水曲柳FmWRKY44基因克隆及表达分析

doi:10.7525/j.issn.1673-5102.2019.04.007

摘要/Abstract

摘要： 克隆水曲柳FmWRKY44基因，探究其在非生物胁迫和激素胁迫中的作用。利用水曲柳干旱转录组序列设计特异引物，克隆FmWRKY44基因的完整ORF序列，并对该序列及其编码产物进行生物信息学分析，采用qRT-PCR技术分析FmWRKY44表达模式。克隆了一个水曲柳WKRY基因，编码区长1 383 bp，编码460氨基酸。对其编码蛋白分析发现其为稳定亲水蛋白，亚细胞定位预测主要在细胞核，进行保守域及同源性分析分析，属于WRKYⅠ类家族，命名为FmWRKY44。qRT-PCR分析发现，FmWRKY44在种子中高度表达，并不同程度响应低温、高温、盐和干旱4种非生物胁迫。同时发现FmWRKY44与 NAA、ABA、GA₃、JA植物激素响应，水曲柳FmWRKY44基因积极响应低温、高温胁迫。

关键词: 水曲柳, WRKY基因, 基因克隆, 表达分析

Abstract: To clone the FmWRKY44 gene from Fraxinus mandshurica and explore its role in abiotic stress and hormone stress, specific primers were designed using the sequences of the variceal drought-transcriptome, the complete ORF sequence of FmWRKY44 gene was cloned, and bioinformatics analysis was carried out on the sequence and its coded products. The expression pattern of FmWRKY44 was analyzed using qRT-PCR. A WKRY gene of F. mandshurica was cloned, encoding 1 383 bp, encoding 460 amino acid. It was found to be a stable hydrophilic protein by the analysis of its encoded protein. The subcellular localization was mainly predicted in the nucleus, and the conserved domain and homology were analyzed. It belonged to the WRKY class I family and was named FmWRKY44. By qRT-PCR analysis, FmWRKY44 was highly expressed in the seeds. Different degrees of response to low temperature, high temperature, salt and drought four abiotic stresses. FmWRKY44 and NAA, ABA, GA₃, JA phytohormone responses were also found. FmWRKY44 gene of F. mandshurica positively responds to low temperature and high temperature stress.

Key words: Fraxinus mandshurica, WRKY gene, gene cloning, expression analysis of gene

中图分类号:

Q949.776.2

李新宇, 何利明, 詹亚光. 水曲柳FmWRKY44基因克隆及表达分析[J]. 植物研究, 2019, 39(4): 529-538.

LI Xin-Yu, HE Li-Ming, ZHAN Ya-Guang. Cloning and Expression Analysis of FmWRKY44 Gene in Fraxinus mandshurica[J]. Bulletin of Botanical Research, 2019, 39(4): 529-538.

参考文献

1. Liu X T,Cao X Q,Shi S C,et al. Comparative RNA-Seq analysis reveals a critical role for brassinosteroids in rose(Rosa hybrida) petal defense against Botrytis cinerea infection[J]. BMC Genet,2018,19:62.
2. 郑超,郑二松,王栩鸣,等. 水稻WRKY转录调控因子研究进展[J]. 生物技术通讯,2018,29(2):286-294.
Zheng C,Zheng E S,Wang X M,et al. Research progress on rice WRKY transcription factors[J]. Letters in Biotechnology,2018,29(2):286-294.
3. Rushton P J,Torres J T,Parniske M,et al. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J]. EMBO Journal,1996,15(20):5690-5700.
4. Liu J J,Xiang Y. In silico mining and PCR-based approaches to transcription factor discovery in non-model plants:gene discovery of the WRKY transcription factors in conifers[J]. Methods in Molecular Biology,2011,754:21-43.
5. Li H E,Xu Y,Xiao Y,et al. Expression and functional analysis of two genes encoding transcription factors,VpWRKY1 and VpWRKY2,isolated from Chinese wild Vitis pseudoreticulata[J]. Planta,2010,232(6):1325-1337.
6. Zhang Y J,Wang L J. The WRKY transcription factor superfamily:its origin in eukaryotes and expansion in plants[J]. BMC Evolutionary Biology,2005,5:1.
7. Eulgem T,Rushton P J,Robatzek S,et al. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science,2000,5(5):199-206.
8. 李珍,华秀婷,张积森. 高等植物WRKY转录因子家族的演化及功能研究进展[J]. 热带作物学报,2018,39(2):405-414.
Li Z,Hua X T,Zhang J S. Evolution and gene function of WRKY transcription factor families in higher plants[J]. Chinese Journal of Tropical Crops,2018,39(2):405-414.
9. Wang C T,Ru J N,Liu Y W,et al. Maize WRKY transcription factor ZmWRKY106 confers drought and heat tolerance in transgenic plants[J]. International Journal of Molecular Sciences,2018,19(10):3046.
10. He L,Wu Y H,Zhao Q,et al. Chrysanthemum DgWRKY2 gene enhances tolerance to salt stress in transgenic chrysanthemum[J]. International Journal of Molecular Sciences,2018,19(7):2062.
11. Bao W Q,Wang X W,Chen M,et al. A WRKY transcription factor,PcWRKY33,from Polygonum cuspidatum reduces salt tolerance in transgenic Arabidopsis thaliana[J]. Plant Cell Reports,2018,37(7):1033-1048.
12. Kim C Y,Vo K T X,Nguyen C D,et al. Functional analysis of a cold-responsive rice WRKY gene,OsWRKY71[J]. Plant Biotechnology Reports,2016,10(1):13-23.
13. 丛庆,张琪,宋丽莉,等. 激素在植物冷胁迫应答中的角色[J]. 核农学报,2016,30(3):614-619.
Cong Q,Zhang Q,Song L L,et al. Roles of phytohormones on cold response in plants[J]. Journal of Nuclear Agricultural Sciences,2016,30(3):614-619.
14. Zhang Y,Yu H J,Yang X Y,et al. 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[J]. Plant Physiology and Biochemistry,2016,108:478-487.
15. 孙丽敏,裴雁曦,刘志强. H2S信号和WRKY在ABA调节根生长和气孔运动中的关系[J]. 植物研究,2016,36(1):97-104.
Sun L M,Pei Y X,Liu Z Q. Relationship between H2S signal and WRKY in ABA regulating root growth and stomatal movement[J]. Bulletin of Botanical Research,2016,36(1):97-104.
16. Li S J,Zhou X,Chen L G,et al. Functional characterization of Arabidopsis thaliana WRKY39 in heat stress[J]. Molecules and Cells,2010,29(5):475-483.
17. Zhang L Y,Gu L K,Ringler P,et al. Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells[J]. Plant Science,2015,236:214-222.
18. Zhang L P,Chen L G,Yu D Q. Transcription factor WRKY75 interacts with DELLA proteins to affect flowering[J]. Plant Physiology,2018,176(1):790-803.
19. 陆爱君,曾凡顺,冯健,等. 我国水曲柳育苗技术研究进展[J]. 辽宁林业科技,2015,(3):43-45,76.
Lu A J,Zeng F S,Feng J,et al. Research progress on mulberry seedling technology in China[J]. Liaoning Forestry Science and Technology,2015,(3):43-45,76.
20. 刘颖,何利明,赵兴堂,等. 水曲柳WRKY40基因的克隆及表达模式分析[J]. 分子植物育种,2017,15(3):833-838.
Liu Y,He L M,Zhao X T,et al. Cloning and expression pattern analysis of FmWRKY40 gene in Fraxinus mandshurica[J]. Molecular Plant Breeding,2017,15(3):833-838.
21. Li D H,Liu P,Yu J Y,et al. Genome-wide analysis of WRKY gene family in the sesame genome and identification of the WRKY genes involved in responses to abiotic stresses[J]. BMC Plant Biology,2017,17:152.
22. 郝林,徐昕. 植物转录因子WRKY家族的结构及功能[J]. 植物生理学通讯,2004,40(2):260-265.
Hao L,Xu X. The structure and function of WRKY superfamily of plant transcription factors[J]. Plant Physiology Communications,2004,40(2):260-265.
23. Finatto T,Viana V E,Woyann L G,et al. Can WRKY transcription factors help plants to overcome environmental challenges?[J]. Genetics and Molecular Biology,2018,41(3):533-544.
24. De Zelicourt A,Colcombet J,Hirt H. The role of MAPK modules and aba during abiotic stress signaling[J]. Trends in Plant Science,2016,21(8):677-685.
25. Jiang J J,Ma S H,Ye N H,et al. WRKY transcription factors in plant responses to stresses[J]. Journal of Integrative Plant Biology,2017,59(2):86-101.
26. Sheikh A H,Eschen-Lippold L,Pecher P,et al. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana[J]. Frontiers in Plant Science,2016,7:61.
27. 孙晓梅. MAPK-WRKY模块调节质体反向信号的研究[D]. 上海:上海师范大学,2018.
Sun X M. Study on MAPK-WRKY module regulates plastid retrograde signaling[D]. Shanghai:Shanghai Normal University,2018.
28. 王娜,张振葆,黄凤珠,等. WRKY转录因子参与植物非生物胁迫应答的研究进展[J]. 核农学报,2014,28(10):1819-1827.
Wang N,Zhang Z B,Huang F Z,et al. Advance on the role of WRKY transcription factors in abiotic stress response[J]. Journal of Nuclear Agricultural Sciences,2014,28(10):1819-1827.
29. Geilen K,Heilmann M,Hillmer S,et al. WRKY43 regulates polyunsaturated fatty acid content and seed germination under unfavourable growth conditions[J]. Scientific Reports,2017,7(1):14235.
30. Wang C T,Ru J N,Liu Y W,et al. The maize WRKY transcription factor ZmWRKY40 confers drought resistance in transgenic Arabidopsis[J]. International Journal of Molecular Sciences,2018,19(9):2580.
31. Cheng M N,Huang Z J,Hua Q Z,et al. The WRKY transcription factor HpWRKY44 regulates CytP450-like1 expression in red pitaya fruit(Hylocereus polyrhizus)[J]. Horticulture Research,2017,4:17039.

[1]	单超然, 陈晓慧, 丁云飞, 赵威, 卢晗, 高尚珠, 齐凤慧, 詹亚光, 曾凡锁. 水曲柳FmCCoAOMT基因在木质素合成及非生物胁迫中的功能分析[J]. 植物研究, 2023, 43(5): 768-778.
[2]	张昊楠, 陈珊珊, 徐建民, 罗萍, 王晓萍, 许志茹, 范春节. 巨桉EgrWAT1基因克隆和功能初步分析[J]. 植物研究, 2023, 43(4): 601-611.
[3]	矫春晶, 李明月, 张鹏. 外源激素浸种与渗透处理对水曲柳种子热休眠的作用[J]. 植物研究, 2023, 43(3): 370-378.
[4]	申晨静, 武文博, 耿露冉, 王福龙, 赵鹏舟, 宋金辉, 詹亚光, 尹静. 水杨酸、纳米氧化锌和促生真菌YZ13-1在水曲柳抵御干旱胁迫中的调控作用[J]. 植物研究, 2023, 43(3): 388-395.
[5]	任悦, 魏骋, 徐添添, 沈海龙, 杨玲. 超低温保存处理对水曲柳胚胎生理生化特征的影响[J]. 植物研究, 2023, 43(3): 396-403.
[6]	宋海云, 张涛, 贺鹏, 郑树芳, 王立丰, 王文林. 澳洲坚果MibZIP1基因克隆及表达规律分析[J]. 植物研究, 2023, 43(1): 131-139.
[7]	黄安瀛, 夏德安, 张洋, 那冬晨, 燕青, 魏志刚. PtrWRKY51基因的克隆及抗旱表达特性分析[J]. 植物研究, 2022, 42(6): 1005-1013.
[8]	陈华峰, 代龙军, 刘明洋, 郭冰冰, 杨洪, 王立丰. 橡胶树胶乳高表达热激蛋白HbHSP90.4基因抗逆功能分析[J]. 植物研究, 2022, 42(6): 1023-1032.
[9]	刘明洋, 肖化兴, 王立丰, 梁晓宇, 张宇, 王萌. 橡胶树热激蛋白HbHSP90.8-1基因的克隆与功能分析[J]. 植物研究, 2022, 42(5): 811-820.
[10]	王宏鹏, 李一丹, 汪耀, 谭晓宇, 陈成彬, 张力鹏. 菊叶薯蓣DcPMK基因克隆及互作蛋白筛选[J]. 植物研究, 2022, 42(5): 855-865.
[11]	齐凤慧, 王雯萱, 刘林, 汤明硕, 宋飞翔, 詹亚光. 基于液—固交替培养的水曲柳快速微繁系统研究[J]. 植物研究, 2022, 42(2): 184-190.
[12]	尹一卜, 李吉祥, 郭樱杰, 芦子廷, 肖英, 刘华领, 詹亚光, 曾凡锁. 白蜡属种间杂交子代木质素含量变异及FmPAL核苷酸多态性关联分析[J]. 植物研究, 2022, 42(2): 191-199.
[13]	杨宇宁, 董昊, 董实伟, 王乃锐, 宋跃, 张含国, 李淑娟. 长白落叶松转录因子LobHLH34克隆及表达分析[J]. 植物研究, 2022, 42(1): 112-120.
[14]	马霜, 王博雅, 曹颖, 胡尚连, 高志民. 毛竹扩展蛋白基因的鉴定及其表达分析[J]. 植物研究, 2022, 42(1): 29-38.
[15]	谭秋锦, 张涛, 韦媛荣, 郑树芳, 汤秀华, 王文林. 澳洲坚果开花相关MiMADS-box基因克隆及表达载体构建[J]. 植物研究, 2021, 41(6): 888-895.