褐藻寡糖诱导CsWRKY46调控黄瓜幼苗响应冷胁迫及转录组分析

doi:10.16420/j.issn.0513-353x.2025-0354

摘要/Abstract

摘要：

为探究褐藻寡糖诱导CsWRKY46基因调控黄瓜幼苗响应冷胁迫的分子机制，以黄瓜‘CU2’野生型和CsWRKY46基因敲除型为材料，通过喷施0.8%褐藻寡糖预处理后进行4 ℃冷胁迫处理，分析褐藻寡糖对黄瓜幼苗抗氧化酶活性、丙二醛含量以及渗透调节物质含量的影响，并进行转录组测序。结果表明，褐藻寡糖预处理显著提高了冷胁迫下黄瓜幼苗的抗氧化能力和耐冷性。转录组测序结果显示，褐藻寡糖诱导的差异表达基因主要富集在植物激素信号转导和蛋白质加工等途径，其中脱落酸和油菜素内酯信号途径相关基因的表达发生显著变化。综上，褐藻寡糖可能通过调控CsWRKY46基因及其下游信号途径，增强黄瓜幼苗对冷胁迫的耐受性。

关键词: 黄瓜, WRKY, 冷胁迫, 褐藻寡糖, 转录组测序

Abstract:

This paper aims to explore the molecular mechanism of CsWRKY46 gene induced by alginate oligosaccharides in cucumber response to cold stress. In this study，cucumber CU2（wild type） and CsWRKY46 gene knockout type were used as materials，and 0.8% alginate oligosaccharides was sprayed before 4 ℃ cold stress treatment. The effects of alginate oligosaccharides on antioxidant enzyme activity，malondialdehyde content and osmotic adjustment substance content of cucumber seedlings were analyzed，and transcriptome sequencing analysis was carried out. The results showed that the pretreatment of alginate oligosaccharide significantly improved the antioxidant capacity and cold tolerance of cucumber seedlings under cold stress. Transcriptome sequencing results showed that the differentially expressed genes induced by alginate oligosaccharides were mainly enriched in plant hormone signal transduction and protein processing，among which the gene expression of abscisic acid and brassinolide signaling pathways was significantly up-regulated. These results suggest that alginate oligosaccharides may enhance the tolerance of cucumber to cold stress by regulating CsWRKY46 gene and its downstream signaling pathway.

Key words: cucumber, WRKY, cold stress, alginate oligosaccharides, transcriptome sequencing

朱芳漪, 苏红玲, 顾翎禾, 张冲, 逄洪波, 佟德利, 张颖. 褐藻寡糖诱导CsWRKY46调控黄瓜幼苗响应冷胁迫及转录组分析[J]. 园艺学报, 2026, 53(5): 1285-1300.

ZHU Fangyi, SU Hongling, GU Linghe, ZHANG Chong, PANG Hongbo, TONG Deli, ZHANG Ying. Alginate Oligosaccharides Induced CsWRKY46 to Regulate Cucumber Response to Cold Stress and Transcriptome Analysis[J]. Acta Horticulturae Sinica, 2026, 53(5): 1285-1300.

图/表 10

参考文献 38

[1]	Anantharaman S, Padmarajaiah N, Al-Tayar N G S, Shrestha A K. 2017. Ninhydrin-sodium molybdate chromogenic analytical probe for the assay of amino acids and proteins. Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,173:897-903.
[2]	Bai Ruyi. 2023. Effects of foliar spraying melatonin on growth,yield and quality of pumpkin seedlings under cold stress[M. D. Dissertation]. Xianyang: Northwest A & F University. (in Chinese)
	白如意. 2023. 叶面喷施褪黑素对低温胁迫下南瓜幼苗生长及产量和品质的影响[硕士论文]. 咸阳: 西北农林科技大学.
[3]	Bai X, Liu P, Zhu F, Zhang C, Pang H, Zhang Y. 2025. CsWRKY46 is involved in the regulation of cucumber salt stress by regulating abscisic acid and modulating cellular reactive oxygen species. Horticulturae, 11 (3):251. doi: 10.3390/horticulturae11030251 URL
[4]	Boriboonkaset T, Theerawitaya C, Yamada N, Pichakum A, Supaibulwatana K, Cha-Um S, Takabe T, Kirdmanee C. 2013. Regulation of some carbohydrate metabolism-related genes,starch and soluble sugar contents,photosynthetic activities and yield attributes of two contrasting rice genotypes subjected to salt stress. Protoplasma, 250 (5):1157-1167. doi: 10.1007/s00709-013-0496-9 pmid: 23558902
[5]	Chen Hongyan, Li Xiao’er, Li Zhongguang. 2022. Sugar signaling and its role in plant response to environmental stress. Biotechnology Bulletin, 38 (7):80-89. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2021-1289
	陈宏艳, 李小二, 李忠光. 2022. 糖信号及其在植物响应逆境胁迫中的作用. 生物技术通报, 38 (7):80-89. doi: 10.13560/j.cnki.biotech.bull.1985.2021-1289
[6]	Chen L, Zhao Y, Xu S, Zhang Z, Xu Y, Zhang J, Chong K. 2018. Osmads57 together with ostb 1 coordinates transcription of its target os94 and d14 to switch its organogenesis to defense for cold adaptation in rice. New Phytologist, 218 (1):219-231. doi: 10.1111/nph.2018.218.issue-1 URL
[7]	Cutler S R, Rodríguez P L, Finkelstein R, Abrams S R. 2010. Abscisic acid:Emergence of a core signaling network. Annual Reviews of Plant Biology,61:651-679.
[8]	Deng X G, Zhu T, Zhang D W, Lin H H. 2015. The alternative respiratory pathway is involved in brassinosteroid-induced environmental stress tolerance in nicotiana benthamiana. Journal of Experimental Botany, 66 (20):6219-6232. doi: 10.1093/jxb/erv328 URL
[9]	Foyer C, Noctor G. 2005. Oxidant and antioxidant signalling in plants:a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell and Environment, 28 (8):1056-1071. doi: 10.1111/pce.2005.28.issue-8 URL
[10]	Gusta L V, Trischuk R, Weiser C J. 2005. Plant cold acclimation:The role of abscisic acid. Journal of Plant Growth Regulation, 24 (4):308-318. doi: 10.1007/s00344-005-0079-x URL
[11]	Huang Xing, Ding Feng, Peng Hongxiang, Pan Jiechun, He Xinhua, Xu Jiongzhi, Li Lin. 2019. Research progress on family of plant WRKY transcription factors. Biotechnology Bulletin, 35 (12):129-143. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2019-0626
	黄幸, 丁峰, 彭宏祥, 潘介春, 何新华, 徐炯志, 李琳. 2019. 植物WRKY转录因子家族研究进展. 生物技术通报, 35 (12):129-143. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0626
[12]	Jose-Santhi J, Sheikh F R, Kalia D, Singh R K. 2023. Sugar metabolism mediates temperature-dependent flowering induction in saffron(crocus sativus l.). Environmental and Experimental Botany,206:105150.
[13]	Koch K. E. 1996. Carbohydrate-modulated gene expression in plants. Annual Review of Plant Physiology & Plant Molecular Biology, 47 (1):509-509.
[14]	Li Hesheng. 2000. Principles and techniques of plant physiological biochemical experiment. Beijing: Higher Education Press:195-197. (in Chinese)
	李合生. 2000. 植物生理生化实验原理和技术. 北京: 高等教育出版社:195-197.
[15]	Li Hongxia, Wang Yu, Zhang Zhanfeng, Peng Huiru, Ni Zhongfu. 2013. Progress in relationship between plant transcription factors and crop stress tolerance. Journal of Triticeae Crops, 33 (3):613-618. (in Chinese)
	李红霞, 汪妤, 张战凤, 彭惠茹, 倪中福. 2013. 植物转录因子与作物抗逆胁迫关系的研究进展. 麦类作物学报, 33 (3):613-618.
[16]	Ling J, Jiang W J, Zhang Y, Yu H J, Mao Z C, Gu X F, Huang S W, Xie B Y. 2011. Genome-wide analysis of WRKY gene family in Cucumis sativus. BMC Genomics, 12 (1):471. doi: 10.1186/1471-2164-12-471
[17]	Liu Hang, Feng Liqiang, Liu Xingjiang. 2015. Effects of oligosaccharides on abscisic acid synthesis in wheat. Acta Agriculturae Zhejiangensis, 27 (1):4. (in Chinese)
	刘航, 冯立强, 刘兴江. 2015. 寡糖对小麦脱落酸合成的影响. 浙江农业学报, 27 (1):4.
[18]	Liu Nan, Han Yike, Zhang Guihua, Cui Xinghua, Wei Aimin, Du Shengli. 2011. Correlation analysis to early maturing traits of cucumber in greenhouse. Journal of Shanxi Agricultural Sciences, 39 (7):655-656,707. (in Chinese)
	刘楠, 韩毅科, 张桂华, 崔兴华, 魏爱民, 杜胜利. 2011. 温室黄瓜早熟性状的相关分析. 山西农业科学, 39 (7):655-656,707.
[19]	Li Xiaofang, Zhang Zhiliang. 2016. Experimental guidance on plant physiology. Beijing: Higher Education Press:91-92. (in Chinese)
	李小芳, 张志良. 2016. 植物生理学实验指导. 北京: 高等教育出版社:91-92.
[20]	Lu Haiqin, Li Na, Jiang Kaixuan, Wang Youping, Jiang Jinjin. 2024. Research advances on NAC transcription factors regulating plant development and stress responses. Plant Physiology Journal, 60 (2):271-283. (in Chinese) doi: 10.1104/pp.60.2.271 URL
	陆海芹, 李娜, 蒋凯旋, 王幼平, 蒋金金. 2024. Nac转录因子调控植物生长发育和胁迫应答的研究进展. 植物生理学报, 60 (2):271-283.
[21]	Luo Yong, Chen Yan, Wen Beibei, Song Xiaofeng, Zhang Fangling, Wang Kunbo. 2022. Research progress of WRKY transcription factors in Camellia sinensis. Molecular Plant Breeding, 20 (11):9. (in Chinese)
	罗勇, 陈燕, 温贝贝, 宋小凤, 张芳玲, 王坤波. 2022. 茶树WRKY转录因子的研究进展. 分子植物育种, 20 (11):9.
[22]	Ng D W K, Abeysinghe J K, Kamali M. 2018. Regulating the regulators:the control of transcription factors in plant defense signaling. International Journal of Molecular Sciences, 19 (12):3737. doi: 10.3390/ijms19123737 URL
[23]	Wang Zhonghua, Li Xiaogang, Yang Qingsong, Li Hui, Kang Jialiang, Wang Jinxing, Wang Hong, Lin Jing, Sheng Baolong, Chang Youhong. 2023. Effects of foliar application of alginate oligosaccharides phosphorus and potassium fertilizers on leaf and fruit characters of early-maturing sand pears. Jiangsu Journal of Agricultural Sciences, 39 (2):498-503. (in Chinese)
	王中华, 李晓刚, 杨青松, 李慧, 阚家亮, 王金星, 王宏, 蔺经, 盛宝龙, 常有宏. 2023. 叶面喷施褐藻寡糖磷钾肥对早熟砂梨叶片与果实性状的影响. 江苏农业学报, 39 (2):498-503.
[24]	Wu Xuexia, Zha Dingshi, Yang Shaojun. 2010. Research advance in cucumber breeding in china. Acta Agriculturae Jiangxi, 22 (9):53-55,59. (in Chinese)
	吴雪霞, 查丁石, 杨少军. 2010. 我国黄瓜育种研究进展. 江西农业学报, 22 (9):53-55,59.
[25]	Wu Yuxin, Cai Changyang, Tang Shibei, Xie Yuhong, Wang Xiaoyan, Zhu Qiang. 2023. Research progress on plant growth and molecular mechanism in response to low temperature. Jiangsu Agricultural Sciences, 51 (19):1-9. (in Chinese)
	吴宇欣, 蔡昌杨, 唐诗蓓, 谢裕红, 王晓艳, 朱强. 2023. 植物响应低温的生长发育及分子机制研究进展. 江苏农业科学, 51 (19):1-9.
[26]	Xiong L, Ishitani M, Lee H, Zhu J K. 2001. The Arabidopsis los5/aba 3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress-and osmotic stress-responsive gene expression. The Plant Cell, 13 (9):2063-2083.
[27]	Yang Y, Liu Q, Wang G X, Wang X D, Guo J Y. 2010. Germination,osmotic adjustment,and antioxidant enzyme activities of gibberellin-pretreated Picea asperata seeds under water stress. New Forests, 39 (2):231-243. doi: 10.1007/s11056-009-9167-2 URL
[28]	Yang Yan, Liu Jun, Zhou Xiaohui, Liu Songyu, Zhuang Yong. 2025. Cloning and functional verification of SmWRKY4 gene in cold tolerance of Solanum melongena. Acta Horticulturae Sinica, 52 (1):101-110. (in Chinese)
	杨艳, 刘军, 周晓慧, 刘松瑜, 庄勇. 2025. 茄子SmWRKY4的克隆及耐冷性功能验证. 园艺学报, 52 (1):101-110. doi: 10.16420/j.issn.0513-353x.2023-0880
[29]	Yao Y X, Dong Q L, You C X, Zhai H, Hao Y J. 2011. Expression analysis and functional characterization of apple mdvhp1 gene reveals its involvement in Na⁺,malate and soluble sugar accumulation. Plant Physiology and Biochemistry, 49 (10):1201-1208. doi: 10.1016/j.plaphy.2011.05.012 URL
[30]	Yu Hui, Meng Jing, Liu Yaqi, Guan Yuxi, Wang Bingwen, Song Wenhao, Tang Xin, Shu Jing. 2022. Effects of EM bacteria soaking seeds on germination and seedling growth of cucumber under low temperature treatment. The Journal of Shandong Agriculture and Engineering University, 39 (9):35-38. (in Chinese)
	尉辉, 孟婧, 刘亚琦, 关玉熙, 王炳文, 宋文豪, 唐欣, 束靖. 2022. Em菌浸种对低温处理黄瓜萌发和幼苗生长的影响. 山东农业工程学院学报, 39 (9):35-38.
[31]	Yu Xianchang, Xing Yuxian, Ma Hong, Wei Min. 1998. Effects of different rootstocks and scions on chilling tolerance in grafted cucumber seedlings. Scientia Agricultura Sinica, 31 (2):41-47. (in Chinese)
	于贤昌, 邢禹贤, 马红, 魏珉. 1998. 不同砧木与接穗对黄瓜嫁接苗抗冷性的影响. 中国农业科学, 31 (2):41-47.
[32]	Zhang Shengping, Dong Shaoyun, Guan Jiantao, Miao Han, Liu Xiaoping, Gu Xingfang. 2025. Research progress on molecular breeding of disease resistance in cucumber. Acta Horticulturae Sinica, 52 (3):773-791. (in Chinese) doi: 10.16420/j.issn.0513-353x.2024-0825
	张圣平, 董邵云, 官健涛, 苗晗, 刘小萍, 顾兴芳. 2025. 黄瓜抗病分子育种研究进展. 园艺学报, 52 (3):773-791. doi: 10.16420/j.issn.0513-353x.2024-0825
[33]	Zhang Ying, Liu Jia, Lian Xiang, Zhang Jinmei, Tong Deli. 2018. cDNA clone and bioinformatics analysis of CsWRKY32 in cucumber. Journal of Shenyang Normal University(Natural Science Edition), 36 (5):441-445. (in Chinese)
	张颖, 刘佳, 廉想, 张金梅, 佟德利. 2018. 黄瓜CsWRKY32基因的cDNA克隆及生物信息学分析. 沈阳师范大学学报(自然科学版), 36 (5):441-445.
[34]	Zhang Y, Yu H, Yang X, Li Q, Ling J, Wang H, Gu X, Huang S, Jiang W. 2016. CsWRKY46,a WRKY transcription factor from cucumber,confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an aba-dependent manner. Plant Physiology and Biochemistry,108:478-487.
[35]	Zhao Yang, Li Yongsheng, Gao Xufeng. 2015. A new method for accurate determination of peroxidase activity based on fluorescence decrease of guaiacol. Chinese Journal of Analytical Chemistry, 43 (7):1040-1046. (in Chinese)
	赵炀, 李永生, 高秀峰. 2015. 基于愈创木酚荧光减量准确测定过氧化物酶活性的新方法. 分析化学, 43 (7):1040-1046.
[36]	Zhu L, Li S, Ouyang M, Yang L, Sun S, Wang Y, Cai X, Wu G, Li Y. 2022. Overexpression of watermelon clwrky20 in transgenic Arabidopsis improves salt and low-temperature tolerance. Scientia Horticulturae, 295:110848. doi: 10.1016/j.scienta.2021.110848 URL
[37]	Zhu X, Qi L, Liu X, Cai S, Xu H, Huang R, Li J, Wei X, Zhang Z. 2014. The wheat ethylene response factor transcription factor pathogen-induced erf 1 mediates host responses to both the necrotrophic pathogen Rhizoctonia cerealis and freezing stresses. Plant Physiology, 164 (3):1499-1514. doi: 10.1104/pp.113.229575 URL
[38]	Zou Qi. 2003. Experimental guidance on plant physiology. Beijing: China Agriculture Press:161-162. (in Chinese)
	邹琦. 2003. 植物生理学实验指导. 北京: 中国农业出版社:161-162.

受害级别 Injury level	总体症状描述 Overall symptom description	具体叶片受害情况 Specific leaf damage situation
S0	无受害症状 No symptoms of victimization	所有叶片均无受害症状。 All leaves had no damage symptoms.
S1	轻微受害 Minor victimization	叶片稍皱缩；下数第1或第2叶叶缘发黄或略失水；第3叶和新叶（心叶）无受害症状。 Leaves slightly shr；the lower 1st or 2nd leaf margin is yellow or slightly dehydrated；the third leaf and new leaf（heart leaf）had no symptoms.
S2	中度受害 Moderate victimization	叶片皱缩；下数第1叶和第2叶叶缘严重失水；第3叶叶缘发黄或略为失水；心叶无明显受害症状。 Leaf shrinkage；the first leaf and the second leaf edge serious water loss；the third leaf margin is yellow or slightly dehydrated；there were no obvious symptoms of heart leaf injury.
S3	较重受害 Heavy victimization	下数第1叶和第2叶中部出现脱水斑；第3叶叶缘严重失水；心叶轻微失水。 Dehydration spots appeared in the middle of the first and second leaves. The third leaf margin is seriously dehydrated；heart leaves slightly dehydrated.
S4	严重受害（部分可恢复） Seriously victimized（partially recoverable）	下数第1叶和第2叶中部脱水斑连接成片，叶片萎蔫；第3叶中部开始显现脱水斑；心叶失水较明显；但幼苗置于常温下，心叶尚能恢复。 The dehydration spots in the middle of the first and second leaves were connected into pieces，and the leaves wilted. Dehydration spots began to appear in the middle of the third leaf；the water loss of the heart leaf is obvious；however，when the seedlings were placed at room temperature，the heart leaves could still recover.
S5	极度严重受害（不可恢复） Extremely serious victimization（unrecoverable）	所有叶片严重失水萎蔫；幼苗即使再置于常温下也不能恢复。 All leaves were severely dehydrated and wilted；the seedlings could not recover even under normal temperature.

受害级别 Injury level	总体症状描述 Overall symptom description	具体叶片受害情况 Specific leaf damage situation
S0	无受害症状 No symptoms of victimization	所有叶片均无受害症状。 All leaves had no damage symptoms.
S1	轻微受害 Minor victimization	叶片稍皱缩；下数第1或第2叶叶缘发黄或略失水；第3叶和新叶（心叶）无受害症状。 Leaves slightly shr；the lower 1st or 2nd leaf margin is yellow or slightly dehydrated；the third leaf and new leaf（heart leaf）had no symptoms.
S2	中度受害 Moderate victimization	叶片皱缩；下数第1叶和第2叶叶缘严重失水；第3叶叶缘发黄或略为失水；心叶无明显受害症状。 Leaf shrinkage；the first leaf and the second leaf edge serious water loss；the third leaf margin is yellow or slightly dehydrated；there were no obvious symptoms of heart leaf injury.
S3	较重受害 Heavy victimization	下数第1叶和第2叶中部出现脱水斑；第3叶叶缘严重失水；心叶轻微失水。 Dehydration spots appeared in the middle of the first and second leaves. The third leaf margin is seriously dehydrated；heart leaves slightly dehydrated.
S4	严重受害（部分可恢复） Seriously victimized（partially recoverable）	下数第1叶和第2叶中部脱水斑连接成片，叶片萎蔫；第3叶中部开始显现脱水斑；心叶失水较明显；但幼苗置于常温下，心叶尚能恢复。 The dehydration spots in the middle of the first and second leaves were connected into pieces，and the leaves wilted. Dehydration spots began to appear in the middle of the third leaf；the water loss of the heart leaf is obvious；however，when the seedlings were placed at room temperature，the heart leaves could still recover.
S5	极度严重受害（不可恢复） Extremely serious victimization（unrecoverable）	所有叶片严重失水萎蔫；幼苗即使再置于常温下也不能恢复。 All leaves were severely dehydrated and wilted；the seedlings could not recover even under normal temperature.

处理Treatment	野生型 Wild type	CsWRKY46-CR1	CsWRKY46-CR2
T1	0.25 b ± 0.0038	0.35 a ± 0.0077	0.40 a ± 0.0067
T2	0.20 c ± 0.0067	0.28 b ± 0.0067	0.32 b ± 0.0067

处理Treatment	野生型 Wild type	CsWRKY46-CR1	CsWRKY46-CR2
T1	0.25 b ± 0.0038	0.35 a ± 0.0077	0.40 a ± 0.0067
T2	0.20 c ± 0.0067	0.28 b ± 0.0067	0.32 b ± 0.0067