1 College of Horticulture, Shanxi Agricultural University, Taigu 030801, China; 2 College of Information Science and Engineering, Shanxi Agricultural University, Taigu 030801, China; 3 Shanxi Key Laboratory of Germplasm Improvement and Utilization in Pomology, Shanxi Agricultural University, Taigu 030801, China
Abstract:WRKY family is widely involved in plant growth and development, regulating substance metabolism, responding to environmental signals and so on. To study the function of grape (Vitis vinifera) VvWRKY26, the VvWRKY26 gene (GenBank No. KX 823961.1) was cloned from 'WuHeZaoHong' grape by homologous sequence method, the bioinformatics, spatio-temporal expression and response salicylic acid (SA) expression were analyzed. The results of bioinformatics analysis showed that the CDS region of VvWRKY26 was 1 434 bp length, encoded 477 amino acids, and its secondary structure was composed of random curl, α-helix and β-fold, which was a non-secreted, non-transmembrane protein distributed in the nucleus. It was highly homologous with Petunia hybrid PhPH3 (GenBank No. AMR43368.1), Brassica napus BnTTG2 (Brassica napus transparent testa glabra2, GenBank No. 106423067) and Arabidopsis thaliana AtTTG2 (Arabidopsis thaliana transparent testa glabra2, GenBank No. 818303), and belongs to the WRKY transcription factor family Ⅰ. Promoter analysis showed that VvWRKY26 promoter had response elements such as light signal, resistance to reactive oxygen stress, SA and gibberellin acid (GA) signals, etc.. qPCR analysis showed that VvWRKY26 was expressed in the flowers, stems, buds, tendrils, young leaves, mature leaves and fruit tissues, and the expression levels were higher during the young fruit stage and color changing stage. It participated in SA signal response and the expression level under the SA treatment at 0.5~8 h was significantly higher than that of the control (P<0.05). This study provides a theoretical basis for the further research on the function of VvWRKY26 in SA signal channel.
[1] 陈建业. 2005. 葡萄酒中酚酸及葡萄果实苯丙烷类代谢途径研究[D]. 博士学位论文, 中国农业大学, 导师: 黄卫东, pp. 10-13.75-93. (Chen J Y.2005. Study on the metabolic pathway of phenolic acid and phenylpropane in grape wine[D].Thesis for Ph.D, China Agriculture University, Supervisor: Huang W D, pp. 13, 93.) [2] 陈锦芬, 顾开元, 贾雨豪, 等. 2021. 外源甜菜碱及水杨酸对锰胁迫下烟草生理特性的影响[J]. 中国烟草学报, 27(02): 79-86. (Chen J F, Gu K Y, Jia Y H, et al.2021. Effects of exogenous betaine and salicylic acid on the physiological properties of tobacco under manganese stress[J]. Chinese Journal of Tobacco, 27(02): 79-86.) [3] 董悦, 王远达, 王志敏, 等. 2021. WRKY12调控植物发育的分子机制[J]. 生物工程学报, 37(01): 142-148. (Dong Y, Wang Y D, Wang Z M, et al.2021. Molecular mechanisms by which WRKY12 regulates plant development[J]. Journal of Bioengineering, 37(01): 142-148.) [4] 高汝勇, 吕亚慈, 时丽冉, 等. 2021. 水杨酸浸种对谷子萌发期抗旱性的影响[J]. 东北农业科学, 46(2): 15-18, 22. (Gao R Y, Lv Y C, Shi L R, et al.2021. Effect of salicylic acid immersion species on drought resistance during millet germination[J]. Northeast Agricultural Science, 46(2): 15-18, 22.) [5] 何庆, 张健, 吴斌,等. 2020. 采前水杨酸喷施对红地球葡萄采后品质的影响[J]. 保鲜与加工, 20(4): 50-56. (He Q, Zhang J, Wu B, et al.2020. Effect of spraying salicylic acid before harvest on postharvest quality of Red Globe grape[J]. Preservation and Processing, 20(4): 50-56.) [6] 何于飞. 2008. 葡萄综合加工[J]. 食品工程, 3: 32-34+58. (He Y F.2008. Grape comprehensive processing[J]. Food Engineering, 3: 32-34+58.) [7] 侯珲, 朱建兰, 周红平, 等. 2006. BTH和水杨酸(SA)对甜瓜抗白粉病的诱导作用[J]. 果树学报, 23(05): 736-739. (Hou H, Zhu J L, Zhou H P, et al.2006. Studis on the resistance induced by BTH and SA against Eiysiphe cucurbitacearum in muskmelon[J]. Journal of Fruit Science, 23(05): 736-739.) [8] 侯廷帅, 韩晓东, 赵江, 等. 2015. 葡萄的加工技术综述[J]. 食品工业, 36(05): 223-228. (Hou T S, Han X D, Zhao J, et al.2015. Summary of grape processing technology[J]. Food Industry, 36(05): 223-228.) [9] 雷瑞祥, 杨冰月, 胡本祥, 等. 2021.水杨酸对秦艽愈伤组织总酚·总黄酮含量及抗氧化酶活性的影响[J]. 安徽农业科学, 49(02): 165-169. (Lei R X, Yang B Y, Hu B X, et al.2021. Effect of salicylic acid on total phenol flavonoids content and antioxidase activity in Gentiana callus[J]. Anhui Agricultural Science, 49(02): 165-169.) [10] 李蕾, 谢丙炎, 戴小枫, 等. 2005. WRKY转录因子及其在植物防御反应中的作用[J]. 分子植物育种, 3(03): 401-408. (Li L, Xie B Y, Dai X F, et al.2005. WRKY transcription factor and its role in plant defense response[J]. Molecular Breeding of Plants, 3(03): 401-408.) [11] 李丽, 罗欢. 2021. 外源水杨酸处理对采后金秋梨贮藏品质的影响[J]. 现代园艺, 44(10): 1-3. (Li L, Luo H.2021. Effect of exogenous salicylic acid treatment on the golden autumn pear storage quality after harvest[J]. Modern Gardening, 44(10): 1-3.) [12] 林晓姿, 李维新, 何志刚, 等. 2007. 葡萄罐头加工的去皮剂筛选及工艺参数优化[J]. 华南热带农业大学学报, 13(04): 17-20. (Lin X Z, Li W X, He Z G, et al.2007. Screening of peeling agents for grape canning and optimization of processing parameters[J]. Journal of South China University of Tropical Agriculture, 13(04): 17-20.) [13] 马培恰, 吴文, 唐小浪, 等, 2009. 采前喷布水杨酸(SA)和赤霉素(GA3)对采后贡柑果实生理及贮藏效果的影响[J]. 果树学报, 26(06): 891-894. (Ma P Q, Wu W, Tang X L, et al.2009. Effects of pre -harvest spraying with SA and GA3 on physiology and storability of post-harvest Gonggan mandarin cultivar fruits[J]. Journal of Fruit Science, 26(06): 891-894.) [14] 潘英杰, 张颖, 武杞蔓,等. 2021. WRKY对糖调控园艺作物冷适应的研究进展[J]. 生物技术通报, 38(3): 12-21. (Pan Y J, Zhang Y, Wu Q M, et al.2021. Research progress of WRKY on sugar regulating cold adaptation of horticultural crops[J]. Journal of Agricultural Biotechnology, 38(3): 12-21.) [15] 乔恒波. 2016. 中国野生毛葡萄WRKY3转录因子基因克隆与功能研究. [D]. 硕士学位论文, 西北农林科技大学, 导师: 范崇辉, pp. 20-22. (Qiao H B.2016. Cloning and functional study of WRKY3 transcription factor gene of wild grape in China[D]. Thesis for M.S., Northwest A&F University, Supervisor: Fan C H, pp. 20-22.) [16] 苏玲, 王鹏飞, 杨阳, 等. 2019. 葡萄全基因组WRKY转录因子鉴定和分析[J]. 黑龙江农业科学, 1: 13-22. (Su L, Wang P F, Yang Y, et al.2019. Genome-wide idntification and analysis of WRKY transcription factors in grape[J]. Heilongjiang Agricultural Sciences, 1: 13-22.) [17] 王海波, 王宝亮, 王孝娣, 等. 2010. 我国葡萄产业现状与存在问题及发展对策[J]. 中国果树, 6: 69-71. (Wang H B, Wang B L,Wang X D, et al.2010. Status, Problems and development countermeasures of grape industry in China[J]. China Fruit, 6: 69-71.) [18] 温鹏飞, 陈建业, 李景明, 等. 2006. 葡萄果实中黄烷-3-醇及其聚合体的HPLC检测[J]. 园艺学报, 33(4): 714-720. (Wen P F, Chen J Y, Li J M, et al.2006. Determination of monomeric and polymeric flavan -3- ols in grape berry by high performance liquid chromatography[J]. Acta Horticulturae Sinica. 33(4): 714-720.) [19] 肖培连, 冯睿杰, 侯丽霞, 等. 2015. 葡萄WRKY18基因的克隆及表达特性分析[J]. 植物生理学报, 51(3): 391-398. (Xiao P L, Feng R J, Hou L X, et al.2015. Analysis of the cloning and expression characteristics of the grape WRKY18 gene[J]. Plant Physiology Journal, 51(3): 391-398.) [20] 于和平. 2013. 葡萄WRKY26、WRKY27基因启动子克隆及功能分析[D]. 硕士学位论文, 辽宁师范大学, 导师: 侯和胜, pp. 31. (Yu H P.2013. Cloning and functional analysis of the promoters of VvWRKY26 and VvWRKY27 genes in grape[D]. Thesis for M.S., Liaoning Normal University, Supervisor: Hou H S, pp. 31.) [21] 翟彩霞, 马春红, 王立安, 等. 2004. 水杨酸(SA)诱导玉米抗小斑病研究[J]. 河北农业科学, 8(02): 57-60. (Zhan C X, Ma C H, Wang L A, et al.2004. Study on resistance of maize to leaf spot induced by salicylic acid[J]. HeBei Agricultural Science, 8(02): 57-60.) [22] 张郎郎. 2018. VaWRKY12和VaWRKY14调控山葡萄低温或干旱应答的机理研究[D]. 博士学位论文, 中国科学院大学, 导师: 李绍华, pp. 45-46, 77-78. (Zhang L L.2018. Study on the mechanism of VaWRKY12 and VaWRKY14 regulating the response of Vitis amurensis to low temperature or drought[D]. Thesis for Ph.D, The University of the Chinese Academy of Sciences, supervisor: Li S H, pp. 45-46, 77-78. ) [23] 张桐, 李智强, 伍国强. 2021. WRKY转录因子在植物逆境响应中的作用[J]. 生物技术通报, 37(10): 203-215. (Zhang T, Li Z Q, Wu G Q.2021. Role of WRKY transcription factor in plant stress response[J]. Journal of Agricultural Biotechnology, 37(10): 203-215.) [24] 朱丹, 马倩, 郝杰, 等. 2016. 葡萄WRKY家族蛋白在非生物胁迫中的功能探讨[J]. 生物技术通报, 32(10): 77-83. (Zhu D, Ma Q, Hao J, et al.2016. Function exploration of grape WRKY family proteins under abiotic stresses[J]. Biotechnology Bulletin, 32(10): 77-83) [25] Amato A, Cavallini E, Zenoni S, et al.2017. A grapevine TTG2-like WRKY transcription factor is involved in regulating vacuolar transport and flavonoid biosynthesis[J]. Froniters in Plant Science, 7: 1979-1998. [26] Amiri S, Nicknam Z, Radi M, et al.2021. Postharvest quality of orange fruit as influenced by salicylic acid, acetic acid, and carboxymethyl cellulose coating[J]. Journal of Food Measurement and Characterization, 2021(15): 3912-3930. [27] Chen T Y, Ferruzzi M G, Wu Q L, et al.2017. Influence of diabetes on plasma pharmacokinetics and brain bioavailability of grape polyphenols and their phase Ⅱ metabolites in the Zucker diabetic fatty rat[J]. Molecular Nutrition & Food Research, 61(10): 1700111-1700140. [28] Dong Q L, Zheng W Q, Duan D Y, et al.2020. MdWRKY30, a group Ⅱa WRKY gene from apple, confers tolerance to salinity and osmotic stresses in transgenic apple callus and Arabidopsis seedlings[J]. Plant Science, 299: 110611-110622. [29] Feizi H, Moradi R, Pourghasemian N, et al.2021. Assessing saffron response to salinity stress and alleviating potential of gamma amino butyric acid, salicylic acid and vermicompost extract on salt damage[J]. South African Journal of Botany, 141(9): 330-343. [30] Huang T, Yang J L, Yu D, et al.2021. Bioinformatics analysis of WRKY transcription factors in grape and their potential roles prediction in sugar and abscisic acid signaling pathway[J]. Journal of Plant Biochemistry and Biotechnology, 1(30): 67-80. [31] Ishida T, Hattori S, Sano R, et al.2007. Arabidopsis TRANSPARENT TESTA GLABRA2 is directly regulated by R2R3 MYB transcription factors and is involved in regulation of GLABRA2 transcription in epidermal differentiation[J]. Plant Cell, 19(8): 2531-2543. [32] Jiang J Z, Xi H F, Dai Z W, et al.2019. VvWRKY8 represses stilbene synthase gene through direct interaction with VvMYB14 to control resveratrol biosynthesis in grapevine[J]. Journal of Experimental Botany, 7(2): 715-729. [33] Johnson C S, Kolevski B, Smyth D R.2002. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor[J]. The Plant Cell Online, 14(6): 73-78. [34] Li Q Y, Yin M, Li Y P, et al.2015. Expression of Brassica napus TTG2, a regulator of trichome development, increases plant sensitivity to salt stress by suppressing the expression of auxin biosynthesis genes[J]. Journal of Experimental Botany. 66(19): 5821-5836. [35] Li W, Wang H P, Yu D Q.2016. Arabidopsis WRKY transcription factors WRKY12 and WRKY13 oppositely regulate flowering under short-day conditions[J]. Molecular Plant (Cell Press), 9(11): 1492-1503. [36] Li X, Zhang Y L.2020. A structural view of salicylic acid perception[J]. Nature Plants, 6(10): 1197-1198. [37] Liang C M, Yang B, Wei Y, et al.2021. SA incubation induced accumulation of flavan-3-ols through activated VvANR expression in grape leaves[J]. Scientia Horticulturae. 287:110296-110396. [38] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-∆∆CT) method[J]. Methods, 25(4): 402-408. [39] Mao Z L, Jiang H Y, Wang S, et al.2021. The MdHY5-MdWRKY41-MdMYB transcription factor cascade regulates the anthocyanin and proanthocyanidin biosynthesis in red-fleshed apple[J]. Plant Science, 306: 110848-110858. [40] Mekoue Nguela J., Vernhet A., Julien-Ortiz A,et al.2019. Effect of grape must polyphenols on yeast metabolism during alcoholic fermentation[J]. Food Research International, 2019(121): 161-175. [41] Peng Y Y, Thrimawithana A H, Cooney J M, et al.2020. The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway[J]. Scientific Reports, 10(1): 14161-14180. [42] Qu C M, Fu F Y, Lu K, et al.2013. Differential accumulation of phenolic compounds and expression of related genes in black- and yellow-seeded Brassica napus[J]. Journal of Experimental Botany, 64(10): 2885-2898. [43] Sajjad R H, Vahid H, Reza M A, et al.2021. Improving the drought tolerance in pistachio (Pistacia vera) seedlings by foliar application of salicylic acid[J]. Gesunde Pflanzen, 73(4): 1-13. [44] Verweij W, Spelt C E, Di Sansebastiano G P, et al.2008. An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour[J]. Nature Cell Biology, 10(12): 1456-1462. [45] Verweij W, Spelt C E, Bliek M.et al.2016. Functionally similar WRKY proteins regulate vacuolar acidification in Petunia and hair development in Arabidopsis[J]. The Plant Cell, 28(3): 786-803. [46] Wang X H, Guo R R, Tu M X, et al.2017. Ectopic expression of the wild grape WRKY transcription factor VqWRKY52 in Arabidopsis thaliana enhances resistance to the biotrophic pathogen powdery mildew but not to the necrotrophic pathogen Botrytis cinerea[J]. Frontiers in Plant Science, 8: 97-109. [47] Yang B, Wei Y, Liang C M, et al.2021. Vvanr silencing promotes expression of vvans and accumulation of anthocyanin in grape berries[J]. Protoplasma, 259(2022): 743-753. [48] Zhang Y, Feng J C.2014. Identification and characterization of the grape WRKY family[J]. BioMed Research International, 2014(18): 787680-787693. [49] Zhao D Y, Simon J E, Wu Q L.2020. A critical review on grape polyphenols for neuroprotection: Strategies to enhance bioefficacy[J]. Critical Reviews in Food Science and Nutrition, 60(4): 597-625. [50] Zhao X Y, Qi C H, Jiang H, et al.2019. MdWRKY46-enhanced apple resistance to botryosphaeria dothidea by activating the expression of MdPBS3.1 in the salicylic acid signaling pathway[J]. Molecular Plant-Microbe Interactions, 32(10): 1391-1401. [51] Zhu D, Che Y M, Xiao P L, et al.2018. Functional analysis of a grape WRKY30 gene in drought resistance[J]. Plant Cell Tissue and Organ Culture, 132(3): 449-459.