番茄SlWRKY46调控低温胁迫响应的功能研究

doi:10.16420/j.issn.0513-353x.2024-0014

摘要/Abstract

摘要：

为了探究SlWRKY46在低温胁迫响应中的功能，分析了从‘Micro-Tom’番茄叶片中克隆到SlWRKY46的CDS序列，该基因属于WRKY转录因子家族的Ⅱa亚族，编码253个氨基酸。亚细胞定位和酵母自激活结果显示，SlWRKY46定位在细胞核，在酵母系统中不具有转录激活活性。酵母双杂交（Y2H）分析发现，该转录因子可形成同源二聚体。实时荧光定量PCR（qRT-PCR）结果表明，SlWRKY46的表达受低温和脱落酸（abscisic acid，ABA）的显著诱导。启动子转基因番茄的GUS活性也受低温和ABA的诱导。番茄过表达SlWRKY46植株的耐低温性提高，低温处理下活性氧积累减少，抗氧化酶活性增加，脯氨酸含量增加，丙二醛含量减少。低温处理下，抗氧化酶和ABA途径相关基因的表达在过表达与野生型植株中存在明显差异。上述结果表明，SlWRKY46正调控番茄的耐低温性，可能是通过抗氧化酶和ABA途径发挥调控作用。

关键词: 番茄, SlWRKY46, 低温, 活性氧, 脱落酸

Abstract:

In order to explore the function of SlWRKY46 in response to low temperature stress，the CDS sequence of SlWRKY46 cloned from tomato‘Micro-Tom’leaves was analyzed. The result showed that SlWRKY46 belonged to Ⅱa group of WRKY family and encoded 253 amino acids. SlWRKY46 was localized in the nucleus by subcellular localization analysis. SlWRKY46 had no transcriptional activation activity in yeast. SlWRKY46 could form homologous dimers by yeast two-hybrid yeast（Y2H）. The results of real-time fluorescence quantitative PCR（qRT-PCR）showed that the expressions of SlWRKY46 were significantly induced by low temperature and abscisic acid（ABA）. The GUS staining of SlWRKY46 promoter transgenic tomato also cloud be induced by low temperature and ABA. The overexpressed SlWRKY46 tomato lines had higher cold tolerance compared with wild-type. The overexpressed SlWRKY46 tomato lines exhibited lower reactive oxygen species accumulation and malondialdehyde levels，and higher antioxidant enzyme activities and proline accumulation compared with wild-type under low temperature treatment. The expression levels of genes related to antioxidant enzyme and ABA showed significant differences between overexpressed SlWRKY46 tomato lines and wild-type under low temperature treatment. Taken together，these results indicated that SlWRKY46 positively regulated the cold tolerance of tomato by antioxidant enzyme and ABA pathways.

Key words: tomato, SlWRKY46, low temperature, reactive oxygen species, ABA

孙会茹, 党峰峰, 任敏, 张嘉宁, 樊蓓, 陈国梁, 程国亭, 王延峰. 番茄SlWRKY46调控低温胁迫响应的功能研究[J]. 园艺学报, 2024, 51(12): 2758-2774.

SUN Huiru, DANG Fengfeng, REN Min, ZHANG Jianing, FAN Bei, CHEN Guoliang, CHENG Guoting, WANG Yanfeng. Function Analysis of SlWRKY46 in Regulating Tomato Response to Low Temperature Stress[J]. Acta Horticulturae Sinica, 2024, 51(12): 2758-2774.

图/表 10

参考文献 57

[1]	Bai Y L, Sunarti S, Kissoudis C, Visser R G F, van der Linden C G. 2018. The role of tomato WRKY genes in plant responses to combined abiotic and biotic stresses. Frontiers in Plant Science,9:801.
[2]	Che Yongmei, Sun Yanjun, Lu Songchong, Zhao Fanggui, Hou Lixia, Liu Xin. 2018. AtWRKY40 functions in drought stress response in Arabidopsis thaliana. Plant Physiology Journal, 54 (3):456-464. (in Chinese)
	车永梅, 孙艳君, 卢松冲, 赵方贵, 侯丽霞, 刘新. 2018. AtWRKY40参与拟南芥干旱胁迫响应过程. 植物生理学报, 54 (3):456-464.
[3]	Chen H, Lai Z B, Shi J W, Xiao Y, Chen Z X, Xu X P. 2010. Roles of Arabidopsis WRKY18,WRKY40 and WRKY 60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biology, 10 (1):281.
[4]	Chen J N, Nolan T M, Ye H X, Zhang M C, Tong H N, Xin P Y, Chu J F, Chu C C, Li Z H, Yin Y H. 2017. Arabidopsis WRKY46,WRKY54,and WRKY 70 transcription factors are involved in brassinosteroid-regulated plant growth and drought responses. The Plant Cell, 29 (6):1425-1439.
[5]	Chen Qingqi, Zhang Hong, Jiang Jingbin, Li Jiangfu. 2018. Partial WRKY genes expression under non-biological stress and analysis of SlWRKY50 gene silencing in tomato. Journal of Northeast Agricultural University, 49 (7):11. (in Chinese)
	陈青奇, 张红, 姜景彬, 李景富. 2018. 番茄部分WRKY基因非生物胁迫表达和SlWRKY50基因沉默分析. 东北农业大学学报, 49 (7):11.
[6]	Chen X J, Li C, Wang H, Guo Z J. 2019. WRKY transcription factors:evolution,binding,and action. Phytopathology Research, 1 (1):13.
[7]	Deng Jiangxia, Zhang Guoliang, Li Bianhao, Huang Zhiwei, Zhao Hongliang, Zhang Ye. 2023. Role of WRKY transcription factors in plant hormone ABA,SA and JA signal transduction. Heilongjiang Agricultural Sciences,10:138-144. (in Chinese)
	邓江霞, 张国良, 李边豪, 黄志炜, 赵宏亮, 张叶. 2023. WRKY类转录因子在植物激素ABA、SA和JA信号转导中的作用. 黑龙江农业科学,10:138-144.
[8]	Diao Yan, Yu Xihong, Jiang Xinmei. 2008. Effects of exogenous substance on membrane lipid peroxidation in tomato seedlings under chilling stress. Journal of Northeast Agricultural University, 39 (5):4. (in Chinese)
	刁艳, 于锡宏, 蒋欣梅. 2008. 低温胁迫下外源物质对番茄幼苗膜脂过氧化作用的影响. 东北农业大学学报, 39 (5):4.
[9]	Du Chao. 2021. Function and application of the WRKY transcription factor superfamily in plant response to stresses. Pratacultural Science, 38 (7):1287-1300. (in Chinese)
	杜超. 2021. WRKY转录因子家族在植物响应逆境胁迫中的功能及应用. 草业科学, 38 (7):1287-1300.
[10]	Du P, Wang Q, Yuan D Y, Chen S S, Su Y N, Li L, Chen S, He X J. 2023. WRKY transcription factors and OBERON histone-binding proteins form complexes to balance plant growth and stress tolerance. EMBO Journal, 42 (19):e113639.
[11]	Duan X Y, Jiang C, Zhao Y P, Gao G, Li M, Qi H Y. 2022. Transcriptome and metabolomics analysis revealed that CmWRKY49 regulating CmPSY1 promotes β-carotene accumulation in orange fleshed oriental melon. Horticultural Plant Journal, 8 (5):650-666.
[12]	Fang Yan, Wu Junyan, Sun Wancang, Yang Xiaojuan, Han Huimin, Han Yawei, Chen Yaping. 2014. Inducing effects of exogenous ABA on seed germination and cold tolerance of winter rape seedlings. Agricultural Research in the Arid Areas, 32 (6):5. (in Chinese)
	方彦, 武军艳, 孙万仓, 杨晓娟, 韩慧敏, 韩亚伟, 陈亚平. 2014. 外源ABA浸种对冬油菜种子萌发及幼苗抗寒性的诱导效应. 干旱地区农业研究, 32 (6):5.
[13]	Fei J, Wang Y S, Cheng H, Su Y B, Zhong Y J, Zheng L. 2022. The Kandelia obovata transcription factor KoWRKY40 enhances cold tolerance in transgenic Arabidopsis. BMC Plant Biology, 22 (1):274.
[14]	Gao Pengfei, Gao Bing, Feng Zhenghong, Wu Jianhui. 2023. Cloning and Cd-resistant analysis of PsWRKY40 in Potentilla sericea. Acta Horticulturae Sinica, 50 (6):1269-1283. (in Chinese) doi: 10.16420/j.issn.0513-353x.2022-0213
	高鹏飞, 高冰, 冯郑红, 吴建慧. 2023. 绢毛委陵菜PsWRKY40的克隆与耐镉功能分析. 园艺学报, 50 (6):1269-1283. doi: 10.16420/j.issn.0513-353x.2022-0213
[15]	Guo M Y, Yang F J, Liu C X, Zou J P, Qi Z Y, Fotopoulos V, Lu G, Yu J Q, Zhou J. 2022. A single-nucleotide polymorphism in WRKY33 promoter is associated with the cold sensitivity in cultivated tomato. New Phytologist, 236 (3):989-1005.
[16]	Han J X, Li X G, Li W H, Yao A Q, Niu C G, Hou R N, Liu W D, Wang Y, Zhang L H, Han D G. 2023. Overexpression of Malus baccata WRKY40 (MbWRKY40)enhances stress tolerance in Arabidopsis subjected to cold and drought. Plant Stress,10:100209.
[17]	Huang H, Zhao W C, Qiao H, Li C H, Sun L L, Yang R, Ma X C, Ma J L, Song S S, Wang S H. 2022. SlWRKY 45 interacts with jasmonate-ZIM domain proteins to negatively regulate defense against the root-knot nematode Meloidogyne incognita in tomato. Horticulture Research,9:uhac197.
[18]	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
[19]	Karim A, Jiang Y, Guo L, Ling Z, Ye S, Duan Y, Li C, Luo K. 2015. Isolation and characterization of a subgroup IIa WRKY transcription factor PtrWRKY 40 from Populus trichocarpa. Tree Physiology, 35 (10):1129-1139.
[20]	Kim C Y, Vo K T X, Nguyen C D, Jeong D H, Lee S K, Kumar M, Kim S R, Park S H, Kim J K, Jeon J S. 2016. Functional analysis of a cold-responsive rice WRKY gene,OsWRKY71. Plant Biotechnology Reports, 10 (1):13-23.
[21]	Kumar S, Stecher G, Tamura K. 2015. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33 (7):1870-1874.
[22]	Lan Liming, Luo Changguo, Wang Sanhong. 2021. Analysis of resistance mechanism to powdery mildew based on transcriptome sequencing in Malus hupehensis. Acta Horticulturae Sinica, 48 (5):860-872. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0578 URL
	兰黎明, 罗昌国, 王三红. 2021. 基于转录组测序的湖北海棠抗白粉病机制分析. 园艺学报, 48 (5):860-872. doi: 10.16420/j.issn.0513-353x.2020-0578 URL
[23]	Li H, Ye K Y, Shi Y T, Cheng J K, Zhang X Y, Yang S H. 2017. BZR1 positively regulates freezing tolerance via CBF-dependent and CBF-independent pathways in Arabidopsis. Molecular Plant, 10 (4):15.
[24]	Li Junming, Xiang Chaoyang, Wang Xiaoxuan, Guo Yanmei, Huang Zejun, Liu Lei, Li Xin, Du Yongchen. 2021. Current situation of tomato industry in China during‘The thirteenth five-year plan’period and future prospect. China Vegetables,(2):13-20. (in Chinese)
	李君明, 项朝阳, 王孝宣, 国艳梅, 黄泽军, 刘磊, 李鑫, 杜永臣. 2021. “十三五”我国番茄产业现状及展望. 中国蔬菜,(2):13-20.
[25]	Li Xiao, Cheng Yufu, Yang Xu. 2017. Research progress of WRKY transcription factors in Solanaceae plants. Acta Horticulturae Sinica, 44 (1):170-178. (in Chinese) doi: 10.16420/j.issn.0513-353x.2016-0703
	李笑, 成玉富, 杨旭. 2017. 茄科植物WRKY转录因子的研究进展. 园艺学报, 44 (1):170-178. doi: 10.16420/j.issn.0513-353x.2016-0703
[26]	Li Xiaohan. 2020. WRKY20 coordinates with ABI5 to regulate the ABA signal pathway in Arabidopsis[M. D. Dissertation]. Tai’an: Shandong Agricultural University. (in Chinese)
	李晓涵. 2020. 拟南芥WRKY20与ABI5协同调控ABA信号通路的机制研究[硕士论文]. 泰安: 山东农业大学.
[27]	Liu Jingyan, Shi Yiting, Yang Shuhua. 2017. CBF:a key factor balancing plant cold stress responses and growth. Chinese Bulletin Botany, 52 (6):689-698. (in Chinese)
	刘静妍, 施怡婷, 杨淑华. 2017. CBF:平衡植物低温应答与生长发育的关键. 植物学报, 52 (6):689-698. doi: 10.11983/CBB17132
[28]	Liu W, Liang X Q, Cai W J, Wang H, Liu X, Cheng L F, Song P H, Luo G J, Han D G. 2022. Isolation and functional analysis of VvWRKY28,a Vitis vinifera WRKY transcription factor gene,with functions in tolerance to cold and salt stress in transgenic Arabidopsis thaliana. International Journal of Molecular Sciences, 23 (21):13418.
[29]	Lu Yanqing, Lin Yanjin, Lu Xinkun. 2023. Cell wall material metabolism and the response to high temperature and water deficiency stresses in pericarp are associated with fruit cracking of‘Duwei’pummelo. Acta Horticulturae Sinica, 50 (8):1747-1768. (in Chinese) doi: 10.16420/j.issn.0513-353x.2022-0653 URL
	卢艳清, 林燕金, 卢新坤. 2023. 果皮细胞壁物质代谢及果皮对高温和水分亏缺逆境的响应与‘度尾文旦柚’裂果相关. 园艺学报, 50 (8):1747-1768.
[30]	Luo D L, Ba L J, Shan W, Kuang J F, Lu W J, Chen J Y. 2017. Involvement of WRKY transcription factors in abscisic-acid-induced cold tolerance of banana fruit. Journal of Agricultural & Food Chemistry, 65 (18):3627-3635.
[31]	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 F, Finer J J, Shen Q J, Rushton P J. 2012. WRKY transcription factors:key components in abscisic acid signalling. Plant Biotechnology Journal, 10 (1):2-11. doi: 10.1111/j.1467-7652.2011.00634.x pmid: 21696534
[32]	Sun S S, Li S P, Zhou X P, Yang X L. 2023. WRKY 1 represses the WHIRLY1 transcription factor to positively regulate plant defense against geminivirus infection. PLoS Pathogens, 19 (4):e1011319.
[33]	Sun X M, Zhang L L, Wong D C J, Wang Y, Zhu Z F, Xu G Z, Wang Q F, Li S H, Liang Z C, Xin H P. 2019. The ethylene response factor VaERF 092 from Amur grape regulates the transcription factor VaWRKY33,improving cold tolerance. The Plant Journal, 99 (5):988-1002.
[34]	Tang J Q, Tian X J, Mei E Y, He M L, Gao J W, Yu J, Xu M, Liu J L, Song L, Li X F, Wang Z Y, Guan Q J, Zhao Z G, Wang C M, Bu Q Y. 2022. WRKY 53 negatively regulates rice cold tolerance at the booting stage by fine-tuning anther gibberellin levels. The Plant Cell, 34 (11):4495-4515.
[35]	Tang Mingjia, Xu Jin, Lin Rui, Song Jianing, Yu Jingquan, Zhou Yanhong. 2022. Advances in physiological and molecular mechanism of tomato responses to light and temperature stress. Acta Horticulturae Sinica, 49 (10):2174-2188. (in Chinese) doi: 10.16420/j.issn.0513-353x.2022-0600 URL
	唐明佳, 徐进, 林锐, 宋珈凝, 喻景权, 周艳虹. 2022. 番茄响应光温逆境的生理分子机制研究进展. 园艺学报, 49 (10):2174-2188. doi: 10.16420/j.issn.0513-353x.2022-0600 URL
[36]	Tian Yisu, Jiang Ming, Zhang Qiyu. 2023. Progress on cold resistance genes and ICE-CBF-COR signaling in plants. Agricultural Engineering, 13 (5):131-137. (in Chinese)
	田一粟, 姜明, 张启宇. 2023. 植物抗寒基因及ICE-CBF-COR信号转导研究进展. 农业工程, 13 (5):131-137.
[37]	Wang H P, Chen W Q, Xu Z Y, Chen M F, Yu D Q. 2023. Functions of WRKYs in plant growth and development. Trends in Plant Science, 28 (6):630-645. doi: 10.1016/j.tplants.2022.12.012 pmid: 36628655
[38]	Wang L H, Chen H, Chen G Y, Luo G B, Shen X Y, Ouyang B, Bie Z L. 2024. Transcription factor SlWRKY 50 enhances cold tolerance in tomato by activating the jasmonic acid signaling. Plant Physiology, 194 (2):1075-1090.
[39]	Wang M Q, Huang Q X, Lin P, Zeng Q H, Li Y, Liu Q L, Zhang L, Pan Y Z, Jiang B B, Zhang F. 2020. The overexpression of a transcription factor gene VbWRKY32 enhances the cold tolerance in Verbena bonariensis. Frontiers in Plant Science,10:1746.
[40]	Wang Y C, Xu H F, Liu W J, Wang N, Qu C Z, Jiang S H, Fang H C, Zhang Z Y, Chen X S. 2019. Methyl jasmonate enhances apple’ cold tolerance through the JAZ-MYC2 pathway. Plant Cell,Tissue and Organ Culture, 136 (1):75-84.
[41]	Wang Yixuan, Meng Qingwei, Ma Nana. 2021. Characterization and analysis of some chilling-response WRKY transcription factors in tomato. Plant Physiology Journal, 57 (6):1349-1362. (in Chinese)
	王艺璇, 孟庆伟, 马娜娜. 2021. 番茄低温响应WRKY转录因子的鉴定和分析. 植物生理学报, 57 (6):1349-1362.
[42]	Wu Dan, Mao Donghai, Zhao Xiaoying. 2022. Advances in molecular mechanism of plant responses to low temperature. Life Science Research, 26 (1):67-75. (in Chinese)
	吴丹, 毛东海, 赵小英. 2022. 植物低温响应的分子机制研究进展. 生命科学研究, 26 (1):67-75.
[43]	Wu J Q, Sun W R, Sun C, Xu C M, Li S, Li P X, Xu H M, Zhu D Y, Li M, Yang L L, Wei J B, Hanzawa A, Tapati S J, Uenoyama R, Miyazaki M, Rahman A, Wu S. 2023. Cold stress induces malformed tomato fruits by breaking the feedback loops of stem cell regulation in floral meristem. New Phytologist, 237 (6):2268-2283.
[44]	Wu Lingyun, Zhou Bo, Li Yuhua. 2014. Roles of WRKY transcription factors in abscisic acid signal transduction. Molecular Plant Breeding, 12 (2):404-410. (in Chinese)
	吴玲云, 周波, 李玉花. 2014. 植物WRKY转录因子在脱落酸信号转导中的作用. 分子植物育种, 12 (2):404-410.
[45]	Xiang Hongtao, Qi Deqiang, Li Wan, Zheng Dianfeng, Wang Yuexi, Wang Tongtong, Wang Lizhi, Zeng Xiannan, Yang Chunjie, Zhou Hang, Zhao Haidong. 2019. Effect of exogenous ABA on the endogenous hormone levels and physiology of chilling resistance in the leaf sheath of rice at the flowering stage under low temperature stress. Acta Prataculturae Sinica, 28 (4):14. (in Chinese)
	项洪涛, 齐德强, 李琬, 郑殿峰, 王月溪, 王彤彤, 王立志, 曾宪楠, 杨纯杰, 周行, 赵海东. 2019. 低温胁迫下外源ABA对开花期水稻叶鞘激素含量及抗寒生理的影响. 草业学报, 28 (4):14.
[46]	Yan J W, Li J, Zhang H P, Liu Y, Zhang A Y. 2022. ZmWRKY 104 positively regulates salt tolerance by modulating ZmSOD4 expression in maize. The Crop Journal, 10 (2):555-564.
[47]	Yang Wenwen, Feng Mengying, Qu Rui, Liu Xinyu, Nie Shuming. 2023. Low temperature induced expression of SlBRI1enhanced cold tolerance of tomato seedlings. Acta Botanica Boreali-Occidentalia Sinica, 43 (11):1842. (in Chinese)
	杨文文, 冯梦颖, 瞿芮, 刘心雨, 聂书明. 2023. 低温诱导表达SlBRI1增强番茄幼苗耐寒能力. 西北植物学报, 43 (11):1842.
[48]	Yang Y, Liu J, Zhou X H, Liu S Y, Zhuang Y. 2020. Identification of WRKY gene family and characterization of cold stress-responsive WRKY genes in eggplant. PeerJ,8:e8777.
[49]	Yuan Y, Ren S Y, Liu X F, Su L Y, Wu Y, Zhang W, Li Y, Jiang Y D, Wang H G, Fu R, Bouzayen M, Liu M C, Zhang Y. 2022. SlWRKY 35 positively regulates carotenoid biosynthesis by activating the MEP pathway in tomato fruit. New Phytologist, 234 (1):164-178.
[50]	Zeng Zexiang, Xiao Xianmei, Tan Xiaoli, Fan Zhongqi, Chen Jianye. 2021. Characteristics of the transcription factor BrWRKY57 and its regulation on BrPPH1and BrNCED3. Acta Horticulturae Sinica, 48 (3):518-530. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0432 URL
	曾泽湘, 肖显梅, 谭小丽, 范中奇, 陈建业. 2021. 菜薹BrWRKY57的特性及其对BrPPH1和BrNCED3的调控. 园艺学报, 48 (3):518-530. doi: 10.16420/j.issn.0513-353x.2020-0432 URL
[51]	Zhang Gaoyuan, Wei Bingqiang. 2020. Identification of WRKY gene family and their expression analysis under low-temperature stress in melon (Cucumis melo). Journal of Agricultural Biotechnology, 28 (10):1761-1775. (in Chinese)
	张高原, 魏兵强. 2020. 甜瓜WRKY基因家族鉴定及其响应低温胁迫的表达分析. 农业生物技术学报, 28 (10):1761-1775.
[52]	Zhang M X, Zhao R R, Huang K, Huang S Z, Wang H T, Wei Z Q, Li Z, Bian M D, Jiang W Z, Wu T, Du X L. 2022. The OsWRKY63-OsWRKY76-OsDREB1B module regulates chilling tolerance in rice. The Plant Journal, 112 (2):383-398.
[53]	Zhang Y, Yu H J, Yang X Y, Li Q, Ling J, Wang H, Gu X F, Huang S W, Jiang W J. 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.
[54]	Zheng J Y, Liu F, Zhu C H, Li X F, Dai X Z, Yang B Z, Zou X X, Ma Y Q. 2019. Identification,expression,alternative splicing and functional analysis of pepper WRKY gene family in response to biotic and abiotic stresses. PLoS ONE, 14 (7):e0219775.
[55]	Zhou Tao, Wang Juan, Wang Lulu, Wang Baike, Hu Jiahui, Lan Haiyan, Yu Qinghui. 2020. Cloning and prokaryotic expression analysis of a transcription factor gene SlWRKY16 in tomato. Acta Horticulturae Sinica, 47 (7):1312-1322. (in Chinese)
	周涛, 王娟, 王露露, 王柏柯, 胡佳蕙, 兰海燕, 余庆辉. 2020. 番茄转录因子基因SlWRKY16的克隆及原核表达分析. 园艺学报, 47 (7):1312-1322. doi: 10.16420/j.issn.0513-353x.2019-0956
[56]	Zhu D, Hou L X, Xiao P L, Guo Y, Deyholos M K, Liu X. 2019. VvWRKY30,a grape WRKY transcription factor,plays a positive regulatory role under salinity stress. Plant Science,280:132-142.
[57]	Zou C S, Jiang W B, Yu D Q. 2010. Male gametophyte-specific WRKY 34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis. Journal of Experimental Botany, 61 (14):3901-3914.

用途 Application	基因名称/ID Gene name/ID	引物序列（5′-3′） Primer sequence
CDS扩增 CDS amplification	SlWRKY46	F：ATGGGAAACAAGTCTTTTTTTA；R：TCAAAAAAGTAATCCTGAGATG
酵母载体构建 Yeast vector construct	SlWRKY46-BD	F：CATGGAGGCCGAATTCATGGGAAACAAGTCTTTTTTTA； R：GCAGGTCGACGGATCCTCAAAAAAGTAATCCTGAGATG
亚细胞定位载体构建Subcellular localization vector construction	SlWRKY46-subcell	F：CAGTGGTCTCACAACATGGGAAACAAGTCTTTTTTTATTGACCTTAAC ACAAATCC； R：CAGTGGTCTCATACAAAAAAGTAATCCTGAGATGGCTGTAGCAACTG
过表达载体构建 Overexpressing vector construction	SlWRKY46-pBI121	F：CACGGGGGACTCTAGAATGGGAAACAAGTCTTTTTTTA； R：GACGGCCAGTGAGCTCTCAAAAAAGTAATCCTGAGATG
启动子扩增 Promoter amplication	SlWRKY46-promoter	F：CAAATAGTTAGCTAGTAGCT；R：ACTTGTGTCTATGGAATATA
启动子载体构建 Promoter vector construction	SlWRKY46-promoter-pBI121	F：TGATTACGCCAAGCTTCAAATAGTTAGCTAGTAGCT； R：GGACTGACCACCCGGGACTTGTGTCTATGGAATATA
内参基因 Reference gene	SlEF1α-RT	F：GACAGGCGTTCAGGTAAGG；R：CCAATGGAGGGTATTCAGC
荧光定量PCR qRT-PCR	SlWRKY46-RT	F：ACAATCATCCCCAACCACCT；R：AAGACGCCATTTGTTCGACT
	SlPYL/Solyc06g050500	F：CGGTGGTGGTAGAGTCGTATG；R：AGCCAAGTTCTGTGCGATTTG
	SlPP2C/Solyc01g065700	F：TGCTTCTCACAACTTGGCTGT；R：AAGGACCATGCCCATCAAACA
	SlPP2C/Solyc05g018300	F：TCATCGAAGTCCTGTTCCTGC；R：CCTCCATGTCACGATTAGGGG
	SlPP2C/Solyc06g051940	F：ACTTGCCAGTGATGGTCTGTG；R：TCTGTACTGCCCCGTGAAATC
	SlNCEB/Solyc01g087260	F：GGAGTGGCGAGAATTGAAGGA；R：GGAGTTTCATCGGTAGGTGCA
	SlNCEB/Solyc08g075490	F：AGACGAACTTCCTCCTACCGA；R：AGCATTCCATCACCGTCGAAT
	SlPOD/Solyc02g079500	F：AACACATGCCCCAACGTATCT；R：AGCACCACTAAAGTTGGCAGT
	SlPOD/Solyc02g083490	F：AACATGCCCTCAAGCTGAAGA；R：AGAAGCATCACAACCCCGTAG
	SlPOD/Solyc05g046020	F：GCCAAGAGTTGTCCAAAAGCA；R：TGCATCACATCCCCTGACAAA
	SlPOD/Solyc07g052510	F：GGAGAGGTCTGTTCCAATCCG；R：GCTTCCTGATTTCACCAGCAC
	SlPOD/Solyc10g084200	F：GCCTAGTATAACGGTGCCACA；R：CCTCGACAGCCTATCACCTTG
	SlSOD/Solyc11g066390	F：TGACCTGGGAAACATAGTGGC；R：CAAGTGCTCGTCCAACAACTG
	SlCAT/Solyc02g082760	F：ACAGGGATGAGGAGATCGACT；R：TGTCTGTCGGGTGTGAATGAG
	SlICE1/Solyc03g118310	F：CAGGCTTTACATGCTGCGAT；R：GGTGTCAGAGGATGGAAGCT
	SlICE1/Solyc06g068870	F：GGAGCACAACCAACCCTTTT；R：TTCCCCACTACCCCACTTTC
	SlDREB/Solyc12g008350	F：AAAAGCCGCTCACATGGATC；R：GAGGATGTAACGCCCGAAAC
	SlDREB/Solyc12g044390	F：TGCGAAGTTGGGGAAAATGG；R：AGGTGAAATTGACGCTGGAC
	SlDREB/Solyc03g120840	F：CCCACCTTCTTCATCCTCGT；R：CCTTCAACCGATTCGCTCAG
	SlDREB/Solyc06g066540	F：ATGGGTGTCTGAAATTCGCG；R：GCAGCTTGAACATCTCTCGG
	SlDREB/Solyc08g066660	F：GCAGCAATGGACAAGTTCGA；R：TGTTGCCAAATCTATCGCCG
	SlCOR47-like/Solyc04g082200	F：TCTAGTAGCTCCAGTGATG；R：TCTCCTCTGTTTCCTCGT
	SlCOR413-like/Solyc02g071210	F：AGCAGCTTATCTGGGTTT；R：AATGATGGCTAGGGTGGC
	SlCBF1/Solyc03g026280	F：GAGTCGGAAGAAGTTTCAGG；R：TGTAGGCATCAGTTTCCAC
	SlCBF2/Solyc03g124110	F：TTTCCTACGGCTGAAATGGC；R：GAGTTGGAGGAAGCAGGGAT
	SlCBF3/Solyc03g026270	F：TGCCGGGTTTACTTACGAAT；R：TCAGCTTCCACATGATCTCC

用途 Application	基因名称/ID Gene name/ID	引物序列（5′-3′） Primer sequence
CDS扩增 CDS amplification	SlWRKY46	F：ATGGGAAACAAGTCTTTTTTTA；R：TCAAAAAAGTAATCCTGAGATG
酵母载体构建 Yeast vector construct	SlWRKY46-BD	F：CATGGAGGCCGAATTCATGGGAAACAAGTCTTTTTTTA； R：GCAGGTCGACGGATCCTCAAAAAAGTAATCCTGAGATG
亚细胞定位载体构建Subcellular localization vector construction	SlWRKY46-subcell	F：CAGTGGTCTCACAACATGGGAAACAAGTCTTTTTTTATTGACCTTAAC ACAAATCC； R：CAGTGGTCTCATACAAAAAAGTAATCCTGAGATGGCTGTAGCAACTG
过表达载体构建 Overexpressing vector construction	SlWRKY46-pBI121	F：CACGGGGGACTCTAGAATGGGAAACAAGTCTTTTTTTA； R：GACGGCCAGTGAGCTCTCAAAAAAGTAATCCTGAGATG
启动子扩增 Promoter amplication	SlWRKY46-promoter	F：CAAATAGTTAGCTAGTAGCT；R：ACTTGTGTCTATGGAATATA
启动子载体构建 Promoter vector construction	SlWRKY46-promoter-pBI121	F：TGATTACGCCAAGCTTCAAATAGTTAGCTAGTAGCT； R：GGACTGACCACCCGGGACTTGTGTCTATGGAATATA
内参基因 Reference gene	SlEF1α-RT	F：GACAGGCGTTCAGGTAAGG；R：CCAATGGAGGGTATTCAGC
荧光定量PCR qRT-PCR	SlWRKY46-RT	F：ACAATCATCCCCAACCACCT；R：AAGACGCCATTTGTTCGACT
	SlPYL/Solyc06g050500	F：CGGTGGTGGTAGAGTCGTATG；R：AGCCAAGTTCTGTGCGATTTG
	SlPP2C/Solyc01g065700	F：TGCTTCTCACAACTTGGCTGT；R：AAGGACCATGCCCATCAAACA
	SlPP2C/Solyc05g018300	F：TCATCGAAGTCCTGTTCCTGC；R：CCTCCATGTCACGATTAGGGG
	SlPP2C/Solyc06g051940	F：ACTTGCCAGTGATGGTCTGTG；R：TCTGTACTGCCCCGTGAAATC
	SlNCEB/Solyc01g087260	F：GGAGTGGCGAGAATTGAAGGA；R：GGAGTTTCATCGGTAGGTGCA
	SlNCEB/Solyc08g075490	F：AGACGAACTTCCTCCTACCGA；R：AGCATTCCATCACCGTCGAAT
	SlPOD/Solyc02g079500	F：AACACATGCCCCAACGTATCT；R：AGCACCACTAAAGTTGGCAGT
	SlPOD/Solyc02g083490	F：AACATGCCCTCAAGCTGAAGA；R：AGAAGCATCACAACCCCGTAG
	SlPOD/Solyc05g046020	F：GCCAAGAGTTGTCCAAAAGCA；R：TGCATCACATCCCCTGACAAA
	SlPOD/Solyc07g052510	F：GGAGAGGTCTGTTCCAATCCG；R：GCTTCCTGATTTCACCAGCAC
	SlPOD/Solyc10g084200	F：GCCTAGTATAACGGTGCCACA；R：CCTCGACAGCCTATCACCTTG
	SlSOD/Solyc11g066390	F：TGACCTGGGAAACATAGTGGC；R：CAAGTGCTCGTCCAACAACTG
	SlCAT/Solyc02g082760	F：ACAGGGATGAGGAGATCGACT；R：TGTCTGTCGGGTGTGAATGAG
	SlICE1/Solyc03g118310	F：CAGGCTTTACATGCTGCGAT；R：GGTGTCAGAGGATGGAAGCT
	SlICE1/Solyc06g068870	F：GGAGCACAACCAACCCTTTT；R：TTCCCCACTACCCCACTTTC
	SlDREB/Solyc12g008350	F：AAAAGCCGCTCACATGGATC；R：GAGGATGTAACGCCCGAAAC
	SlDREB/Solyc12g044390	F：TGCGAAGTTGGGGAAAATGG；R：AGGTGAAATTGACGCTGGAC
	SlDREB/Solyc03g120840	F：CCCACCTTCTTCATCCTCGT；R：CCTTCAACCGATTCGCTCAG
	SlDREB/Solyc06g066540	F：ATGGGTGTCTGAAATTCGCG；R：GCAGCTTGAACATCTCTCGG
	SlDREB/Solyc08g066660	F：GCAGCAATGGACAAGTTCGA；R：TGTTGCCAAATCTATCGCCG
	SlCOR47-like/Solyc04g082200	F：TCTAGTAGCTCCAGTGATG；R：TCTCCTCTGTTTCCTCGT
	SlCOR413-like/Solyc02g071210	F：AGCAGCTTATCTGGGTTT；R：AATGATGGCTAGGGTGGC
	SlCBF1/Solyc03g026280	F：GAGTCGGAAGAAGTTTCAGG；R：TGTAGGCATCAGTTTCCAC
	SlCBF2/Solyc03g124110	F：TTTCCTACGGCTGAAATGGC；R：GAGTTGGAGGAAGCAGGGAT
	SlCBF3/Solyc03g026270	F：TGCCGGGTTTACTTACGAAT；R：TCAGCTTCCACATGATCTCC