKY21基因沉默增强棉花的耐热性A：GhWRKY21沉默棉花在高温胁迫处理（42 ℃，24 h）后的典型表型；B：GhWRKY21沉默棉花中GhWRKY21的表达水平；C：GhWRKY21沉默棉花在高温处理（42 ℃，24 h）后叶片的典型表型

Fig. 3 Silencing of GhWRKY21 enhances heat tolerance in cottonA: Representative phenotypes of GhWRKY21-silenced cotton plants after heat stress (42 ℃, 24 h). B: Relative expression of GhWRKY21 in GhWRKY21-silenced cotton. C: Representative leaf phenotypes of GhWRKY21-silenced cotton plants after heat treatment (42 ℃, 24 h)

图4 GhWRKY21过表达植株对热胁迫的敏感性增加A、B：空载体对照和转基因植株烟草种子在热处理（42 ℃）6 h后的萌发情况；C、D：空载体对照和转基因植株种子在萌发后热处理（42 ℃）6 d后的根长表型及数据统计；E：8周龄空载体对照和转基因植株在高温处理（42 ℃）12 h后的表型；F：8周龄空载体对照和转基因植株在高温处理（42 ℃）12 h后常规培养存活情况；G、H：高温处理下（42 ℃，12 h）空载体对照和转基因植株气孔闭合程度

Fig. 4 Overexpression of GhWRKY21 increases the sensitivity to heat stress in transgenic plantsA, B: Seed germination phenotype and germination rates of Vec and OE tobacco lines exposed to heat stress (42 ℃, 6 h). C, D: Root length phenotype and statistical analysis of empty vector control and transgenic seeds after germination, exposed to heat stress (42 ℃) for 6 days. E: Phenotypes of Vec and OE plants grown to 8 weeks after 12 h high-temperature treatment (42 ℃). F: Survival rates of 8-week-old Vec and OE after a 12 h high-temperature treatment (42 ℃) followed by recovery under normal growth conditions. G, H: Stomatal closure degree of Vec and OE lines under heat stress (42 ℃, 12 h)

图5 过表达GhWRKY21的转基因烟草对氧化应激敏感A：空载体对照和转基因植株叶片经高温处理后DAB和NBT染色的典型表型；B：空载体对照和转基因烟草植株在含不同浓度MV中培养时的表型；C：对图B中不同浓度下空载体对照和转基因烟草植株叶圆片叶绿素含量进行测定；经Tukey’s HSD检验，*P<0.05，** P<0.01，***P<0.001

Fig. 5 Overexpression of GhWRKY21 increases the sensitivity of transgenic tobacco plants to oxidative stressA: Representative phenotypes of leaves from Vec and OE plants showing DAB and NBT staining after heat treatment. B: Phenotypes of leaf discs obtained from Vec and OE plants after being incubated in different concentrations of MV. C: The chlorophyll content of tobacco leaf discs from empty vector control and transgenic plants under different concentrations was quantified in Fig. B. Tukey’s HSD test, * P<0.05, ** P<0.01, and *** P<0.001

参考文献 24

[1]	Yamasaki K, Kigawa T, Inoue M, et al. Structures and evolutionary origins of plant-specific transcription factor DNA-binding domains [J]. Plant Physiol Biochem, 2008, 46(3): 394-401.
[2]	Eulgem T, Rushton PJ, Robatzek S, et al. The WRKY superfamily of plant transcription factors [J]. Trends Plant Sci, 2000, 5(5): 199-206.
[3]	Jiang YJ, Liang G, Yu DQ. Activated expression of WRKY57 confers drought tolerance in Arabidopsis [J]. Mol Plant, 2012, 5(6): 1375-1388.
[4]	Yan HR, Jia HH, Chen XB, et al. The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production [J]. Plant Cell Physiol, 2014, 55(12): 2060-2076.
[5]	Yan Y, Jia HH, Wang F, et al. Overexpression of GhWRKY27a reduces tolerance to drought stress and resistance to Rhizoctonia solani infection in transgenic Nicotiana benthamiana [J]. Front Physiol, 2015, 6: 265.
[6]	胡启瑞, 宋桂成, 王雪姣, 等. 高温对陆地棉花粉萌发及棉铃发育的影响 [J]. 新疆农业大学学报, 2017, 40(1): 1-7.
	Hu QR, Song GC, Wang XJ, et al. Effects of high temperature on pollen germination and boll development in upland cotton [J]. J Xinjiang Agric Univ, 2017, 40(1): 1-7.
[7]	刘群, 陈振, 张巨松, 等. 高温胁迫对海岛棉光合生理及棉铃发育的影响 [J]. 西北植物学报, 2020(9): 1574-1581.
	Liu Q, Chen Z, Zhang JS, et al. Effect of high temperature stress on photosynthetic physiology and boll development of island cotton [J]. Acta Bot Boreali Occidentalia Sin, 2020(9): 1574-1581.
[8]	Cottee NS, Tan DKY, Bange MP, et al. Multi-level determination of heat tolerance in cotton (Gossypium hirsutum L.) under field conditions [J]. Crop Sci, 2010, 50(6): 2553-2564.
[9]	Song GC, Wang MM, Zeng B, et al. Anther response to high-temperature stress during development and pollen thermotolerance heterosis as revealed by pollen tube growth and in vitro pollen vigor analysis in upland cotton [J]. Planta, 2015, 241(5): 1271-1285.
[10]	Min L, Li YY, Hu Q, et al. Sugar and auxin signaling pathways respond to high-temperature stress during anther development as revealed by transcript profiling analysis in cotton [J]. Plant Physiol, 2014, 164(3): 1293-1308.
[11]	Pettigrew WT. The effect of higher temperatures on cotton lint yield production and fiber quality [J]. Crop Sci, 2008, 48(1): 278-285.
[12]	王佳玉. 棉花转录因子GhWRKY21调控植株抗旱和耐高温的分子机理研究 [D]. 泰安: 山东农业大学, 2020.
	Wang JY. Moleculor mechanism of cotton GhWRKY21 transcription factor regulating plant drought and high temperature resistance [D]. Tai’an: Shandong Agricultural University, 2020.
[13]	Journot-Catalino N, Somssich IE, Roby D, et al. The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana [J]. Plant Cell, 2006, 18(11): 3289-3302.
[14]	Cai RH, Zhao Y, Wang YF, et al. Overexpression of a maize WRKY58 gene enhances drought and salt tolerance in transgenic rice [J]. Plant Cell Tiss Organ Cult, 2014, 119(3): 565-577.
[15]	Wang CT, Ru JN, Liu YW, et al. Maize WRKY transcription factor ZmWRKY106 confers drought and heat tolerance in transgenic plants [J]. Int J Mol Sci, 2018, 19(10): 3046.
[16]	Ma JL, Wang YY, Hong YH, et al. SlWRKY55 coordinately acts with SlVQ11 to enhance tomato thermotolerance by activating SlHsfA2 [J]. Plant J, 2024, 119(6): 2904-2918.
[17]	Zhang ZJ, Yang C, Xi J, et al. The MdHSC70-MdWRKY75 module mediates basal apple thermotolerance by regulating the expression of heat shock factor genes [J]. Plant Cell, 2024, 36(9): 3631-3653.
[18]	Shi WN, Hao LL, Li J, et al. The Gossypium hirsutum WRKY gene GhWRKY39-1 promotes pathogen infection defense responses and mediates salt stress tolerance in transgenic Nicotiana benthamiana [J]. Plant Cell Rep, 2014, 33(3): 483-498.
[19]	Wang JY, Wang LJ, Yan Y, et al. GhWRKY21 regulates ABA-mediated drought tolerance by fine-tuning the expression of GhHAB in cotton [J]. Plant Cell Rep, 2021, 40(11): 2135-2150.
[20]	Mittler R, Zandalinas SI, Fichman Y, et al. Reactive oxygen species signalling in plant stress responses [J]. Nat Rev Mol Cell Biol, 2022, 23(10): 663-679.
[21]	Averill-Bates D. Reactive oxygen species and cell signaling. Review [J]. Biochim Biophys Acta BBA Mol Cell Res, 2024, 1871(2): 119573.
[22]	Li SH, Liu S, Zhang Q, et al. The interaction of ABA and ROS in plant growth and stress resistances [J]. Front Plant Sci, 2022, 13: 1050132.
[23]	Waszczak C, Carmody M, Kangasjärvi J. Reactive oxygen species in plant signaling [J]. Annu Rev Plant Biol, 2018, 69: 209-236.
[24]	Miller G, Suzuki N, Ciftci-Yilmaz S, et al. Reactive oxygen species homeostasis and signalling during drought and salinity stresses [J]. Plant Cell Environ, 2010, 33(4): 453-467.